Human antibody against bone morphogenetic protein 6

Abstract

The present invention provides antibodies that bind to BMP6 and methods of use. According to certain embodiments of the present invention, the antibodies are fully human antibodies that bind to BMP6. The antibodies of the present invention are useful for inhibiting the binding of BMP6 to hemojuvelin receptors, thereby downregulating the transcription and expression of hepcidin, thereby providing a means for preventing or treating iron deficiency anemia or iron deficiency-related disorders. In some embodiments, the antibodies of the present invention are used to treat at least one symptom or complication of iron deficiency anemia or iron deficiency-related disorders.

Description

[Technical Field]

[0001] Priority Data This application claims priority to U.S. Provisional Application No. 62 / 860,597, filed June 12, 2019, entitled "HUMAN ANTIBODIES TO BONE MORPHOGENETIC PROTEIN 6," which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listing This application incorporates by reference the Sequence Listing, which was filed in computer readable format as file 0431_31PCT_ST25, created on June 5, 2019, and contains 43.2 kilobytes.

[0003] The present invention relates to human antibodies and antigen-binding fragments thereof that specifically bind to bone morphogenetic protein 6 (BMP6), and to therapeutic and diagnostic methods using such antibodies and fragments. [Background technology]

[0004] BMP6 is a member of the TGF-β superfamily of bone morphogenetic proteins, or BMPs. BMP6 has been shown to be involved in a variety of physiological processes, including in particular the regulation of iron levels.

[0005] Nutritional iron uptake is mediated by ferric iron reductase, which converts Fe 3+ reduction of Fe and the subsequent transport of Fe across the apical membrane of enterocytes 2+ Transport of Fe from enterocytes to plasma 2 + Ferroportin-mediated efflux of iron is critical for systemic iron homeostasis. This process is negatively regulated by hepcidin, a liver-derived peptide hormone that binds to ferroportin and promotes its phosphorylation, internalization, and lysosomal degradation.

[0006] Hepcidin expression is transcriptionally regulated. Iron-dependent induction of hepcidin requires BMP (bone morphogenetic protein) signaling. Iron triggers BMP6 expression in liver sinusoidal endothelial cells due to binding to BMP receptors on the surface of hepatocytes. BMP6 signaling leads to phosphorylation of SMAD1 / 5 / 8 and their translocation to the nucleus, along with SMAD4, where they promote hepcidin transcription upon binding to proximal and distal sites on its promoter.

[0007] Hemojuvelin (HJV, HFE2) is a co-receptor for BMP6 that enhances BMP6 signaling in the liver to induce hepcidin expression. Mutations in hemojuvelin (HJV) have been found to lead to low hepcidin levels and excessive iron accumulation in the liver.

[0008] Antibodies that block BMP6 binding to hemojuvelin or its type II BMP receptor have shown that reduction of such binding reduces plasma hepcidin levels, which in turn reduces the amount of Fe transport from enterocytes into plasma. 2+ This promotes ferroportin-mediated efflux of iron, thus offering a promising treatment for conditions associated with low plasma iron levels. Summary of the Invention [Means for solving the problem]

[0009] The present invention provides fully human monoclonal antibodies (mAbs) and antigen-binding fragments thereof that specifically bind to bone morphogenetic protein 6 (BMP6). Such antibodies may be useful for treating conditions associated with low plasma iron levels. The antibodies act to reduce the transcription of hepcidin, which then reduces the uptake of Fe from enterocytes into plasma. 2+Such antibodies can promote ferroportin-mediated efflux of BMP6. Such antibodies can prevent, halt the progression of, or reduce the severity of, conditions associated with low plasma iron levels, or ameliorate at least one symptom associated with such conditions, including, but not limited to, extreme fatigue, weakness, pale skin, chest pain, rapid heartbeat, heart palpitations, shortness of breath, headache, dizziness, lightheadedness, cold hands, cold feet, tongue inflammation, and restless legs. In some cases, the antibodies can be used to prevent or treat conditions or symptoms associated with low plasma iron levels, such as iron deficiency anemia or iron deficiency-related disorders. Such antibodies can be used alone or in combination with a second agent useful in treating iron deficiency anemia or iron deficiency-related disorders. In certain embodiments, antibodies specific for BMP6 can be given therapeutically in combination with a second agent to prevent, halt the progression of, or reduce the severity of conditions associated with low plasma iron levels, or to ameliorate at least one symptom associated with such conditions. In certain embodiments, antibody can be used as a preventive monotherapy to protect patients at risk of developing iron deficiency anemia or iron deficiency-related disorders.For example, certain patient groups, including elderly patients or patients who have experienced blood loss, may be at risk of developing iron deficiency anemia or iron deficiency-related disorders.All of these patient groups can benefit from the treatment with the antibody of the present invention when given alone or in combination with a second drug.

[0010] The antibodies of the present invention can be used to treat iron deficiency anemia or iron deficiency-related disorders in patients. The antibodies can be full-length (e.g., IgG1 or IgG4 antibodies) or can contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and can be modified to affect functionality, for example, to eliminate residual effector function (Reddy et al., (2000), J. Immunol. 164:1925-1933) or to increase mAb half-life (Zalevsky et al., (2010), Nature Biotechnology 28:157-159). The present invention provides antibodies comprising a V antibody identified herein linked to a heavy chain constant region (e.g., a human constant region), such as gamma (e.g., gamma-1, gamma-2, gamma-3, or gamma-4), delta, alpha, mu, or epsilon. H and / or any V identified herein linked to a light chain constant region (e.g., a human constant region), such as lambda or kappa. L The present invention includes any antibody or antigen-binding fragment thereof, including any region thereof.

[0011] Accordingly, in a first aspect, the present invention provides an isolated fully human monoclonal antibody or antigen-binding fragment thereof that specifically binds to BMP6.

[0012] In one embodiment, the isolated human antibody or antigen-binding fragment thereof has a denaturing activity of 10 or more, as measured by surface plasmon resonance. -7 K below M D It binds to BMP6.

[0013] In some embodiments, the isolated antibody or antigen-binding fragment thereof has (a) a binding dissociation equilibrium constant (K) of less than about 2 nM at 37°C, as measured by surface plasmon resonance. D ), (b) binds to human BMP6 with a dissociation half-life (t) of greater than about 130 minutes at 37°C as measured by surface plasmon resonance; and (c) a K of less than about 1 nM at 25°C as measured by surface plasmon resonance.D (d) binds to human BMP6 with a t of greater than about 180 minutes at 25°C as measured by surface plasmon resonance; (e) binds to human BMP6 with a binding dissociation equilibrium constant (K) of less than about 10 nM at 37°C as measured by surface plasmon resonance. D ), (f) binds to mouse BMP6 with a dissociation half-life (t) of greater than about 70 minutes at 37°C as measured by surface plasmon resonance; (g) a K of less than about 4 nM at 25°C as measured by surface plasmon resonance. D and (h) binds to mouse BMP6 with a t1 / 2 of greater than about 80 minutes at 25°C as measured by surface plasmon resonance.

[0014] In some cases, the isolated human antibody or antigen-binding fragment thereof that binds to BMP6 comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 1 and 3, and / or three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 2 and 4. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within particular heavy chain variable region (HCVR) and / or light chain variable region (LCVR) amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., (1997), J. Mol. Biol. 273:927-948; and Martin et al., (1989), Proc. Natl. Acad. Sci. USA 86:9268-9272. Public databases for identifying CDR sequences within antibodies are also available.

[0015] In some embodiments, the isolated human antibody or antigen-binding fragment thereof that binds to BMP6 comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3.

[0016] In some embodiments, the isolated human antibody or antigen-binding fragment thereof that binds to BMP6 comprises an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4.

[0017] In some cases, the isolated human antibody or antigen-binding fragment thereof that binds to BMP6 comprises (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3, and (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4.

[0018] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to BMP6 is (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 11; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 12; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 13; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 14; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 15; and (f) comprises an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 16.

[0019] In various embodiments, the present invention provides fully human monoclonal antibodies or antigen-binding fragments thereof that bind to BMP6, wherein the antibodies or fragments thereof exhibit one or more of the following characteristics: (i) comprising an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) comprising an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) comprising an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 11, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 14, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. (iv) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 12, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 13, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 14, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 15, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 16, orand / or (v) an LCDR3 domain having a substantially similar sequence thereof, having at least 98%, or at least 99%, sequence identity thereto, as measured by surface plasmon resonance, of 10, -7 K below M D It binds to BMP6.

[0020] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that competes for binding to BMP6 with a reference antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) complementarity-determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3, and a light chain variable region (LCVR) CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4.

[0021] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to the same epitope on BMP6 as a reference antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a CDR of a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3, and a CDR of a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4.

[0022] In some embodiments, the present invention provides an isolated human antibody or antigen-binding fragment thereof that binds to BMP6, wherein the antibody or fragment thereof comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1 / 3 and 2 / 4.

[0023] In another aspect, the present invention provides a nucleic acid molecule encoding an anti-BMP6 antibody or a fragment thereof. Recombinant expression vectors carrying the nucleic acids of the present invention and host cells into which such vectors have been introduced are also contemplated by the present invention, as is a method for producing the antibody by culturing the host cells under conditions that allow antibody production and recovering the produced antibody.

[0024] In some embodiments, the present invention provides an antibody or fragment thereof comprising an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17 and 19, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0025] In some embodiments, the antibody or fragment thereof further comprises an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 18 and 20, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0026] In some cases, the present invention provides an antibody or antigen-binding fragment of an antibody comprising an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23 and 29, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 32, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0027] In some embodiments, the present invention provides an antibody or fragment thereof, further comprising: an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21 and 27, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22 and 28, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24 and 30, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 25 and 31, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0028] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to BMP6 described herein can be linked to a detectable label, eg, a radionuclide label or an MRI-detectable label.

[0029] In another aspect, the present invention provides a pharmaceutical composition comprising an isolated fully human monoclonal antibody or antigen-binding fragment thereof that binds to BMP6 as described above or herein, and a pharmaceutically acceptable carrier or diluent.

[0030] In some embodiments, the pharmaceutical composition comprises a fully human monoclonal antibody that binds to BMP6 and has any one or more of the characteristics described above or herein. In one embodiment, the antibody is -7 K below M Dand binds to BMP6 at SEQ ID NO: 1 / 3. In various embodiments, the composition comprises an antibody that binds to BMP6 and has an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1 / 3 and 2 / 4. The present invention also provides an isolated human antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: (i) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 34 and a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 33, (ii) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 36 and a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0031] In some cases, the present invention features a composition that is a combination of the antibody or antigen-binding fragment of the antibody of the present invention and a second therapeutic agent. The second therapeutic agent can be a small molecule drug, a protein / polypeptide, an antibody, a nucleic acid molecule, such as an antisense oligonucleotide, or an siRNA. The second therapeutic agent can be synthetic or naturally derived. The second therapeutic agent can be any agent that is advantageously combined with the antibody or fragment thereof of the present invention.

[0032] In certain embodiments, the second therapeutic agent may be an agent that serves to counteract or reduce any possible side effects associated with the antibody or antigen-binding fragment of the antibody of the invention, if such side effects occur.

[0033] It will also be understood that the antibodies and pharmaceutically acceptable compositions of the present invention can be used in combination therapy, i.e., the antibodies and pharmaceutically acceptable compositions can be administered simultaneously with, before, or after one or more other desired therapeutic agents or medical procedures. The particular combination of therapies (therapeutics or procedures) used in a combination regimen will take into account the compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It will also be understood that the therapies used may achieve a desired effect for the same disorder (e.g., an antibody may be administered simultaneously with another agent used to treat the same disorder) or may achieve a different effect (e.g., control of any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease or condition are appropriate for the disease or condition being treated. When multiple therapeutic agents are administered simultaneously, dosages may be adjusted accordingly, as recognized in the relevant art.

[0034] In another aspect, the present invention provides a method for preventing, treating, or managing iron deficiency anemia or an iron deficiency-related disorder. In certain embodiments, the present invention provides a method for treating a patient suffering from iron deficiency anemia or an iron deficiency-related disorder, the method comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof that binds to BMP6, or a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof that binds to BMP6, such that iron deficiency anemia or iron deficiency-related disorder is prevented or reduced in severity and / or duration, or at least one symptom or complication associated with the condition or disease is prevented or alleviated, or the frequency and / or duration, or severity of iron deficiency anemia or iron deficiency-related disorder is reduced.

[0035] In some embodiments of the method, a pharmaceutical composition comprising an antibody of the invention is administered to the patient in combination with a second therapeutic agent.

[0036] In embodiments of the invention, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody, is administered subcutaneously, intravenously, intradermally, orally, or intramuscularly.

[0037] In related embodiments, the invention includes the use of an isolated anti-BMP6 antibody or antigen-binding portion of an antibody of the invention in the manufacture of a medicament for the prevention or treatment of a disease or disorder associated with or caused by iron deficiency anemia or an iron deficiency-related disorder. The invention also includes the use of an isolated anti-BMP6 antibody or antigen-binding portion thereof for the prevention or treatment of a disease or disorder associated with or caused by iron deficiency anemia or an iron deficiency-related disorder. In one embodiment, the invention includes the use of an isolated anti-BMP6 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of iron deficiency anemia or an iron deficiency-related disorder. In some cases, the invention includes the use of an anti-BMP6 antibody or antigen-binding fragment thereof described above or discussed herein to treat a patient suffering from or at risk of developing iron deficiency anemia or an iron deficiency-related disorder.

[0038] Other embodiments will be apparent from the detailed description that follows. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. An isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human bone morphogenetic protein 6 (BMP6), wherein the antibody or antigen-binding fragment comprises: (a) a binding-dissociation equilibrium constant (K) of less than about 2 nM at 37°C as measured by surface plasmon resonance; D ) binds to human BMP6, (b) binds to human BMP6 with a dissociation half-life (t) of greater than about 130 minutes at 37°C, as measured by surface plasmon resonance; (c) a K of less than about 0.4 nM at 25°C as measured by surface plasmon resonance D binds to human BMP6 at (d) binds to human BMP6 with a t of greater than about 180 minutes at 25°C, as measured by surface plasmon resonance; and (e) an isolated human monoclonal antibody or antigen-binding fragment thereof that exhibits one or more properties selected from the group consisting of: blocking the interaction between human BMP6 and human hemojuvelin (HJV). (Item 2) An isolated human antibody or antigen-binding fragment thereof that specifically binds to human BMP6, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 1 and 3, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 2 and 4. (Item 3) 3. The isolated human antibody or antigen-binding fragment thereof of item 1 or 2, wherein the antibody or antigen-binding fragment comprises an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3. (Item 4) 4. The isolated human antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the antibody or antigen-binding fragment comprises an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4. (Item 5) 5. The isolated human antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the antibody or antigen-binding fragment comprises: (a) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3; and (b) an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4. (Item 6) the antibody or antigen-binding fragment: (a) an HCDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 11; (b) an HCDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 12; (c) an HCDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 13; (d) an LCDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 14; (e) an LCDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 15, and / or (f) The isolated human antibody or antigen-binding fragment thereof according to any one of items 1 to 5, comprising an LCDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 16. (Item 7) 7. The isolated human antibody or antigen-binding fragment of any one of items 1 to 6, wherein the antibody or antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1 / 3 and 2 / 4. (Item 8) the antibody or antigen-binding fragment: (i) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 34, and a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 33, and / or (ii) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 36, and 8. The isolated human antibody or antigen-binding fragment of any one of items 1 to 7, comprising a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 35. (Item 9) The antigen-binding fragment may be a Fab fragment, F(ab') 2 9. The antigen-binding fragment of any one of items 1 to 8, which is a fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) molecule, or a dAb fragment. (Item 10) An isolated antibody or antigen-binding fragment thereof that binds to the same epitope on human BMP6 as a reference antibody, comprising a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3, and a CDR of a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4. (Item 11) An isolated antibody or antigen-binding fragment thereof that competes with a reference antibody for binding to human BMP6, the antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3, and a complementarity-determining region (CDR) of a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4. (Item 12) A method for producing the antibody or antigen-binding fragment according to any one of items 1 to 11, comprising: (i) introducing into a host cell one or more polynucleotides encoding the immunoglobulin light chain of said antibody or fragment and the immunoglobulin heavy chain of said antibody or fragment; (ii) culturing the host cells in a growth medium under conditions favorable to expression of the polynucleotide; (iii) optionally isolating said antibody or fragment from said host cell and / or the medium in which said host cell is growing. (Item 13) 13. An antibody or antigen-binding fragment that is the product of the method described in item 12. (Item 14) 14. An injection device or container comprising the antibody or antigen-binding fragment of any one of items 1 to 11 and 13. (Item 15) A pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof that binds to human BMP6 described in any one of items 1 to 11 and 13, a pharmaceutically acceptable carrier or diluent, and optionally one or more additional therapeutic agents. (Item 16) 16. The pharmaceutical composition of item 15, comprising the additional therapeutic agent being an iron supplement. (Item 17) 17. A method for preventing or treating iron deficiency anemia or an iron deficiency-related disorder in a patient in need thereof, comprising administering to the patient an effective amount of the antibody or antigen-binding fragment thereof described in any one of items 1 to 11 and 13, or the pharmaceutical composition described in item 15 or 16. (Item 18) 18. The method of claim 17, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, orally, or intramuscularly. (Item 19) the iron deficiency anemia or iron deficiency related disorder results in a condition selected from the group consisting of extreme fatigue, weakness, pale skin, chest pain, rapid heartbeat, heart palpitations, shortness of breath, headache, dizziness, lightheadedness, cold hands, cold feet, inflamed tongue, and restless legs; 19. The method of item 17 or 18, wherein administration of the antibody or antigen-binding fragment treats the condition or reduces the severity of one or more symptoms of the condition. (Item 20) 14. The antibody or antigen-binding fragment thereof according to any one of items 1 to 11 and 13 for use in treating a patient with iron deficiency anemia or an iron deficiency-related disorder. (Item 21) 14. A composition comprising one or more antibodies or antigen-binding fragments thereof according to any one of items 1 to 11 and 13 for use in treating iron deficiency anemia or an iron deficiency-related disorder. (Item 22) 14. Use of the isolated antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 11 and 13 in the manufacture of a medicament for treating a patient with iron deficiency anemia or an iron deficiency-related disorder. [Brief explanation of the drawings]

[0039] [Figure 1] 1 shows that mice receiving the BMP6 antibody H4H17855P of the present invention had increased serum iron compared to mice receiving an isotype control antibody at 10 mg / kg. There was a dose-dependent effect of the BMP6 antibody, with an increase in serum iron at 20 mg / kg compared to 10 mg / kg. [Figure 2] 10 shows that mice receiving the BMP6 antibody H4H17855P of the present invention had reduced serum hepcidin compared to mice receiving an isotype control antibody at 10 mg / kg. There was a dose-dependent effect of the BMP6 antibody, with a reduction in hepcidin at 20 mg / kg compared to 10 mg / kg. [Figure 3] 1 shows that mice receiving the BMP6 antibody of the present invention, H4H17871P, had increased serum iron compared to mice receiving an isotype control antibody. [Figure 4] 1 shows that mice receiving the BMP6 antibody of the present invention, H4H17871P, had reduced serum hepcidin compared to mice receiving an isotype control antibody. DETAILED DESCRIPTION OF THE INVENTION

[0040] Before describing the methods of the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, the preferred methods and materials are described here. All publications mentioned herein are incorporated herein by reference in their entirety.

[0042] definition The terms "BMP6" and "bone morphogenetic protein 6" refer interchangeably to a 57 kDa monomeric protein. BMP6 is a member of the transforming growth factor beta (TGF-beta) superfamily of regulatory molecules. The amino acid sequence of human BMP6 is set forth in SEQ ID NO: 40. The amino acid sequence of mouse BMP6 is set forth in SEQ ID NO: 42. Unless otherwise specified, reference to BMP6 refers to the human form.

[0043] The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule (i.e., a "full antibody molecule") composed of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ") and heavy chain constant region (domain C H 1. C H 2, and C H Each light chain is composed of a light chain variable region ("LCVR" or "VVR"). L ") and the light chain constant region (CL ) V H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0044] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies that can allocate one or two CDRs for binding have been described in the scientific literature. Padlan et al. (FASEB J. 1995, 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0045] CDR residues that do not contact antigen can be identified based on previous research, by molecular modeling and / or empirically.When a CDR or its residue is omitted, it is usually replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such a sequence.The position for substitution within the CDR and the amino acid to be substituted can also be selected empirically.Empirical substitutions can be conservative or non-conservative substitutions.

[0046] The fully human BMP6 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be easily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can easily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence; for example, the mutated residues are found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or the mutated residues are found only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention can contain any combination of two or more germline mutations within the framework and / or CDR regions; for example, certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the scope of the present invention.

[0047] The present invention also includes fully human anti-BMP6 monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes anti-BMP6 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, or 4 or less conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0048] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present invention may include, for example, amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) are grafted onto human FR sequences.

[0049] The terms "specifically bind" or "specifically binds to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is defined as binding to an antigen with a specific binding affinity of at least about 1 x 10 -6 M or less (e.g., a smaller K D (Indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to BMP6 have been identified by surface plasmon resonance, e.g., BIACORE™. Furthermore, multispecific antibodies that bind to one domain of BMP6, and one or more additional antigens or bispecific antibodies that bind to two different regions of BMP6, are still considered to be "specifically binding" antibodies as used herein.

[0050] The term "high affinity" antibody refers to an antibody that has an affinity of at least 10 for BMP6 as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -7 M, preferably 10 -8 M, more preferably 10 -9 M, more preferably 10 -10 M, even more preferably 10 -11 Binding affinity (K DThe term refers to those mAbs with a nucleotide sequence (denoted as nucleotide sequence).

[0051] The term "slow off rate," "Koff," or "kd" refers to a slow off rate of 1×10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 s -1 Less than 1 × 10 -4 s -1 It is meant to describe an antibody that dissociates from BMP6 with the following rate constant:

[0052] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to BMP6.

[0053] In certain embodiments, the antibodies or antibody fragments of the invention may be conjugated to a therapeutic moiety (an "immunoconjugate") such as an antibiotic, a second anti-BMP6 antibody, or an antibody against a cytokine such as IL-1, IL-6, or TGF-β, or any other therapeutic moiety useful in treating iron deficiency anemia or an iron deficiency-related disorder.

[0054] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds to BMP6 is substantially free of Abs that specifically bind to antigens other than BMP6).

[0055] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0056] As used herein, "K D The term "antibody-antigen interaction" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0057] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B and / or T cells respond. It also refers to the region of an antigen bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes, possessing those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural and / or specific charge characteristics.

[0058] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0059] When applied to polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that, when optimally aligned by a program such as GAP or BESTFIT using a predetermined gap weight, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Preferably, non-identical residue positions differ only by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331 (incorporated herein by reference). Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:144345, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0060] The sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG Version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0061] In certain embodiments, antibodies or antibody fragments for use in the methods of the invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for epitopes of two or more target polypeptides.

[0062] The phrase "therapeutically effective amount" means the amount for which it is administered that produces the desired effect. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0063] overview Bone morphogenetic protein 6 (BMP6) is a 57 kDa protein that has been shown to be involved in various physiological processes, including the regulation of iron levels. BMP6 signaling leads to the phosphorylation of SMAD1 / 5 / 8 and the translocation of SMAD4 to the nucleus, where it promotes hepcidin (a negative regulator of serum iron) transcription upon binding to proximal and distal sites on its promoter. Hemojuvelin (HJV, HFE2) is a coreceptor for BMP6 that enhances signal transduction. Reduced binding of BMP6 to HJV reduces hepcidin transcription, thereby promoting serum iron levels.

[0064] The antibodies described herein exhibit specific binding to BMP6 and, in some embodiments, may be useful in treating patients suffering from iron deficiency anemia or iron deficiency-related disorders. They may be used alone or as adjunctive therapy with other therapeutic moieties or modalities known in the art for treating iron deficiency anemia or iron deficiency-related disorders, such as, but not limited to, iron supplementation with iron supplements, dietary changes to promote serum iron and / or intravenous delivery of iron, blood transfusions, and iron-promoting drugs. They may be used in combination with additional antibodies specific for antigens other than BMP6, or may be combined with other types of treatment.

[0065] In some embodiments, the antibodies described herein may be useful in the prevention, treatment, or management of iron deficiency anemia or iron deficiency-related disorders.

[0066] In certain embodiments, antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as native full-length human BMP6 (SEQ ID NO: 40) or a BMP6 fragment, followed by immunization with a secondary immunogen or with an immunogenically active fragment of BMP6.

[0067] The immunogen can be an immunogenic fragment of BMP6 or DNA encoding the fragment. The immunogen can be BMP6 bound to a histidine tag and / or a fragment of the Fc region of an antibody.

[0068] The amino acid sequence of full-length human BMP6 is shown as SEQ ID NO: 40. The full-length amino acid sequence of mouse BMP6 is shown as SEQ ID NO: 42.

[0069] In certain embodiments, antibodies that specifically bind to BMP6 can be prepared using fragments of the above regions or peptides extending beyond the designated regions by about 5 to about 20 amino acid residues from either or both of the N- and C-termini of the regions described herein. In certain embodiments, any combination of the above regions or fragments thereof can be used to prepare BMP6-specific antibodies. In certain embodiments, any one or more of the above regions of BMP6 or fragments thereof can be used to prepare monospecific, bispecific, or multispecific antibodies.

[0070] Antigen-binding fragment of an antibody Unless otherwise specified, the term "antibody," as used herein, should be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "intact antibody molecules"), as well as antigen-binding fragments thereof. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment," refers to one or more fragments of an antibody that retain the ability to specifically bind to BMP6. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard techniques, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0071] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides), or limited FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0072] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V associated with the domain H In an antigen-binding fragment having a domain, V H and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H- V H , V H- V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may include a domain.

[0073] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) V H -CH 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be linked to each other and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domain (e.g., via disulfide bonds).

[0074] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be suitable for use in connection with the antigen-binding fragments of antibodies of the present invention, using routine techniques available in the art.

[0075] The present invention includes anti-BMP6 antibodies and antigen-binding fragments having immunoglobulin chains comprising the amino acid sequences described herein, as well as variants with cellular and / or in vitro post-translational modifications. For example, the present invention includes antibodies and antigen-binding fragments thereof (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3) that specifically bind to BMP6, comprising the heavy and / or light chain amino acid sequences described herein, as well as antibodies and fragments in which one or more amino acid residues are glycosylated, antibodies and fragments in which one or more Asn residues are deamidated, antibodies and fragments in which one or more residues (e.g., Met, Trp, and / or His) are oxidized, antibodies and fragments in which the N-terminal Gln is pyroglutamic acid (pyroE), and / or antibodies and fragments lacking the C-terminal lysine.

[0076] The present invention includes recombinant methods for producing an anti-BMP6 antibody or antigen-binding fragment thereof, or immunoglobulin chains thereof, of the present invention, the methods comprising: (i) producing an immunoglobulin light chain and / or heavy chain (e.g., a heavy chain or a V H or an immunoglobulin comprising HCDR1, HCDR2, and HCDR3 thereof, and / or a light chain or V thereof L(ii) culturing host cells (e.g., Chinese hamster ovary (CHO) cells or Pichia cells or Pichia pastoris cells) under conditions favoring expression of the polynucleotides; and (iii) optionally isolating the antibody or fragment or chain from the host cells and / or the medium in which the host cells are growing. When producing antibodies or antigen-binding fragments comprising two or more immunoglobulin chains, e.g., antibodies comprising two immunoglobulin heavy chains and two immunoglobulin light chains, co-expressing the chains in a single host cell results in the association of the chains intracellularly or on the cell surface or extracellularly, e.g., when such chains are secreted, to form the antibody or antigen-binding fragment molecule. The methods include those in which only immunoglobulin heavy chains or only immunoglobulin light chains (e.g., any of those discussed herein, including mature fragments and / or variable domains thereof) are expressed. Such chains are useful, for example, as intermediates in the expression of antibodies or antigen-binding fragments comprising such chains. The invention relates to the products of such expression methods (e.g., antibodies, antigen-binding fragments, V H , or V L ) is included.

[0077] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to generate human antibodies that specifically bind to BMP6.

[0078] Using VELOCIMMUNE® technology (e.g., US Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, a high-affinity chimeric antibody against BMP6 having a human variable region and a mouse constant region is first isolated. VELOCIMMUNE® technology involves the generation of transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0079] Generally, VELOCIMMUNE® mice are inoculated with an antigen of interest, and lymphocytes (such as B cells) expressing antibodies are collected from the mice. Lymphocytes can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions can be isolated and linked to the desired heavy and light chain isotype constant regions. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

[0080] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. The antibody is characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with a desired human constant region to generate a fully human antibody of the present invention, such as a wild-type or modified IgG1 or IgG4. The constant region selected can vary depending on the specific application, but the characteristics of high-affinity antigen binding and target specificity reside in the variable region.

[0081] In general, the antibodies of the present invention have very high affinities, as measured by binding to antigen either immobilized on a solid phase or in solution phase, typically on the order of 10 -12 ~about 10 -7 K of M D The mouse constant region is replaced with the desired human constant region to generate the fully human antibody of the present invention. The constant region selected may vary depending on the particular application, but the characteristics of high affinity antigen binding and target specificity reside in the variable region.

[0082] biological equivalent The anti-BMP6 antibodies and antibody fragments of the present invention include proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to BMP6. Such variant antibodies and antibody fragments may contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit essentially the same biological activity as the described antibodies. Similarly, DNA sequences encoding the antibodies of the present invention include sequences that encode antibodies or antibody fragments that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but are essentially biologically equivalent to the antibodies or antibody fragments of the present invention.

[0083] Two antigen-binding proteins or antibodies are considered bioequivalents or pharmaceutical substitutes if, for example, they exhibit no significant difference in the rate and extent of absorption when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies are considered bioequivalents or pharmaceutical substitutes if their extent of absorption is equivalent but their absorption rate is not, and such differences in absorption rate are nevertheless considered bioequivalent because they are intentional, reflected in the labeling, are not essential for achieving effective body drug concentrations for long-term use, and are not considered medically significant for the particular drug product being studied.

[0084] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0085] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy, compared to continuous therapy without switching.

[0086] In one embodiment, two antigen binding proteins are biologically equivalent if they both act by a common mechanism or mode of action for the condition(s) of use, to the extent that such mechanism is known.

[0087] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Methods for measuring bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0088] Biologically equivalent variants of the antibodies of the present invention can be constructed, for example, by various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antibodies can include antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0089] Anti-BMP6 antibodies containing Fc variants According to certain embodiments of the present invention, there are provided anti-BMP6 antibodies comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present invention provides an anti-BMP6 antibody comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThe present invention also includes anti-BMP6 antibodies containing mutations in the 3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH ranging from about 5.5 to about 6.0). Such mutations can increase the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification, a 428L, a 259I (e.g., V259I), and a 308F (e.g., V308F) modification, a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification, a 252, a 254, and a 256 (e.g., 252Y, 254T, and 256E) modification, a 250Q and a 428L modification (e.g., T250Q and M428L), and a 307 and / or a 308 modification (e.g., 308F and / or 308P). In yet another embodiment, the modifications include a 265A (e.g., D265A) and / or a 297A (e.g., N297A) modification.

[0090] For example, the present invention provides 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E), 428L and 434S (e.g., M428L and N434S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A, and 434A (e.g., T307A, E380A, and N434A), and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated within the scope of the present invention.

[0091] The present invention also provides chimeric heavy chain constant (C H ) region, and a chimeric C H The region is composed of C H For example, the antibodies of the present invention may comprise segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C derived from human IgG1, human IgG2, or human IgG4 molecules combined with some or all of the three domains H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies of the present invention may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein may be H Antibodies comprising the region may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Provisional Application No. 61 / 759,578, filed February 1, 2013, the disclosure of which is incorporated herein by reference in its entirety).

[0092] Biological characteristics of antibodies Generally, the antibodies of the present invention may function by binding to BMP6. In some embodiments, the antibodies of the present invention may bind to another antigen (cross-reactive antibody).

[0093] In certain embodiments, the antibodies of the present invention may be bispecific antibodies. Bispecific antibodies of the present invention may bind to one epitope in one domain and also to one epitope in a second domain of BMP6. In certain embodiments, bispecific antibodies of the present invention may bind to two different epitopes in the same domain.

[0094] In one embodiment, the present invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds to BMP6, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) comprises an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) comprises an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 13, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 16, or a sequence of at least 90%, at least 95%, at least 98%, or at least 99% identity. (iv) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 11, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 12, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 14, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 15, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; -7 The following K D It binds to BMP6.

[0095] Certain anti-BMP6 antibodies of the present invention can bind to and neutralize the activity of BMP6, as determined by in vitro or in vivo assays. The ability of antibodies of the present invention to bind to and neutralize the activity of BMP6 can be measured using any standard method known to those skilled in the art, including the binding or activity assays described herein.

[0096] Peptides can be modified to include the addition or substitution of certain residues for tagging or for conjugation to carrier molecules such as KLH. For example, cysteine ​​can be added to either the N-terminus or C-terminus of the peptide, or a linker sequence can be added to prepare the peptide for conjugation to KLH for immunization, for example.

[0097] Antibodies specific for BMP6 may not contain additional labels or moieties, or they may contain N- or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which it binds. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin will be oriented so that the C-terminal portion of the peptide is distal to the surface. In one embodiment, the label can be a radionuclide, a fluorescent dye, or an MRI-detectable label. In certain embodiments, such labeled antibodies can be used in diagnostic assays, including imaging assays.

[0098] Epitope mapping and related techniques The present invention includes anti-BMP6 antibodies that interact with one or more amino acids found in one or more regions of BMP6. The epitope to which the antibody binds can consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the aforementioned regions of the BMP6 molecule (e.g., a linear epitope within a domain). Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located within either or both of the aforementioned regions of the BMP6 molecule (e.g., a conformational epitope).

[0099] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol Biol 248:443-63), peptide truncation analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange, detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo deuterium-hydrogen back exchange at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface can retain deuterium and therefore exhibit a relatively high mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing peptides containing deuterium-labeled residues, including specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0100] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B cell epitopes can be formed from both contiguous amino acids juxtaposed by tertiary folding of a protein or noncontiguous amino acids. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more usually at least five or 8-10 amino acids in a unique spatial conformation.

[0101] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying multiple monoclonal antibodies (mAbs) directed against the same antigen according to the similarity of each antibody's binding profile to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is either distinct or partially overlapping with the epitope represented by another category. This technique allows for rapid filtering of genetically identical antibodies, so that characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones producing mAbs with desired characteristics. MAP can be used to sort the antibodies of the present invention into groups of antibodies that bind to different epitopes.

[0102] In certain embodiments, the anti-BMP6 antibody or antigen-binding fragment thereof binds to an epitope within any one or more of the regions exemplified by human BMP6 as exemplified by SEQ ID NO: 40, or a fragment thereof.

[0103] The present invention includes human anti-BMP6 antibodies that bind to the same epitope or portion of an epitope as any of the specific exemplary antibodies described herein, or antibodies having the CDR sequences of any of the exemplary antibodies described herein. Similarly, the present invention also includes anti-BMP6 antibodies that compete for binding to BMP6 or a BMP6 fragment with any of the specific exemplary antibodies described herein, or antibodies having the CDR sequences of any of the exemplary antibodies described herein.

[0104] Whether an antibody binds to the same epitope as a reference anti-BMP6 antibody or competes for binding with a reference anti-BMP6 antibody can be easily determined by using conventional methods known in the art.For example, to determine whether a test antibody binds to the same epitope as a reference anti-BMP6 antibody of the present invention, the reference antibody is bound to BMP6 protein or peptide under saturating conditions.Then, the ability of the test antibody to bind to BMP6 molecule is evaluated.If the test antibody can bind to BMP6 after saturating binding with the reference anti-BMP6 antibody, it can be concluded that the test antibody binds to a different epitope from the reference anti-BMP6 antibody.On the other hand, if the test antibody cannot bind to BMP6 protein after saturating binding with the reference anti-BMP6 antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-BMP6 antibody of the present invention.

[0105] To determine whether an antibody competes with a reference anti-BMP6 antibody for binding, the above-mentioned binding method is carried out in two directions.In the first direction, the reference antibody is bound to BMP6 protein under saturating conditions, and then the binding of the test antibody to the BMP6 molecule is evaluated.In the second direction, the test antibody is bound to BMP6 molecule under saturating conditions, and then the binding of the reference antibody to the BMP6 molecule is evaluated.In both directions, if only the first (saturating) antibody can bind to the BMP6 molecule, it is concluded that the test antibody and the reference antibody compete for binding to BMP6.As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0106] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0107] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0108] Immunoconjugates The present invention encompasses human anti-BMP6 monoclonal antibodies conjugated to a therapeutic moiety (an "immunoconjugate"), such as a drug capable of reducing the severity of iron deficiency anemia or an iron deficiency-related disorder or ameliorating at least one symptom associated with iron deficiency anemia or an iron deficiency-related disorder. As used herein, the term "immunoconjugate" refers to an antibody chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, or therapeutic agent. The antibody may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, or therapeutic agent at any position along the molecule, as long as it is capable of binding to its target. An example of an immunoconjugate is an antibody-drug conjugate. In some embodiments, the drug may be a second, different antibody against BMP6 or a cytokine such as IL-1 or IL-6, or a chemokine such as TGF-β. The type of therapeutic moiety that can be conjugated to an anti-BMP6 antibody takes into account the condition being treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art, see, for example, WO05 / 103081. The preparation of immunoconjugates and immunotoxins is generally well known in the art (see, for example, U.S. Patent No. 4,340,535). Immunoconjugates are described in detail in, for example, U.S. Patent No. 7,250,492, U.S. Patent No. 7,420,040, and U.S. Patent No. 7,411,046, each of which is incorporated herein in its entirety.

[0109] multispecific antibodies The antibodies of the present invention can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of a single target polypeptide or can contain antigen-binding domains specific for two or more target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The antibodies of the present invention can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical bond, genetic fusion, noncovalent association, or other method) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity. For example, the present invention includes bispecific antibodies in which one immunoglobulin arm is specific for the N-terminal region of BMP6 or a fragment thereof, and the other immunoglobulin arm is specific for the C-terminal region of BMP6 or a second therapeutic target, or is conjugated to a therapeutic moiety. An exemplary bispecific antibody format that can be used in the context of the present invention is H3 Domain and second Ig C H3 The first and second Ig C domains are involved in the H3 The domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to BMP6 compared to a bispecific antibody without the amino acid difference. H3 The domain binds to BMP6 and binds to the second IgC H3 The domain contains a mutation that reduces or abolishes BMP6 binding, such as the H95R modification (according to IMGT exon numbering, H435R in EU numbering). H3 may further comprise a Y96F modification (Y436F in EU according to IMGT). H3Further modifications that may be found within include D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in EU by IMGT), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by IMGT). Variations on the above bispecific antibody formats are contemplated within the scope of the present invention.

[0110] Other exemplary bispecific formats that may be used in the context of the present invention include, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (such as common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats are included (for a review of such formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugates, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and shape. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0111] Therapeutic Administration and Formulations The present invention provides therapeutic compositions comprising the anti-BMP6 antibodies or antigen-binding fragments thereof discussed herein. Therapeutic compositions according to the present invention can be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Many suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN®), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0112] The antibody dosage may vary depending on the age and size of the subject, the target disease, condition, route of administration, etc. When the antibodies of the present invention are used to prevent or treat iron deficiency anemia or iron deficiency-related disorders, they are advantageously administered intravenously in a single dose of typically about 0.1 to about 100 mg / kg body weight, more preferably about 5 to about 100, about 10 to about 90, or about 20 to about 70 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention may be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, about 100 mg, or about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be about the same as or less than the initial dose, with the subsequent doses being separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0113] Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions of the present invention (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, or absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249:1527-1533).

[0114] The use of nanoparticles for delivering the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods for their preparation and use are described in more detail in Arruebo, M., et al. 2009 ("Antibody-conjugated nanoparticles for biomedical applications" in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi:10.1155 / 2009 / 439389) (incorporated herein by reference). Nanoparticles for drug delivery are also described in, for example, US8277812, US8258256, US8257740, US8246995, and US8236330 (each of which is incorporated herein in its entirety).

[0115] In certain circumstances, the pharmaceutical composition can be delivered in a sustained release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the sustained release system can be placed proximal to the target of the composition, thus requiring only a fraction of the systemic dose.

[0116] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, glucose-containing isotonic solutions, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable preparations prepared in this manner are preferably filled into appropriate ampoules.

[0117] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are easily adapted to deliver the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0118] Numerous reusable pen and autoinjector delivery devices are adapted for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include the AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG® pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN® I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD pen (Becton Dickinson, Franklin), to name a few. (Sanofi-Aventis, Frankfurt, Germany), OPTIPEN®, OPTIPEN® PRO, OPTIPEN® STARLET, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices applicable to subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR® pen (Sanofi-Aventis), FLEXPEN® (Novo Nordisk), and KWIKPEN® (HUMALOG®), the SURECLICK® autoinjector (PENLET (Haselmeier, Stuttgart, Germany), EPIPEN® (Mylan®), and the HUMIRA® pen (Abbott Labs, Abbott Park, IL), to name a few.

[0119] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a unit-dose dosage form suitable for the dosage of the active ingredient. Examples of such unit-dose dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories. The amount of the antibody contained is generally about 5 to about 500 mg per unit-dose dosage form. In particular, in the form of an injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and for other dosage forms, about 10 to about 250 mg. The present invention also includes an injection device (e.g., a pre-filled syringe or a pre-filled autoinjector) or a vial (e.g., a glass or plastic vial) containing the antibody or antigen-binding fragment of the present invention, or a pharmaceutical composition thereof containing a pharmaceutically acceptable carrier.

[0120] Therapeutic Uses of Antibodies In certain embodiments of the present invention, the antibodies are useful for treating iron deficiency anemia or iron deficiency-related disorders, or at least one symptom associated with iron deficiency anemia or iron deficiency-related disorders. The antibodies of the present invention are also intended for prophylactic use in patients at risk of developing iron deficiency anemia or iron deficiency-related disorders. These patients include elderly patients or patients with immunocompromised immune systems due to disease or treatment with immunosuppressive therapeutic agents. It is contemplated that the antibodies of the present invention can be used alone or in combination with a second or third agent to treat iron deficiency anemia or iron deficiency-related disorders or to alleviate at least one symptom or complication associated with iron deficiency anemia or iron deficiency-related disorders. The second or third agent can be delivered simultaneously with the antibody of the present invention, or they can be administered separately, either before or after the antibody of the present invention. Patients who may receive an antibody or antigen-binding fragment of the invention, or a pharmaceutical composition thereof, include, for example, animals such as mammals, such as humans (e.g., elderly humans, e.g., 65 years of age or older), rabbits, mice, rats, cows, pigs, dogs, primates, horses, or sheep.

[0121] In a further embodiment of the invention, the antibody is used in the preparation of a pharmaceutical composition for treating a patient suffering from iron deficiency anemia or an iron deficiency related disorder.

[0122] Combination therapy The combination therapy can include an anti-BMP6 antibody of the present invention and any additional therapeutic agent that can be advantageously combined with an antibody of the present invention or a biologically active fragment of an antibody of the present invention.

[0123] The antibodies may be used in combination with other therapies such as moieties or modalities known in the art for treating iron deficiency anemia or iron deficiency-related disorders, including, but not limited to, iron replacement with iron supplements, dietary modifications to enhance serum iron and / or intravenous delivery of iron, blood transfusions, and iron-promoting medications.

[0124] The additional therapeutically active component may be administered prior to, simultaneously with, or after administration of the anti-BMP6 antibody of the present invention. For purposes of this disclosure, such administration regimens are considered to be administration of the anti-BMP6 antibody "in combination with" one or more additional therapeutically active components.

[0125] Diagnostic Uses of Antibodies The anti-BMP6 antibody of the present invention can also be used to detect and / or measure BMP6 in a sample, for example, for diagnostic purposes. An exemplary diagnostic assay for BMP6 can include, for example, contacting a sample obtained from a patient with an anti-BMP6 antibody of the present invention, where the anti-BMP6 antibody is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate BMP6 from the patient sample. Alternatively, an unlabeled anti-BMP6 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P 、35 S, or 125The BMP6 may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure BMP6 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0126] The samples that can be used in the BMP6 diagnostic assay of the present invention include any tissue or body fluid sample that can be obtained from a patient and that contains a detectable amount of BMP6 or any of its fragments under normal or pathological conditions.Generally, the level of BMP6 in a specific sample obtained from a healthy patient (for example, a patient who does not suffer from iron deficiency anemia or iron deficiency-related disorders) is measured to first establish a baseline or standard level of BMP6.Then, this baseline level of BMP6 can be compared with the level of BMP6 measured in a sample obtained from an individual suspected of having a condition related to iron deficiency anemia or iron deficiency-related disorders, or a symptom related to such a condition.

[0127] Antibodies specific for BMP6 may not contain additional labels or moieties, or they may contain N- or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which it binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented so that the C-terminal portion of the peptide is distal to the surface. In some embodiments, the label can be a detectable label, such as a radionuclide, a fluorescent dye, or an MRI-detectable label. The detectable label can be linked to an antibody, and such an antibody can be used in imaging assays. [Example]

[0128] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as the invention. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0129] Example 1. Generation of human antibodies against BMP6 Human antibodies against BMP6 were generated in VELOCIMMUNE® mice, which contain DNA encoding human immunoglobulin heavy and kappa light chain variable regions. Mice were immunized with stabilized full-length BMP6 protein.

[0130] The antibody immune response was monitored by BMP6-specific immunoassay. When the desired immune response was achieved, the splenocytes were collected and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce BMP6-specific antibodies. Using the cell lines, several anti-BMP6 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained.

[0131] Exemplary antibodies generated as disclosed above were designated H4H17855P and H4H17871P. The biological properties of exemplary antibodies generated by the methods of this example are further described in the Examples below.

[0132] Example 2. Amino acid sequences of heavy and light chain variable regions Table 1 shows the heavy and light chain variable region amino acid sequence pairs of selected antibodies specific to BMP6, as well as their corresponding antibody identifiers. Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H"), followed by a numerical identifier (e.g., "7855" shown in Table 1), followed by the suffix "P." Thus, according to this nomenclature, an antibody may be referred to, for example, as "H4H17855P." The H4H prefix in antibody names used herein indicates the specific Fc region of the antibody. For example, an "H4H" antibody has a human IgG4 Fc. [Table 1]

[0133] Example 3. Binding kinetics of human monoclonal antibodies to BMP6 The equilibrium dissociation constants (K DThe binding activity (RI) was determined using a real-time surface plasmon resonance (SPR) biosensor system, MASS-1. All binding studies were performed at 25°C and 37°C in a running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 1 μg / mL heparin, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-EHT). The HCA sensor surface was first derivatized by amine-coupling a monoclonal mouse anti-human Fc antibody (GE, #BR100839), followed by the individual capture of an anti-BMP6 monoclonal antibody. Different concentrations of human BMP6 reagent (hBMP6; R&D Systems, Cat. No. 507-BP; 60 nM to 0.94 nM; 4-fold serial dilutions) or mouse BMP6 (mBMP6; R&D Systems, Cat. No. 6325-BM; 60 nM and 15 nM) prepared in HBS-EHT running buffer were injected over the captured anti-BMP6 monoclonal antibody at a flow rate of 30 μL / min for 4 min, while dissociation of the BMP6 reagent bound to the captured anti-BMP6 monoclonal antibody was monitored for 10 min in HBS-EHT running buffer. Kinetic association (k a ) and dissociation (k ) were calculated by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c software. d The binding-dissociation equilibrium constants (K) of different anti-BMP6 monoclonal antibodies were determined. D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

number

[0134] The binding kinetic parameters of hBMP6 or mBMP6 bound to different anti-BMP6 monoclonal antibodies of the present invention at 25°C and 37°C are shown in Tables 2 to 5.

[0135] As shown in Table 2, at 25°C, both antibodies of the present invention bind to human BMP6 and DAs shown in Table 3, at 37°C, both antibodies of the present invention bound to human BMP6 with K D As shown in Table 4, at 25°C, both antibodies of the present invention bound to mouse BMP6 with K D As shown in Table 5, at 37°C, both antibodies of the present invention bound to mouse BMP6 with K values ​​of 3.39 nM and 3.55 nM. D The values ​​were 4.0 nM and 6.46 nM. [Table 2] NB: No binding was observed under the current experimental conditions. [Table 3] NB: No binding was observed under the current experimental conditions. # Under the current experimental conditions, no dissociation of hBMP6 from the captured anti-BMP6 monoclonal antibody was observed, and k d The value was manually fixed at 1.00E-05 when fitting real-time binding sensorgrams. [Table 4] NB: No binding was observed under the current experimental conditions. [Table 5] *NB indicates that no binding was observed under the current experimental conditions.

[0136] Example 4. Blockade of BMP6 receptor binding by anti-BMP6 monoclonal antibodies Blockade of BMP6 binding to its receptors, hemojuvelin, ActR2A, or ActR2B, by anti-BMP6 monoclonal antibodies (mAbs) was determined using a real-time surface plasmon resonance (SPR) biosensor instrument, Biacore 3000. All binding studies were performed at 25°C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 1 μg / mL heparin, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-EHT) buffer.

[0137] Approximately 10,500, 5,000, and 5,000 RU of human hemojuvelin expressed with a human Fc tag (hHJV-hFc; SEQ ID NO: 37), human ActR2A extracellular domain expressed with a C-terminal human Fc tag (hActR2A-hFc; SEQ ID NO: 38), and human ActR2B extracellular domain expressed with a C-terminal human Fc tag (hActR2B-hFc; SEQ ID NO: 39) were immobilized individually onto different flow cells of a CM4 sensor surface using EDC / NHS surface chemistry, while activated / unactivated surfaces were used as reference control surfaces. A concentration of 10 nM human BMP-6 was premixed with 400 nM anti-BMP-6 mAb for at least 2 hours before the start of the experiment. The mixture of BMP6 and anti-BMP6 mAb was injected over the different immobilized sensor surfaces at a flow rate of 5 μL / min for 10 minutes. Binding of 10 nM BMP6 to the immobilized surface was used to assess blocking rates, while nonspecific binding of anti-BMP6 mAb without BMP6 was also tested.

[0138] As shown in Table 6, the anti-BMP6 antibodies of the present invention showed partial blocking of BMP6 binding to hHJV-hFc. The anti-BMP6 antibodies of the present invention showed enhanced binding of BMP6 to the surfaces of hActR2A-hFc and hActR2B-hFc. [Table 6]

[0139] Example 5. Anti-BMP6 antibodies that block BMP6 binding to hemojuvelin and activin R2A The ability of anti-BMP6 monoclonal antibodies to block the binding of human BMP6 to two natural binding partners, the coreceptor hemojuvelin (HJV) and the type II binding receptor, activin receptor 2a (ActR2a), was measured using two competitive sandwich ELISAs.

[0140] The human BMP6 protein used in the experiments was purchased from R&D systems (hBMP6; catalog number 507-BP / CF) and biotinylated (biot-hBMP6) for detection purposes. Because BMP6 protein is naturally dimeric, a molecular weight of 30 kDa was used for calculations. The HJV protein used in the experiments consisted of a portion of the human HJV extracellular domain (aa Gln36-Ser399) expressed with a linker sequence and the Fc portion of human IgG1 at the C-terminus (hHJV-hFc; SEQ ID NO: 37). Activin R2a protein was purchased from R&D Systems (hActR2a-hFc; R&D Systems, catalog number 340-RC2). An isotype antibody control was included, along with a commercially available goat anti-hBMP6 positive blocking control antibody (R&D Systems, catalog number AF507).

[0141] The experiment was performed using the following procedure. Receptors were separately coated onto 96-well microtiter plates overnight at 4°C in Hank's Balanced Salt Solution (HBSS) at a concentration of 5 μg / mL for hHJV-hFc and 2.5 μg / mL for hActR2a-hFc. Subsequently, nonspecific binding sites were blocked using a 1.0% (w / v) solution of BSA in HBSS. In other microtiter plates, a fixed amount of 2.5 nM biot-BMP6 (for HJV capture) or 1.5 nM biot-BMP6 (for ActR2a capture) protein was titrated with anti-BMP6 antibodies or isotype control antibodies ranging from 5.1 pM to 300 nM in serial dilutions in HBSS containing 1.0% BSA and 3.33 μg / mL heparin. After 1 hour of incubation, these antibody-protein complexes were transferred to microtiter plates coated with hHJV-hFc or hActR2a-hFc. After 2 hours of incubation at room temperature, the wells were washed, and plate-bound biot-BMP6 was detected with neutravidin conjugated to horseradish peroxidase (HRP) (Thermo Scientific, Cat. No. 31030). The plates were then developed using TMB substrate solution (BD Biosciences, Cat. No. 555214) according to the manufacturer's recommendations, and the absorbance at 450 nm was measured using a Victor X5 plate reader.

[0142] Data analysis with Prism (商標) The calculation was performed using a sigmoidal dose-response model within the software (GraphPad). The calculated IC was defined as the concentration of antibody required to reduce 50% of biot-BMP6 binding to hHJV-hFc or hActR2a-hFc. 50The values ​​were used as an indicator of blocking efficacy. The blocking percentage at the indicated concentration of the tested antibody was calculated as an indicator of the antibody's ability to block the binding of 2.5 nM or 1.5 nM biot-BMP6 to hHJV-hFc or hActR2a-hFc on the plate, respectively. In the calculation, the binding signal of a given biot-BMP6 sample in the absence of antibody in each assay was referenced as 100% binding or 0% blocking, and the baseline signal of a buffer-only sample in the absence of biot-BMP6 was referenced as 0% binding or 100% blocking.

[0143] Blockade of each antibody at the highest antibody concentration of 300 nM was calculated and compared. In addition, blockade of BMP6 binding to ActR2a was calculated for each antibody at 11.1 nM and reported to reflect the enhanced binding signal of biot-BMP6 in the presence of some antibodies in this assay.

[0144] The blocking results of the two assays are summarized in Table 7. The two antibodies of the present invention blocked >90% of 2.5 nM biot-BMP6 protein binding to hHJV-hFc at 300 nM antibody. The blocking potency (IC) of H4H17855P was 50 The IC value of H4H17871P to block BMP6 protein binding to hHJV-hFc was calculated to be 0.522 nM, which was below the lower limit of quantitation of the assay, 1.25 nM. 50 The potency of the positive control anti-BMP6 commercial Ab was 2.32 nM, and 93% was blocked by 300 nM of antibody. The reference Ab had an IC of 2.99 nM. 50 The maximum blocking rate was 88%. As expected, the isotype control antibody blocked <30% at antibody concentrations up to 300 nM.

[0145] Two test antibodies, H4H17855P and H4H17871P, enhanced biot-BMP6 protein binding to hActR2a-hFc by 29.8% and 15.5%, respectively, at 11.1 nM, while blocking it by 19.5% and 37.3% at 300 nM. A positive control anti-BMP6 commercial Ab had an IC of 11.8 nM. 50 The reference Ab blocked >30% at 300 nM but did not have a sigmoidal curve, so the IC 50 Values ​​not reported. As expected, the isotype control antibody blocked <30%. [Table 7] IC is an uncertain IC due to a non-sigmoidal cutoff curve. 50 Show values NB showed non-blocking (<30%) * indicates values ​​below the lower limit of quantitation of 1.25E-09M for the BMP6-blocking HJV assay.

[0146] Example 6. Binding cross-reactivity of anti-BMP6 monoclonal antibodies. The binding cross-reactivity of anti-BMP6 monoclonal antibodies to BMP6 family members (human: BMP6, BMP5, BMP7, BMP8A, BMP9, BMP10, BMP12, BMP14, BMP3b, activin A, and GDF3; mouse: BMP6 and GDF6) was determined using a real-time biolayer interferometry (BLI) biosensor with an Octet HTX instrument. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 1 mg / mL BSA, 50 μg / mL heparin, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-EBHT) buffer at 25°C with plate shaking at 1000 rpm. To assess binding cross-reactivity, a Protein A-coated Octet biosensor (Pall ForteBio Corp., #18-5010) was first immersed in a well containing 20 μg / mL of anti-BMP6 monoclonal antibody for 4 minutes, followed by immersion in a well containing 100 nM of a different BMP6 family member for 4 minutes. The biosensor was washed in HBS-EBHT buffer between each step of the experiment. At the end of each cycle, the Protein A biosensor was regenerated using three alternating 5-second immersions in 10 mM glycine pH 2.0 and 10-second immersions in HBS-EBHT buffer. Real-time binding responses were monitored throughout the course of the experiment, and the binding responses at the end of each step were recorded and tabulated as shown in Tables 8A and 8B. [Table 8] [Table 9]

[0147] Example 7. Antibody inhibition test of BMP6 activation in a bioassay using Hep3B / BRE-luc cells (human BMP6) or W-20-17 / BRE-luc cells (mouse BMP6) Cell lines were engineered to stably express a luciferase reporter [BMP-responsive element (BRE(2x)-luciferase-IRES-GFP)] and screened for high GFP expression to detect modulation of BMP6 signaling. To test human BMP6 (hBMP6), we used Hep3B2.1-7 cells (hereafter referred to as Hep3B cells), a human hepatocellular carcinoma cell line. To test mouse BMP6 (mBMP6), we used W-20-17 cells, a mouse bone marrow stromal cell line previously shown to be responsive to BMP2 (Thies et al., 1992). The resulting reporter cell lines are designated Hep3B / BRE-luc and W-20-17 / BRE-luc. Hep3B / BRE-luc cells were maintained in a medium consisting of MEM, 10% FBS, penicillin / streptomycin, L-glutamine, NEAA, and sodium pyruvate (this medium is referred to as Hep3B medium), and W-20-17 / BRE-luc cells were maintained in a medium consisting of 10% FBS, DMEM, penicillin / streptomycin / L-glutamine, and 200 μg / ml G418 (this medium is referred to as W-20-17 medium).

[0148] For the hBMP6 bioassay, Hep3B / BRE-luc cells were seeded at 10,000 cells / well into a 96-well assay plate in Hep3B medium and incubated overnight at 37°C in 5% CO2. The next day, the Hep3B medium was removed from the Hep3B cells and replaced with medium consisting of MEM, 1% FBS, penicillin / streptomycin, L-glutamine, NEAA, and sodium pyruvate. After an additional 6 hours of incubation at 37°C in 5% CO2, BMP6 and antibodies diluted in assay medium (MEM, 0.1% BSA, penicillin / streptomycin + L-glutamine) were added to the cells. For the mBMP6 bioassay, W-20-17 / BRE-luc cells were seeded at 10,000 cells / well into a 96-well assay plate in W-20-17 medium and incubated overnight at 37°C in 5% CO2. BMP6 and antibodies were added to the cells the next day in assay medium consisting of DMEM, 0.1% BSA, and penicillin / streptomycin / L-glutamine.

[0149] For BMP6 activation, human BMP6 (hBMP6; R&D Systems, Catalog No. 507-BP / CF) or mouse BMP6 (mBMP6; R&D Systems, Catalog No. 6325-BM / CF) was serially diluted 1:3 from 300 nM to 0.005 nM and added to cells without a BMP6 control for dose response. For antibody inhibition of BMP6, antibodies were serially diluted 1:3 from either 1000 nM to 0.02 nM or 100 nM to 0.002 nM and mixed with either 1 nM hBMP6 or 5 nM mBMP6. No antibody control was included in all antibody dose responses. These antibody / BMP6 mixtures were then incubated at 25°C for 30 min and added to cells. Cells were incubated overnight at 37°C and 5% CO2 for Hep3B / BRE-luc cells or for 5.5 hours for W-20-17 / BRE-luc cells. At the end of these incubations, cells were incubated at 25°C for 15 minutes, followed by the addition of OneGlo™ reagent (Promega E6130) to measure the amount of luciferase present in the cells. Plates were read for luminescence on a Victor™ X instrument (Perkin Elmer) 4 minutes after the addition of OneGlo™, and results were analyzed using nonlinear regression (four-parameter logistics) with Prism 6 software (GraphPad) to determine EC 50 and IC 50 Values ​​were obtained. Antibody inhibition was calculated such that 0-100% inhibition was the range of inhibition for either 1-1 nM hBMP6 with no inhibitor or 0-5 nM mBMP6.

[0150] As shown in Table 9, both anti-BMP6 antibodies of the present invention showed complete inhibition of hBMP6-mediated activation of Hep3B / BRE-luc cells at 1 nM. IC of hBMP6 inhibition 50 The values ​​ranged from 0.25 nM to 1.6 nM. The reference Ab had an IC of 0.53 nM. 50 EC values ​​of 0.94 nM and 0.33 nM showed complete inhibition of hBMP6 at 1 nM. 50Dose response of hBMP6-activated Hep3B / BRE-luc cells with values.

[0151] As shown in Table 9, both anti-BMP6 antibodies of the present invention showed complete inhibition of mBMP6-mediated activation of W-20-17 / BRE-luc cells at 5 nM. IC of mBMP6 inhibition 50 The values ​​ranged from 1.9 nM to 11 nM. The reference Ab had an IC of 1.9 nM. 50 showed complete inhibition of mBMP6 at 5 nM with EC values ​​of 1.2 nM and 1.3 nM. 50 Dose response of mBMP6-activated W-20-17 / BRE-luc cells with values.

[0152] The isotype control antibody showed no inhibition of either human or mouse BMP6. [Table 10]

[0153] Example 8. In vivo mouse experiments, serum hepcidin and iron levels after Bmp6 antibody treatment (H4H17855P) To determine the effectiveness of the BMP6 antibodies of the present invention in increasing serum hepcidin and decreasing serum hepcidin, in vivo experiments were performed in mice homozygous for expression of human BMP6 and HJV instead of mouse BMP6 and HJV. For the study, 5 or 6 mice per group received two doses of antibody at either 10 or 20 mg / kg on days 1 and 3. On day 5, the mice were sacrificed, serum was collected, and hepcidin and iron levels were measured.

[0154] As shown in Table 10 and Figures 1 and 2, mice receiving the BMP6 antibody of the present invention, H4H17855P, had increased serum iron and decreased serum hepcidin compared to mice receiving an isotype control antibody at 10 mg / kg. There was a dose-dependent effect of the BMP6 antibody, with increased serum iron and decreased hepcidin at 20 mg / kg compared to 10 mg / kg. [Table 11]

[0155] Example 9. In vivo mouse experiments, serum hepcidin and iron levels after Bmp6 antibody treatment (H4H17871P) To determine the effectiveness of the BMP6 antibody of the present invention in increasing serum hepcidin and decreasing serum hepcidin, an in vivo experiment was carried out in mice homozygous for the expression of human BMP6 and HJV instead of mouse BMP6 and HJV. For the study, seven mice per group received a 1 s.c. dose of 5 mg / kg of antibody on day 1. On day 5, the mice were sacrificed, and serum was collected to measure hepcidin and iron levels.

[0156] Serum iron levels were measured using the QuantiChrom iron assay kit (BioAssay Systems DIFE-250), and serum hepcidin was measured using the Hepcidin Murine-Compete ELISA kit (Intrinsic Lifesciences HMC-001).

[0157] As shown in Table 11 and Figures 3 and 4, mice receiving the BMP6 antibody of the present invention, H4H17871P, had increased serum iron and decreased serum hepcidin compared to mice receiving an isotype control antibody. [Table 12]

Claims

1. 1. An isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human bone morphogenetic protein 6 (BMP6), wherein the antibody or antigen-binding fragment comprises: (i) (a) an HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 5; (b) an HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 6; (c) an HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 7; (d) an LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 8; (e) an LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 9; and (f) an LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 10; or (ii) (a) an HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 11; (b) an HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 12; (c) an HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 13; (d) an LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 14; (e) an LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 15; and (f) an LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 16; An isolated human monoclonal antibody or antigen-binding fragment thereof, comprising:

2. 2. The isolated human antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1 / 2 and 3 / 4.

3. The antibody (i) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 34; and a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 33, and / or (ii) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 36; and 3. The isolated human antibody or antigen-binding fragment of claim 1 or 2, comprising a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:

35.

4. The antigen-binding fragment may be a Fab fragment, F(ab') ２ 4. The antigen-binding fragment of any one of claims 1 to 3, which is a fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) molecule, or a dAb fragment.

5. A method for producing the antibody or antigen-binding fragment of any one of claims 1 to 4, comprising: (i) introducing into a host cell one or more polynucleotides encoding the immunoglobulin light chain of the antibody or antigen-binding fragment and the immunoglobulin heavy chain of the antibody or antigen-binding fragment; (ii) culturing said host cell in a growth medium under conditions favorable for expression of said polynucleotide.

6. (iii) isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is growing. The method of claim 5 further comprising:

7. An injection device or container comprising the antibody or antigen-binding fragment of any one of claims 1 to 4.

8. A pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof that binds to human BMP6 according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier or diluent.

9. 10. The pharmaceutical composition of claim 8, further comprising one or more additional therapeutic agents.

10. 10. The pharmaceutical composition of claim 9, wherein the additional therapeutic agent is an iron supplement.

11. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 4, or the pharmaceutical composition of any one of claims 8 to 10, for preventing or treating iron deficiency anemia or an iron deficiency-related disorder in a patient in need thereof.

12. 12. The composition of claim 11, which is administered subcutaneously, intravenously, intradermally, orally, or intramuscularly.

13. the iron deficiency anemia or iron deficiency related disorder results in a condition selected from the group consisting of extreme fatigue, weakness, pale skin, chest pain, rapid heartbeat, heart palpitations, shortness of breath, headache, dizziness, lightheadedness, cold hands, cold feet, inflamed tongue, and restless legs; 13. The composition of claim 11 or 12, wherein administration of the composition treats the condition or reduces the severity of one or more symptoms of the condition.

14. A composition comprising one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 4 for use in treating iron deficiency anemia or an iron deficiency-related disorder.

15. 10. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 4 in the manufacture of a medicament for treating a patient with iron deficiency anemia or an iron deficiency-related disorder.

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