RU2790023C2 - Methods for treatment of disease, using bone morphogenetic protein 6 (bmp6) inhibitors - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine, namely, to a method for the treatment of anemia in a patient, using a BMP6 antagonist, providing for selective administration of therapeutically effective amount of the BMP6 antagonist to the patient based on that a biological sample of the patient is characterized by a ferritin level greater than or equal to 500 ng/ml. In this case, the specified BMP6 antagonist is an antibody to BMP6 or its antigen-binding fragment, containing (i) HCDR1, HCDR2, and HCDR3 sequences under SEQ ID NO: 69, 70, and 71, respectively, and LCDR1, LCDR2, and LCDR3 sequences under SEQ ID NO: 79, 80, and 81, respectively; (ii) HCDR1, HCDR2, and HCDR3 sequences under SEQ ID NO: 72, 73, and 74, respectively, and LCDR1, LCDR2, and LCDR3 sequences under SEQ ID NO: 82, 83, and 84, respectively; (iii) HCDR1, HCDR2, and HCDR3 sequences under SEQ ID NO: 29, 30, and 31, respectively, and LCDR1, LCDR2, and LCDR3 sequences under SEQ ID NO: 39, 40, and 41, respectively; (iv) HCDR1, HCDR2, and HCDR3 sequences under SEQ ID NO: 32, 33, and 34, respectively, and LCDR1, LCDR2, and LCDR3 sequences under SEQ ID NO: 42, 43, and 44, respectively; (v) HCDR1, HCDR2, and HCDR3 sequences under SEQ ID NO: 49, 50, and 51, respectively, and LCDR1, LCDR2, and LCDR3 sequences under SEQ ID NO: 59, 60, and 61, respectively; (vi) HCDR1, HCDR2, and HCDR3 sequences under SEQ ID NO: 52, 53, and 54, respectively, and LCDR1, LCDR2, and LCDR3 sequences under SEQ ID NO: 62, 63, and 64, respectively; (vii) HCDR1, HCDR2, and HCDR3 sequences under SEQ ID NO: 9, 10, and 11, respectively, and LCDR1, LCDR2, and LCDR3 sequences under SEQ ID NO: 19, 20, and 21, respectively, or (viii) HCDR1, HCDR2, and HCDR3 sequences under SEQ ID NO: 12, 13, and 14, respectively, and LCDR1, LCDR2, and LCDR3 sequences under SEQ ID NO: 22, 23, and 24, respectively. The group of inventions also relates to a method for selective treatment of anemia in a patient, using a BMP6 antagonist. The group of inventions also relates to a method for prediction of the probability that a patient with anemia will respond to the treatment, using a BMP6 antagonist. The group of inventions also relates to the use of a BMP6 antagonist in the treatment of anemia in a patient.

EFFECT: group of inventions provides the implementation of improved methods for the treatment of anemia.

71 cl, 3 ex, 15 dwg, 8 tbl

Description

SEQUENCE LIST

This application contains a sequence listing that has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on May 23, 2017, is named PAT057354-WO-PCT_SL.txt and is 82879 bytes in size.

INTRODUCTION

The present invention relates to methods for treating anemia using bone morphogenetic protein 6 (BMP6) inhibitors.

BACKGROUND OF THE INVENTION

Anemia is common in patients with chronic kidney disease (CKD) and is associated with lower quality of life and higher risk of adverse outcomes, including cardiovascular disease and death. Several ways to control anemia in patients with CKD include the use of erythropoiesis-stimulating agents (ESAs), supplemental oral and intravenous iron, and blood transfusions. However, many patients do not respond well to these treatments or require higher doses of ESA and/or iron. High doses of iron can also cause toxicity associated with the formation of oxygen radicals and allergic reactions. These treatments may not be effective because they do not fully address the root cause of anemia, ie, impaired iron absorption and mobilization of iron from body stores.

Currently, attempts are being made to control erythropoietin resistance by co-administering a high dose of parenteral iron. However, most intravenous iron is first processed by macrophages, and its utilization for erythropoiesis depends on ferroportin-mediated iron export.

In many anemic patients, ferroportin-mediated iron export is suppressed by high levels of hepcidin. Additional evidence suggests that elevated hepcidin levels correlate with poor susceptibility to ESA during hemodialysis. Therefore, hepcidin-lowering agents may be an effective strategy to ameliorate ESA-refractory anemia in this patient population and in other forms of anemia due to chronic disease (ACD) characterized by iron deficiency.

Therefore, methods that reduce circulating levels of circulating hepcidin in the bloodstream should enhance iron absorption, promote the release of sequestered iron, and stimulate erythropoiesis in the ESA-refractory anemia present in patients with chronic kidney disease.

Despite current treatment options for the treatment of diseases and disorders associated with anemia, there remains a need for improved treatments for anemia that are effective and well tolerated.

BRIEF DESCRIPTION OF THE INVENTION

Ferritin is an intracellular protein that is an indicator of stored iron. Ferritin levels can be used as an indirect marker of the total amount of iron stored in the body. Without wishing to be bound by theory, it is believed that BMP6 inhibitors can lead to the release of stored iron from cells, eg macrophages and/or enterocytes. Again, without wishing to be bound by theory, the present invention is based in part on the discovery that BMP6 inhibitors may be effective in treating conditions associated with sequestered iron, such as anemia, in patients with high ferritin levels (for example, pre-treatment ferritin levels of more than 500 ng/mL), such as serum ferritin, and therefore high levels of stored iron.

In a first aspect, the present invention relates to a method for selectively

a. inhibition of BMP6;

b. elevations in serum iron levels, transferrin saturation (TAST), reticulocyte hemoglobin (CHr), reticulocyte count, red blood cell count, hemoglobin, or hematocrit;

c. decreased activity or levels of hepcidin;

d. anemia treatment or

e. increase or maintain hemoglobin levels

in a patient in need thereof, which comprises selectively administering to the patient a therapeutically effective amount of a BMP6 antagonist based on a biological sample from the patient having a ferritin level of ≤ 2000 ng/mL. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a method of treating an anemic patient with a BMP6 antagonist, comprising selectively administering to the patient a therapeutically effective amount of the BMP6 antagonist based on a biological sample from the patient having a ferritin level of ≤2000 ng/mL. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a method for selectively treating a patient having anemia with a BMP6 antagonist, comprising:

a) performing an analysis of a biological sample from the patient in relation to the level of ferritin and

b) thereafter selectively administering to the patient a therapeutically effective amount of a BMP9 antagonist wherein the ferritin level is ≤ 2000 ng/mL. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a method for selectively treating a patient having anemia with a BMP6 antagonist, comprising:

a) performing an analysis of a biological sample from the patient in relation to the level of ferritin;

b) thereafter selecting a patient for treatment with a BMP6 antagonist based on a biological sample from the patient having a ferritin level of ≤ 2000 ng/mL; And

c) thereafter administering to the patient a therapeutically effective amount of the BMP9 antagonist. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a method for selectively

a. inhibition of BMP6;

b. elevations in serum iron levels, transferrin saturation (TAST), reticulocyte hemoglobin (CHr), reticulocyte count, red blood cell count, hemoglobin, or hematocrit;

c. decreased activity or levels of hepcidin;

d. anemia treatment or

e. increase or maintain hemoglobin levels

in a patient in need thereof, which comprises selectively administering to the patient a therapeutically effective amount of a BMP6 antagonist based on a biological sample from the patient having a ferritin level of ≥ 500 ng/mL.

In another aspect, the present invention relates to a method of treating an anemic patient with a BMP6 antagonist comprising selectively administering to the patient a therapeutically effective amount of the BMP6 antagonist based on a biological sample from the patient having a ferritin level of ≥500 ng/mL.

In another aspect, the present invention relates to a method for selectively treating a patient having anemia with a BMP6 antagonist, comprising:

a) performing an analysis of a biological sample from the patient in relation to the level of ferritin and

b) thereafter selectively administering to the patient a therapeutically effective amount of a BMP9 antagonist wherein the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a method for selectively treating a patient having anemia with a BMP6 antagonist, comprising:

a) performing an analysis of a biological sample from the patient in relation to the level of ferritin;

b) thereafter selecting a patient for treatment with a BMP6 antagonist based on a biological sample from the patient having a ferritin level of ≥ 500 ng/mL; And

c) thereafter administering to the patient a therapeutically effective amount of the BMP9 antagonist.

In the embodiments included in any of the above aspects, the ferritin level is the ferritin protein level.

In embodiments included in any of the above aspects, the step of performing the assay comprises a technique selected from the group consisting of immunoassay, immunohistochemistry, ELISA, flow cytometry, Western blotting, HPLC, and mass spectrometry.

In another aspect, the present invention relates to a BMP6 antagonist for use in the treatment of a patient having anemia, characterized in that a therapeutically effective amount of the BMP6 antagonist is to be administered to the patient based on a biological sample from the patient having a ferritin level of ≤ 2000 ng/mL. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a BMP6 antagonist for use in the treatment of a patient having anemia, characterized in that a therapeutically effective amount of the BMP6 antagonist is to be administered to the patient based on a biological sample from the patient having a ferritin level of ≥500 ng/mL.

In another aspect, the present invention relates to a BMP6 antagonist for use in the treatment of a patient having anemia, characterized in that

a) selection of a patient for treatment with a BMP6 antagonist is based on a biological sample from the patient having a ferritin level of ≤ 2000 ng/mL; And

b) thereafter, a therapeutically effective amount of the BMP6 antagonist is administered to the patient. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a BMP6 antagonist for use in the treatment of a patient having anemia, characterized in that

a) selection of a patient for treatment with a BMP6 antagonist is based on a biological sample from the patient having a ferritin level of ≥ 500 ng/mL; And

b) thereafter, a therapeutically effective amount of the BMP6 antagonist is administered to the patient.

In another aspect, the present invention relates to a BMP6 antagonist for use in the treatment of a patient having anemia, characterized in that

a) a biological sample from the patient is to be analyzed for ferritin; And

b) a therapeutically effective amount of the BMP6 antagonist is to be selectively administered to a patient based on a biological sample from the patient having a ferritin level of ≤ 2000 ng/mL. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a BMP6 antagonist for use in the treatment of a patient having anemia, characterized in that

a) a biological sample from the patient is to be analyzed for ferritin; And

b) a therapeutically effective amount of the BMP6 antagonist is to be selectively administered to a patient based on a biological sample from the patient having a ferritin level of ≥ 500 ng/mL.

In another aspect, the present invention relates to a BMP6 antagonist for use in the treatment of a patient having anemia, characterized in that

a) a biological sample from the patient is to be analyzed for ferritin;

b) the patient is selected for treatment with a BMP6 antagonist on the basis that a biological sample from the patient has a ferritin level of ≤ 2000 ng/mL; And

c) a therapeutically effective amount of the BMP6 antagonist is selectively administered to the patient. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a BMP6 antagonist for use in the treatment of a patient having anemia, characterized in that

a) a biological sample from the patient is to be analyzed for ferritin;

b) the patient is selected for treatment with a BMP6 antagonist on the basis that a biological sample from the patient has a ferritin level of ≥ 500 ng/mL; And

c) a therapeutically effective amount of the BMP6 antagonist is selectively administered to the patient.

In another aspect, the present invention relates to a method for predicting the likelihood that a patient having anemia will respond to treatment with a BMP6 antagonist, comprising assaying a biological sample from the patient for ferritin, where a ferritin level ≤ 2000 ng/mL is indicative of an increased likelihood that the patient will respond to treatment with a BMP6 antagonist. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention relates to a method for predicting the likelihood that a patient having anemia will respond to treatment with a BMP6 antagonist, comprising performing analysis of a biological sample from the patient for ferritin, where a ferritin level ≥ 500 ng/ml is indicative of an increased likelihood that the patient will respond to treatment with a BMP6 antagonist.

In embodiments, including embodiments of any of the above aspects and embodiments, the method or use further comprises the step of obtaining a biological sample from a patient, the obtaining step being performed before the step of performing the analysis.

In embodiments, including embodiments of any of the above aspects and embodiments, the ferritin level is a ferritin protein level.

In embodiments, including embodiments of any of the above aspects and embodiments, the step of performing the assay comprises a technique selected from the group consisting of immunoassay, immunohistochemistry, ELISA, flow cytometry, Western blotting, HPLC, and mass spectrometry. .

In another aspect, the present invention provides a method for obtaining a transferable form of information for predicting the susceptibility of a patient having anemia to treatment with a BMP6 antagonist, comprising:

a) determining an increased likelihood of a patient's response to treatment with a BMP6 antagonist based on the presence of a ferritin level of ≤ 2000 ng/mL in a biological sample from the patient, and

b) recording the result from the determination stage on a tangible or intangible medium for use in transmission. In embodiments, the ferritin level is ≥ 500 ng/mL.

In another aspect, the present invention provides a method for obtaining a transferable form of information for predicting the susceptibility of a patient having anemia to treatment with a BMP6 antagonist, comprising:

a) determining an increased likelihood of a patient's response to treatment with a BMP6 antagonist based on the presence of a ferritin level of ≥ 500 ng/mL in a biological sample from the patient, and

b) recording the result from the determination stage on a tangible or intangible medium for use in transmission.

In embodiments, including embodiments of any of the above aspects and embodiments, the patient is anemic. In embodiments, the anemia is anemia associated with a chronic disease. In embodiments, the chronic disease is chronic kidney disease, cancer, or inflammation.

In embodiments, including embodiments of any of the above aspects and embodiments, the patient is or has been treated with an erythropoiesis stimulating agent (ESA), such as erythropoietin (EPO).

In embodiments, including embodiments of any of the above aspects and embodiments, the anemia is an EPO hyporesponsive anemia.

In embodiments, including embodiments of any of the above aspects and embodiments, the anemia is iron deficiency anemia, such as functional iron deficiency anemia.

In embodiments, including embodiments of any of the above aspects and embodiments, the patient is a patient on chronic hemodialysis.

In embodiments, including embodiments of any of the above aspects and embodiments, the method or use further comprises providing a reduction in a patient's iron requirement, a reduction in a patient's EPO dose, or a reduction in both the patient's iron requirement and the patient's requirement. at an EPO dose relative to said EPO dose requirement and/or iron dose requirement in the absence of treatment with a therapeutically effective amount of a BMP6 antagonist. In embodiments, including embodiments of any of the above aspects and embodiments, the method or use further comprises or results in a reduction in the ESA Resistance Index (ERI) in a patient.

In embodiments, including embodiments of any of the above aspects and embodiments, the biological sample is a synovial fluid, blood, serum, feces, blood plasma, urine, tear, saliva, cerebrospinal fluid, a leukocyte sample, or a tissue sample.

In embodiments, including embodiments of any of the above aspects and embodiments, the biological sample is blood serum or blood.

In embodiments, including embodiments of any of the above aspects and embodiments, the biological sample is blood serum.

In embodiments, including embodiments of any of the above aspects and embodiments, the BMP6 antagonist is a BMP6 binding molecule.

In embodiments, including embodiments of any of the above aspects and embodiments, the BMP6 antagonist is an anti-BMP6 antibody or antigen-binding fragment thereof, such as those described in Table 1 or Table 14.

In embodiments, including embodiments of any of the above aspects and embodiments, an anti-BMP6 antibody or antigen-binding fragment thereof comprises:

(a) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively;

(b) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively;

(c) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively;

(d) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively;

(e) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively;

(f) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively;

(g) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or

(h) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody or antigen-binding fragment thereof comprises:

(a) the VH sequence under SEQ ID NO: 75;

(b) the VH sequence under SEQ ID NO: 35;

(c) the VH sequence under SEQ ID NO: 55 or

(d) VH sequence under SEQ ID NO: 15.

In embodiments, including embodiments of any of the above aspects and embodiments, an anti-BMP6 antibody or antigen-binding fragment thereof comprises:

(a) the VL sequence under SEQ ID NO: 85;

(b) the VL sequence under SEQ ID NO: 45;

(c) the VL sequence under SEQ ID NO: 65 or

(d) VL sequence under SEQ ID NO: 25.

In embodiments, including embodiments of any of the above aspects and embodiments, an anti-BMP6 antibody or antigen-binding fragment thereof comprises:

(a) the VH sequence of SEQ ID NO: 75 and the VL sequence of SEQ ID NO: 85;

(b) the VH sequence of SEQ ID NO: 35 and the VL sequence of SEQ ID NO: 45;

(c) the VH sequence of SEQ ID NO: 55 and the VL sequence of SEQ ID NO: 65, or

(d) the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 25.

In embodiments, including embodiments of any of the above aspects and embodiments, an anti-BMP6 antibody or antigen-binding fragment thereof comprises:

(a) the sequence of the heavy chain under SEQ ID NO: 77;

(b) the sequence of the heavy chain under SEQ ID NO: 37;

(c) the heavy chain sequence of SEQ ID NO: 57 or

(d) the heavy chain sequence of SEQ ID NO: 17.

In embodiments, including embodiments of any of the above aspects and embodiments, an anti-BMP6 antibody or antigen-binding fragment thereof comprises:

(a) the light chain sequence of SEQ ID NO: 87;

(b) the light chain sequence of SEQ ID NO: 47;

(c) a light chain sequence of SEQ ID NO: 67 or

(d) the light chain sequence of SEQ ID NO: 27.

In embodiments, including embodiments of any of the above aspects and embodiments, an anti-BMP6 antibody or antigen-binding fragment thereof comprises:

(a) the heavy chain sequence of SEQ ID NO: 77 and the light chain sequence of SEQ ID NO: 87;

(b) the heavy chain sequence of SEQ ID NO: 37 and the light chain sequence of SEQ ID NO: 47;

(c) a heavy chain sequence of SEQ ID NO: 57 and a light chain sequence of SEQ ID NO: 67, or

(d) the heavy chain sequence of SEQ ID NO: 17 and the light chain sequence of SEQ ID NO: 27.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, binds to human BMP6 with a KD of ≦1 nM.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, binds to human BMP6 with a KD of ≦0.1 nM.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, has an affinity for human BMP6 that is at least about 100-fold greater than that for human BMP7.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, has an affinity for human BMP6 that is at least about 100-fold greater than that for human BMP2, human BMP5, or BMP7. person.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, has an affinity for human BMP6 that is at least about 500-fold greater than that for human BMP2, human BMP5, or BMP7. person.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, is characterized by no detectable binding to human BMP2 and/or BMP7 in ELISA.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, comprises a backbone selected from IgM and IgG.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody or antigen-binding fragment thereof is an IgG selected from IgG1, IgG2 and IgG3 or IgG4.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a single chain antibody, a Fab, and a scFv.

In embodiments, including embodiments of any of the above aspects and embodiments, an anti-BMP6 antibody or antigen-binding fragment thereof is a component of an immunoconjugate.

In embodiments, including embodiments of any of the above aspects and embodiments, the anti-BMP6 antibody, or antigen-binding fragment thereof, has an altered effector function due to a mutation in the Fc region.

In embodiments, including embodiments of any of the above aspects and embodiments, an anti-BMP6 antibody or antigen-binding fragment thereof binds to a human BMP6 epitope comprising the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 98), e.g., consisting of it.

In embodiments, including embodiments of any of the above aspects and embodiments, the antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.001 mg/kg to 0.1 mg/kg.

In embodiments, including embodiments of any of the above aspects and embodiments, the antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.0063 to 0.1 mg/kg.

In embodiments, including embodiments of any of the above aspects and embodiments, the antibody or antigen-binding fragment thereof is administered at a dose of 0.001 mg/kg, 0.0016 mg/kg, 0.0025 mg/kg, 0.0040 mg/kg, 0.0063 mg/kg, 0.01 mg/kg, 0.016 mg/kg, 0.025 mg/kg, 0.040 mg/kg, 0.063 mg/kg or 0.1 mg/kg.

In embodiments, including embodiments of any of the above aspects and embodiments, the antibody or antigen-binding fragment thereof is administered intravenously or subcutaneously.

In embodiments, including embodiments of any of the above aspects and embodiments, the antibody or antigen-binding fragment thereof is administered intravenously.

In embodiments, including embodiments of any of the above aspects and embodiments, administration is by infusion over a period of about 30 to about 60 minutes.

In embodiments, including embodiments of any of the above aspects and embodiments, the ferritin level is ≤ 1900 ng/mL, ≤ 1800 ng/mL, ≤ 1700 ng/mL, ≤ 1600 ng/mL, ≤ 1500 ng/mL , ≤ 1400 ng/mL, ≤ 1300 ng/mL, ≤ 1200 ng/mL, ≤ 1100 ng/mL, ≤ 1000 ng/mL, ≤ 900 ng/mL, ≤ 800 ng/mL, ≤ 700 ng/mL, ≤ 600 ng/ml or ≤ 500 ng/ml.

In embodiments, including embodiments of any of the above aspects and embodiments, the ferritin level is ≤ 1500 ng/mL. In embodiments, including embodiments of any of the above aspects and embodiments, the ferritin level is ≤ 1000 ng/mL.

In embodiments, including embodiments of any of the above aspects and embodiments, the ferritin level is ≥ about 200 ng/mL, ≥ about 250 ng/mL, ≥ about 300 ng/mL, ≥ about 350 ng/mL, ≥ approximately 400 ng/mL, ≥ approximately 450 ng/mL, ≥ approximately 500 ng/mL, ≥ approximately 600 ng/mL, ≥ approximately 700 ng/mL, ≥ approximately 800 ng/mL, ≥ approximately 900 ng/mL, ≥ approximately 1000 ng/mL, ≥ approximately 1100 ng/mL, ≥ approximately 1200 ng/mL, ≥ approximately 1300 ng/mL, ≥ approximately 1400 ng/mL, ≥ approximately 1500 ng/mL, ≥ approximately 1600 ng/mL, ≥ approximately 1700 ng/ml, ≥ about 1800 ng/ml or ≥ about 1900 ng/ml.

In embodiments, including embodiments of any of the above aspects and embodiments, the ferritin level is ≥ about 500 ng/mL, ≥ about 600 ng/mL, ≥ about 700 ng/mL, ≥ about 800 ng/mL, ≥ about 900 ng/ml, ≥ about 1000 ng/ml, ≥ about 1100 ng/ml, ≥ about 1200 ng/ml, ≥ about 1300 ng/ml, or ≥ about 1400 ng/ml.

In embodiments, including embodiments of any of the above aspects and embodiments, the ferritin level is ≥ about 500 ng/mL.

DEFINITIONS

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains.

"BMP6" as used herein means a bone morphogenetic protein 6 (BMP6) protein or a gene or nucleic acid encoding BMP6. Hahn et al. 1992 Genomics 14: 759-62; Sauermann et al. 1993 J. Neurosci. Res. 33:142-7; NCBI Gene ID: 654. BMP6 is also known as BMP-6; VGR; VGR1; ID in third-party systems: OMIM: 112266; MGI: 88182; Homologene: 1300; GeneCards: BMP6 gene. Orthologues: species: human: Entrez: 654; Ensemble: ENSG00000153162; UniProt: P22004; RefSeq (mRNA): NM_001718; RefSeq (protein): NP_001709; position (UCSC): hr. 6: 7.73-7.88 Mbps; type: mouse: Entrez: 12161; Ensemble: ENSMUSG00000039004; UniProt: P20722; RefSeq (mRNA): NM_007556; RefSeq (protein): NP_031582; position (UCSC): hr. 13: 38.35-38.5 Mbps. As described herein, an antibody, an antigen-binding fragment thereof that binds to BMP6 binds to the BMP6 protein.

"BMP2" as used herein means a bone morphogenetic protein 2 (BMP2) protein or a gene or nucleic acid encoding BMP2. BMP2 is also known as BDA2 and BMP2A; ID in third party systems: OMIM: 112261; MGI: 88177; Homologene: 926; GeneCards: BMP2 gene. Kind: human; Entrez: 650; Ensemble: ENSG00000125845; UniProt: P12643; RefSeq (mRNA): NM_001200; RefSeq (protein): NP_001191; position (UCSC): hr. 20: 6.75-6.76 Mbps. Kind: mouse; Entrez: 12156; Ensemble: ENSMUSG00000027358; UniProt: P21274; RefSeq (mRNA): NM_007553; RefSeq (protein): NP_031579; position (UCSC): hr. 2: 133.55-133.56 Mbps. As described herein, an antibody, an antigen-binding fragment thereof that binds to BMP2 binds to the BMP2 protein.

"BMP5" as used herein means a bone morphogenetic protein 5 (BMP5) protein or a gene or nucleic acid encoding BMP5. BMP5 is also known as MGC34244; ID in third-party systems: OMIM: 112265; MGI: 88181; HomoloGene: 22412; GeneCards: BMP5 gene. Kind: human; Entrez: 653; Ensemble: ENSG00000112175; UniProt: P22003; RefSeq (mRNA): NM_021073; RefSeq (protein): NP_066551; position (UCSC): hr. 6: 55.62-55.74 Mb. Kind: mouse; Entrez: 12160; Ensemble: ENSMUSG00000032179; UniProt: P49003; RefSeq (mRNA): NM_007555; RefSeq (protein): NP_031581; position (UCSC): hr. 9: 75.78-75.9 Mbps. As described herein, an antibody, an antigen-binding fragment thereof that binds to BMP5 binds to the BMP5 protein.

"BMP7" as used herein means a bone morphogenetic protein 7 (BMP7) protein or a gene or nucleic acid encoding BMP7. BMP7 is also known as osteogenic protein-1; OP-1; ID in third-party systems: OMIM: 112267; MGI: 103302; HomoloGene: 20410; GeneCards: BMP7 gene. Kind: human; Entrez: 655; Ensemble: ENSG00000101144; UniProt: P18075; RefSeq (mRNA): NM_001719; RefSeq (protein): NP_001710; position (UCSC): hr. 20: 55.74-55.84 Mb. Kind: mouse; Entrez: 12162; Ensemble: ENSMUSG00000008999; UniProt: P23359; RefSeq (mRNA): NM_007557; RefSeq (protein): NP_031583; position (UCSC): hr. 2: 172.87-172.94 Mb. As described herein, an antibody, an antigen-binding fragment thereof, that binds to BMP7 binds to the BMP7 protein.

"Hepcidin" means the hepcidin gene or hepcidin protein, a peptide hormone. Hepcidin is also known as HAMP (antimicrobial protein or peptide hepcidin); HEPC; HFE2B; LEAP1 (LEAP-1); PLTR; OMIM: 606464; HomoloGene: 81623; GeneCards: HAMP gene; Entrez: 57817; Ensemble: ENSG00000105697; UniProt: P81172; RefSeq (mRNA): NM_021175; RefSeq (protein): NP_066998; position (UCSC): hr. 19: 35.77-35.78 Mbps. Krause et al. FEBS Lett. 480: 147-150; and Pigeon et al. 2001 J. Biol. Chem. 276:7811-9. See also Ganz 2003 Blood 102: 783-8; Roy et al. 2005 Curr. Opin. hemat. 12:107-111; Fleming et al. 2006 Semin. Liver Dix. 25:411-9; Park et al. 2001 J. Biol. Chem. 276:7806-10; Majore et al. 2002 Haematologica 87: 221-2; Kluver et al. 2002 J. Pept. Res. 59:241-8; Hunter et al. 2002 J. Biol. Chem. 277:37597-603; Weinstein et al. 2003 Blood 100: 3776-81; Nemeth et al. 2003 Blood 101: 2461-3; Roetto et al. 2003 Nat. Genet. 33:21-2; Strausberg et al. 2003 Proc. Natl. Acad. Sci USA 99: 16899-903; Gehrke et al. 2003 Blood 102: 371-6; Merryweather-Clarke et al. 2004 Human Mol. Genet. 12:2241-7; Clark et al. 2003 Genome Res. 13:2265-70; Roetto et al. 2004 Blood 103: 2407-9; Jacolot et al. 2004 Blood 103: 2835-40; and Ota et al. 2004 Nat. Genet. 36:40-45.

"BMP6 antagonist", as used herein, refers to a molecule capable of antagonizing (e.g., reducing, inhibiting, reducing, delaying) the functioning, expression and/or signaling of BMP6 (e.g., by blocking BMP6 binding to a receptor BMP6). Non-limiting examples of BMP6 antagonists include BMP6 binding molecules and BMP6 receptor binding molecules. In some embodiments of the disclosed methods, regimens, kits, processes, routes of administration, and compositions, a BMP6 antagonist is used.

"BMP6 binding molecule" as used herein refers to any molecule capable of binding to a human BMP6 antigen, either alone or associated with other molecules. The binding response can be shown using standard methods (qualitative assays), including, for example, a binding assay, a competitive assay, or a biological assay to determine inhibition of BMP6 binding to the BMP6 receptor, or any type of binding assay compared to a negative control assay in which an antibody with unrelated specificity is used, but ideally of the same isotype. Non-limiting examples of BMP6 binding molecules include small molecules, BMP6 decoy receptors, and antibodies that bind to BMP6 produced by B cells or hybridomas, and chimeric antibodies, CDR-grafted antibodies, or human antibodies, or any fragment thereof, for example , F(ab')2 and Fab fragments, as well as single chain or single domain antibodies. Preferably, the BMP6 binding molecule counteracts (eg, reduces, inhibits, reduces, delays) BMP6 function, expression and/or signaling. In some embodiments of the disclosed methods, regimens, kits, processes, uses, and compositions, a BMP6 binding molecule is used.

"BMP6 receptor binding molecule" means any molecule capable of binding to the human BMP6 receptor, either alone or associated with other molecules. The binding response can be shown using standard methods (qualitative assays), including, for example, a binding assay, a competitive assay, or a biological assay to determine inhibition of BMP6 receptor binding to BMP6, or any type of binding assay compared to a negative control assay in which an antibody with unrelated specificity is used, but ideally of the same isotype. Non-limiting examples of BMP6 receptor binding molecules include small molecules, BMP6 decoys, and anti-BMP6 receptor antibodies produced by B cells or hybridomas, and chimeric, CDR-grafted, or human antibodies, or any fragment thereof, such as F( ab')2 and Fab fragments, as well as single chain or single domain antibodies. Preferably, the BMP6 receptor binding molecule counteracts (eg, reduces, inhibits, reduces, delays) BMP6 function, expression and/or signaling. In some embodiments of the disclosed methods, regimens, kits, processes, uses, and compositions, a BMP6 receptor binding molecule is used.

"Anemia", as used herein, means a decrease in the number of red blood cells or a decrease in the amount of hemoglobin or iron in the blood with a decrease in the blood's ability to carry oxygen.

"EPO resistance index" or "ERI", as used interchangeably herein, refers to the change in dose of ESA, eg EPO (in units per kg of body weight), depending on the level of hemoglobin. In embodiments, the ESA dose/hemoglobin level is measured weekly. In embodiments, the ESA dose/hemoglobin level is measured monthly. In embodiments, the ESA dose/hemoglobin level is compared for multiple measurements to obtain an ERI.

Anemia can be diagnosed by any method known in the art, including, but not limited to, in males based on a hemoglobin level of less than about 130-140 g/L (13-14 g/dL), and in women, less than about 120-130 g/l (12-13 g/dl). Janz et al. 2013 Emerge. Med. Pract. 15:1-15; and Smith 2010 Am. J. Man. Care 16 Supp. S59-66.

As used herein, the terms "anti-BMP6 antibody", "anti-human BMP6 antibody", "BMP6-binding antibody", "anti-BMP6 antagonist antibody" and the like (and antigen-binding fragments thereof) include antibodies (and antigen-binding fragments thereof) that bind to the BMP6 protein.

As used herein, the terms "antibody," "antigen-binding fragment thereof," "antigen-binding portion," and the like include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or individual chains thereof. A naturally occurring "antibody" is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that alternate with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

As used herein, the terms "antigen-binding fragment", "antigen-binding fragment thereof", "antigen-binding portion" of an antibody, and the like, refer to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., BMP6). The antigen-binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include the Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; F(ab) ₂ fragment, bivalent fragment containing two Fab-fragment connected via a disulfide bridge in the hinge region; Fd fragment consisting of VH and CH1 domains; Fv fragment, consisting of the VL and VH domains of one antibody arm; a single domain antibody (dAb) fragment (Ward et al., 1989 Nature 341:544-546) which consists of a VH domain; and a dedicated complementarity determining region (CDR).

In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined using recombinant methods using an artificial peptide linker, which allows them to be converted into a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-chain Fvs (scFvs); see, for example, Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883) . Such single chain antibodies include one or more "antigen-binding portions" of the antibody. These antibody fragments are prepared using conventional techniques known to those skilled in the art and the fragments are screened for suitability in the same manner as intact antibodies.

Antigen-binding moieties can also be included in single domain antibodies, maxibody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR and bis-scFv (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126- 1136). Antigen-binding portions of antibodies can be grafted onto polypeptide backbones such as type III fibronectin (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

The antigen-binding moieties can be incorporated into single-stranded molecules containing a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8 (10 ):1057-1062; and US Pat. No. 5,641,870).

As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at individual antigenic sites. Within each antigenic site, the antibody "arm" variable region interacts with the antigen through weak non-covalent forces at multiple sites; the more interactions, the stronger the affinity.

As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of an antigen-antibody complex. This process is controlled by three main factors: the affinity of the antibody epitope; the valency of both the antigen and the antibody and the structural arrangement of the interacting parts. Ultimately, these factors determine the specificity of an antibody, in other words, the likelihood that a particular antibody will bind to a particular antigen epitope.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are the amino acids encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, gamma-carboxyglutamate and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. an alpha carbon that is bonded to a hydrogen, carboxylic acid group, amino group, and R group, e.g., homoserine, norleucine, methionine sulfoxide , methionine methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

As used herein, the term "binding specificity" refers to the ability of a single antigen-binding antibody active site to react with only one antigenic determinant. The antigen-binding active site of an antibody is located in the Fab portion of the molecule and is formed from the hypervariable regions of the heavy and light chains. The binding affinity of an antibody is the strength of the reaction between one antigenic determinant and one antigen-binding active site of an antibody. It is the sum of the attractive and repulsive forces acting between the antigenic determinant and the antigen-binding active site of an antibody.

Specific binding between two molecules means binding with an equilibrium constant (KA or K _A ) of at least 1×10 ⁷ M ^-1 , 10 ⁸ M ^-1 , 10 ⁹ M ^-1 , 10 ¹⁰ M ^-1 or 10 ¹¹ M ^-1 . The phrase "specifically (or selectively) binds" to an antibody (eg, an antibody that binds BMP6) refers to a binding reaction that is determined by the presence of a recognizable antigen (eg, human BMP6 protein) in a heterogeneous population of proteins and other biological materials. In addition to the equilibrium constant (KA) above, the BMP6-binding antibody of the present invention typically also has a dissociation rate constant (Kd or KD or _KD ) of approximately 1×10 ^-2 s ^-1 . ×10 ^-3 s ^-1 or less, and binds to BMP6 with an affinity that is at least two times higher than its affinity in the case of binding to a non-specific antigen (for example, BMP2, BMP5 or BMP7). The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

Specific binding between two molecules means binding with an equilibrium constant (KA) (kon/koff) of at least 10 ² M ^-1 , at least 5×10 ² M ^-1 , at least 10 ³ M ^-1 , at least 5×10 ³ M ^-1 , at least 10 ⁴ M ^-1 , at least 5×10 ⁴ M ^-1 , at least 10 ⁵ M ^-1 , at least 5×10 ⁵ M ^-1 , at least 10 ⁶ M ^-1 , at least 5×10 ⁶ M ^-1 , at least 10 ⁷ M ^-1 , at least 5×10 ⁷ M ^-1 , at least 10 ⁸ M ^-1 , at least 5 X 10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 5×10 ⁹ M ^-1 , at least 10 ¹⁰ M ^-1 , at least 5×10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 5×10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , at least 5×10 ¹² M ^-1 , at least 10 ¹³ M ^-1 , at least 5×10 ¹³ M ^-1 , at least 10 ¹⁴ M ^-1 , at least 5×10 ¹⁴ M ^-1 , at least 10 ¹⁵ M ^-1 or at least 5×10 ¹⁵ M ^-1 .

The term "chimeric antibody" (or an antigen-binding fragment thereof) is an antibody molecule (or an antigen-binding fragment thereof) in which (a) a constant region or portion thereof has been altered, replaced, or exchanged such that the antigen-binding site (variable region) associated with a constant region of a different or altered class, with a different or altered effector function and/or from a different or altered species, or with a completely different molecule that confers new properties on the chimeric antibody, e.g., enzyme, toxin, hormone, growth factor, drug etc.; or (b) the variable region, or a portion thereof, is altered, replaced, or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. By changing to a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in humans compared to the original mouse antibody.

The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. With regard to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or in the case where the nucleic acid does not encode an amino acid sequence, they refer to essentially identical sequences. Due to the degeneracy of the genetic code, any given protein is encoded by a large number of functionally identical nucleic acids. For example, all of the codons GCA, GCC, GCG, and GCU code for the amino acid alanine. Thus, at each position where alanine is determined by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one kind of conservatively modified variations. Each nucleic acid sequence that encodes a polypeptide also describes herein each possible silent variation of the nucleic acid. One skilled in the art will appreciate that every codon in a nucleic acid (with the exception of AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, every silent variation of a nucleic acid that encodes a polypeptide is meant in every described sequence.

In the case of polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions in a polypeptide sequence that result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, cross-species homologues and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins (1984)). In one embodiment, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or change the binding characteristics of an antibody containing an amino acid sequence.

As used herein, the term "blocks" refers to stopping or preventing an interaction or process, such as stopping ligand-dependent or ligand-independent signaling.

As used herein, the term "recognizes" refers to an antibody, an antigen-binding fragment thereof, that finds and interacts (eg, binds) to its conformational epitope.

The terms "cross-blocking", "cross-blocking", "cross-blocking", "compete", "cross-compete" and related terms are used interchangeably herein to refer to the ability of an antibody or other binding agent to prevent other antibodies or binding agents from binding to BMP6 in standard competitive binding assay.

The ability or degree to which an antibody or other binding agent is able to interfere with the binding of another antibody or binding molecule to BMP6, and therefore may be considered cross-blocking according to the present invention, can be determined using standard competitive binding assays. One suitable assay involves the use of Biacore technology (eg, by using the BIAcore 3000 device (Biacore, Uppsala, Sweden)), by which the degree of interactions can be measured using surface plasmon resonance technology. Another assay uses an ELISA-based approach to measure cross-blocking.

The term "neutralizes" means that the antibody, after binding to its target, reduces the activity, level, or stability of the target; for example, an anti-BMP6 antibody, upon binding to BMP6, neutralizes BMP6, at least partially reducing the activity, level, or stability of BMP6, such as in signaling or its role in hepcidin levels and anemia.

The term "epitope" means a protein determinant capable of specifically binding to an antibody. Epitopes usually consist of reactive surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structure characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that the binding of the former, but not the latter, is lost in the presence of denaturing solvents.

The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or otherwise interacting with a molecule. Epitope determinants typically consist of reactive surface groups of molecules such as BMP6 amino acids or carbohydrate or sugar side chains, and they may have specific three-dimensional structure characteristics as well as specific charge characteristics. An epitope may be "linear" or "conformational".

The term "linear epitope" refers to an epitope in which all of the points of interaction between a protein and an interacting molecule (such as an antibody) are linear along the protein's primary amino acid sequence (contiguous).

As used herein, the term "high affinity" for an IgG antibody refers to an antibody that has a KD of 10 ^-8 M or less, 10 ^-9 M or less, or 10 ^-10 M, or 10 ^-11 M or less, in against the target antigen, for example, BMP6. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody that has a KD of 10 ^-7 M or less, or 10 ^-8 M or less.

As used herein, the term "human antibody" (or antigen-binding fragment thereof) is intended to include antibodies (and antigen-binding fragments thereof) having variable regions in which both the framework and CDR regions are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, for example, human germline sequences or mutated variants of human germline sequences. The human antibodies and antigen-binding fragments thereof of the present invention may contain amino acid residues not encoded by human sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation).

As used herein, the phrase "monoclonal antibody" or "monoclonal antibody composition" (or antigen-binding fragments thereof) refers to polypeptides, including antibodies, antibody fragments, bispecific antibodies, etc., that have a substantially identical amino acid sequence. or derived from the same genetic source. The term also includes preparations of antibody molecules of the same molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

The term "human monoclonal antibody" (or antigen-binding fragment thereof) refers to antibodies (and antigen-binding fragments thereof) exhibiting a single binding specificity that have variable regions in which both framework and CDR regions are derived from human sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that provides a B cell derived from a transgenic non-human animal, e.g., a transgenic mouse, with a genome containing a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

As used herein, the phrase "recombinant human antibody" (or antigen-binding fragment thereof) includes all human antibodies (and antigen-binding fragments thereof) that are produced, expressed, generated, or isolated by recombinant methods, such as antibodies isolated from an animal (e.g., mice). ) that is transgenic or transchromosomal for human immunoglobulin genes, or hybridomas derived therefrom, antibodies isolated from a host cell transformed to express a human antibody, such as from a transfectome, antibodies isolated from a recombinant combinatorial human antibody library, and antibodies obtained, expressed, created or isolated using any other methods that include splicing all or part of the human immunoglobulin gene, sequences into other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal that is transgenic for human Ig sequences, somatic in vivo mutagenesis is used), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibodies are are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the spectrum of human germline antibodies in vivo.

A "humanized" antibody (or antigen-binding fragment thereof), as used herein, is an antibody (or antigen-negative fragment thereof) that retains non-human antibody reactivity while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their corresponding parts of human antibodies (ie, the constant region as well as the framework parts of the variable region). See, for example, Morrison et al., Proc. Natl. Acad. sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988; Padlan, Molec. Immun. 28:489-498, 1991; and Padlan, Molec. Immun., 31:169-217, 1994. Other examples of human molecular engineering technology include, without limitation, the Xoma technology disclosed in US Pat. No. 5,766,886.

The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a certain percentage of amino acid residues or nucleotides that are the same (i.e. 60% identical, optionally 65%, 70%, 75%, 80%, 85% identical, 90%, 95%, or 99% in a specific area or, if specified, in the entire sequence) when compared and aligned to achieve the best fit in the comparison window or specific area, as measured using one of the following sequence comparison algorithms or using alignment in manual mode and visual inspection. Optionally, identity exists in a region that is at least about 50 nucleotides (or 10 amino acids) long, or more preferably in a region that is 100-500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) long. Optionally, identity exists in a region that is at least 50 nucleotides (or 10 amino acids) long, or more preferably in a region that is 100-500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) long.

For sequence comparisons, usually one sequence acts as a reference sequence against which the test sequences are compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the coordinates of the subsequences are set, and the software parameters of the algorithm for sequence analysis are set. You can use the default software options, or you can set alternative options. The sequence comparison algorithm then calculates percent sequence identity values for the test sequences compared to the reference sequence based on the program parameters.

"Comparison window", as used herein, includes reference to a segment with any of a number of adjacent positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, in in which the sequence can be compared with a reference sequence with the same number of contiguous positions after performing an optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the local homology search algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, using the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. sci. USA 85:2444, 1988, using computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA as part of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or using manual alignment mode and visual inspection (see, for example, Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described respectively in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990. BLAST analysis software is publicly available from the National Center for Biotechnology Information. This algorithm first identifies high scoring sequence pairs (HSPs) by identifying short words of length W in the requested sequence that either match or satisfy some positively evaluated threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word threshold score (Altschul et al., supra). These initial matches for adjacent words act as "seeds" to begin searches to find longer HSPs containing them. Matches for words are extended in both directions along each sequence by as much as the total alignment score can be increased. Total scores are calculated using, in the case of nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). In the case of amino acid sequences, the scoring matrix is used to calculate the total score. The extension of matches for words in each direction stops when the total alignment score decreases by the amount of X from its maximum achieved value; the total score tends to zero or lower due to the accumulation of negatively evaluated alignments of one or more residues; or the end of either sequence is reached. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to word length (N) 11, expectation (E) or 10, M=5, N=-4, and comparison of both strands. In the case of amino acid sequences, the BLASTP program defaults to a word length of 3 and an expectation (E) of 10 and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989 ), equalize (B) 50, wait (E) 10, M=5, N=-4 and compare both chains.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides a measure of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

Percent identity between two amino acid sequences can also be determined using the algorithm according to E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988), which was included in the ALIGN program (version 2.0) using the table weight of PAM120 residues, a gap extension penalty of 12, and a gap opening penalty of 4. In addition, percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970), which was included in the GAP program as part of the GCG software package (available at www.gcg.com), using either the Blossom 62 matrix or the PAM250 matrix, and a gap opening penalty of 16, 14, 12, 10, 8, 6 or 4 and a gap extension penalty of 1, 2, 3, 4, 5, or 6.

In addition to the percent sequence identity mentioned above, another indication that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies induced by the polypeptide encoded by the second nucleic acid. acid as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example, if the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules, or their complementary strands, hybridize to each other under stringent conditions, as described below. Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

As used herein, the term "isolated antibody" (or antigen-binding fragment thereof) refers to an antibody (or antigen-binding fragment thereof) that is substantially free of other antibodies having differing antigenic specificities (e.g., an isolated antibody that specifically binds to BMP6 is substantially free of contains antibodies that specifically bind to antigens other than BMP6). Moreover, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "isotype" refers to the class of antibody (eg, IgM, IgE, IgG, such as IgG1 or IgG4) that is provided by heavy chain constant region genes. An isotype also includes modified variants of one of these classes that have been modified to alter Fc function, for example to increase or decrease effector functions or binding to Fc receptors.

As used herein, the terms "Kass" or "Ka" or "KA" or "K _A " are intended to refer to the rate of association for a particular antibody-antigen interaction, while the terms "Kdiss." or "Kd" is intended to refer to the rate of dissociation for a particular antibody-antigen interaction. In one embodiment, the term "K _D " as used herein is intended to refer to a dissociation constant that is derived from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M). Antibody K _D values can be determined using methods well known in the art. The method for determining the K _D of an antibody is carried out using surface plasmon resonance or using a biosensor system such as the Biacore® system.

As used herein, the terms "monoclonal antibody" (or antigen-binding fragment thereof) or "composition based on a monoclonal antibody (or antigen-binding fragment)" refers to a preparation of antibody molecules (or antigen-binding fragment thereof) of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers based on them in single or double stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring or non-naturally occurring, that have binding properties similar to the reference nucleic acid, and that are metabolized like the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptidonucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also potentially encompasses its conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as well as the sequence itself. Specifically, as detailed below, degenerate codon substitutions can be achieved by creating sequences in which the third position in one or more selected (or all) codons is replaced by any of the canonical nucleosides and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

The term "operably linked" refers to a functional relationship between two or more segments of a polynucleotide (eg, DNA). Generally, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. In general, transcriptional control promoter sequences that are operably linked to the transcribed sequence are physically adjacent to the transcribed sequence, ie, they are cis-acting. However, some transcriptional control sequences, such as enhancers, do not need to be physically contiguous or in close proximity to the coding sequences whose transcription they enhance.

As used herein, the term "optimized" means that a nucleotide sequence has been altered such that it encodes an amino acid sequence using codons that are preferred in the producing cell or organism, usually a eukaryotic cell, e.g., Pichia cell, Chinese Hamster Ovary cell (CHO) or human cell. The optimized nucleotide sequence is designed in such a way as to completely or as far as possible preserve the amino acid sequence originally encoded by the original nucleotide sequence, which is also known as the "primary" sequence. Optimized sequences within the context of this document have been designed to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic cells or prokaryotic cells is also contemplated within the context of this document. Amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to polymers of amino acids in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids and a polymer of non-naturally occurring amino acids. Unless otherwise indicated, a particular polypeptide sequence also potentially encompasses conservatively modified variants thereof.

As used herein, the term "recombinant human antibody" (or antigen-binding fragment thereof) includes all human antibodies (and antigen-binding fragments thereof) that are produced, expressed, generated, or isolated by recombinant methods, such as antibodies isolated from an animal (e.g., mouse ) that is transgenic or transchromosomal for human immunoglobulin genes, or hybridomas derived therefrom, antibodies isolated from a host cell transformed to express a human antibody, such as from a transfectome, antibodies isolated from a recombinant combinatorial human antibody library, and antibodies obtained, expressed, created or isolated using any other methods that include splicing all or part of the human immunoglobulin gene, sequences into other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in one embodiment, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when an animal that is transgenic for human Ig sequences, somatic in vivo mutagenesis) is used, and hence the VH- and VL- recombinant antibody regions are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the spectrum of human germline antibodies in vivo.

The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to a particular cell-subject, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are included within the scope of the term "host cell" as used herein.

The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, eg mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Except where noted, the terms "patient" or "subject" are used interchangeably herein.

The term "treatment" includes administering compositions or antibodies to prevent or delay the onset of symptoms, complications, or biochemical signs of a disease (eg, anemia), alleviate symptoms, or arrest or suppress further progression of a disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the onset of its clinical or subclinical symptoms) or therapeutic suppression or relief of symptoms after the onset of the disease.

The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is the viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of a host cell after introduction into the host cell and thereby replicate along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In this description, "plasmid" and "vector" can be used interchangeably, since the plasmid is the most widely used form of the vector. However, the present invention is intended to include other forms of expression vectors such as, for example, viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

As used herein, the term "hematocrit" or "hematocrit index" is also known as erythrocyte sedimentation volume (PCV) or erythrocyte volume fraction (EVF) and is the volume (%) of erythrocytes in the blood. Normally, it is approximately 45% for men and approximately 50% for women. It is considered an integral part of an individual's CBC results, along with hemoglobin concentration, white blood cell count, and platelet count. In one embodiment, anemia refers to an abnormally low hematocrit, as opposed to polycythemia, which is an abnormally high hematocrit.

The term "performing analysis" is used to refer to the act of identifying, screening, probing, measuring, or determining in tests, and this act can be performed by any conventional means. For example, a sample can be analyzed for the presence of a particular genetic or protein marker using ELISA, Northern blotting, imaging, serotyping, cell typing, gene sequencing, phenotyping, haplotyping, immunohistochemistry, Western blotting, mass spectrometry, etc. The term "revealing" (and the like) means the act of extracting specific information from a given source, which may be direct or indirect. In some embodiments of the predictive methods disclosed herein, the presence or level of a given entity (eg, protein level, etc.) is indirectly detected in a biological sample, such as by querying a database for information. The terms "analysing" and "determining" involve the transformation of a material, for example, the transformation of a biological sample, such as a blood sample or other tissue sample, from one state to another by subjecting that sample to physical testing.

The term "acquisition" means to acquire, for example, to be made available in any way, for example, through physical intervention (for example, biopsy, blood sampling) or other than physical intervention (for example, transmission of information through a server), etc.

The phrase "performing the analysis of a biological sample..." etc. is used to mean that the sample can be subjected to tests (either directly or indirectly) for either the presence or level of a given marker (eg, protein). It will be understood that if the level of a substance indicates a likelihood, then the level of such substance can be used as a basis for choosing a treatment. For example, it is possible to determine the level of ferritin in a patient by analyzing its presence using quantitative or comparative qualitative methods (eg, levels compared to levels in other samples). The disclosed methods include, among other things, determining the level of a particular marker, such as ferritin, in a patient.

As used herein, the terms "selection" and "selected" as used in relation to a patient mean that a particular patient is specifically selected from a larger group of patients based on (due to) that a particular patient is characterized by predetermined criteria, for example, the patient has a specific ferritin level, such as that described herein. Similarly, "selective treatment" refers to providing treatment to a patient having a particular disease, where that patient is specifically selected from a larger group of patients on the basis that the particular patient has predetermined criteria, e.g., an anemic patient is specifically selected for treatment because that the patient has a particular ferritin level, such as that described herein. Similarly, "selective administration" refers to the administration of a drug to a patient that is specifically selected from a larger group of patients based on (due to) that a particular patient is characterized by predetermined criteria, such as a specific genetic or other biological marker, such as a specific level ferritin, such as those described herein. By selection, selective treatment, and selective administration is meant that a patient receives personalized therapy based on the specific biological characteristics of the patient, rather than receiving standard treatment solely on the basis that the patient has a particular disease. Selection, in relation to a method of treatment in the context of this document, does not refer to random treatment of a patient who has a particular marker, but rather refers to a weighted choice of administering a BMP6 antagonist to a patient based on the fact that the patient has a particular marker, e.g. a particular ferritin level, e.g. described in this document. Thus, selective treatment differs from standard treatment, in which all patients receive a particular drug, regardless of their marker status.

As used herein, "prediction" indicates that the methods described herein provide information to allow a healthcare professional to determine the likelihood that an individual having a disease, such as anemia, will respond or will more favorably respond for treatment with a BMP6 binding molecule. This term does not refer to the ability to predict a response with 100% accuracy. Instead, one of ordinary skill in the art will appreciate that this refers to increased probability.

As used herein, "probability" and "probably" are a measure of the likelihood that an event will occur. They can be used interchangeably with the term "opportunity". Probability refers to the possibility that a statement is more than a hypothesis, but less than a certain fact. Thus, an event is likely if a reasonable person, using general principles, training, or experience, concludes that, given the circumstances, the event is possible. In some embodiments, once the likelihood has been established, the patient may be treated (either treatment continued or treated at a dose escalation) with a BMP6 binding molecule, or the patient may not be treated (either treatment stopped or treatment given using low dose) with a BMP6 binding molecule.

The phrase "increased probability" refers to an increase in the likelihood that an event will occur. For example, some of the methods provided herein provide the ability to predict whether a patient will have an increased likelihood of responding to treatment with a BMP6 binding molecule or an increased likelihood of a better response to treatment with a BMP6 binding molecule compared to a patient. with the same or similar disease that does not have a marker (eg, the ferritin level described herein).

BRIEF DESCRIPTION OF GRAPHICS

In FIG. 1A shows inhibition of BMP activity by antagonist antibodies 5, 6 and 7 in a reporter gene assay. Shown activity against BMP2, BMP5, BMP6 and BMP7. In FIG. 1B shows binding of antibody 7 to human BMP6, human BMP7, human BMP5, mouse BMP6, hBaffR, BSA and Neu in an ELISA binding assay. In this figure and various other figures, as well as elsewhere in this description, Ab 5=antibody 5; Ab 6=antibody 6 and Ab 7=antibody 7.

In FIG. 2 shows pharmacodynamic profiles obtained in a single dose PK triage study in rats. Antibodies 5, 6 and 7 were used. Serum hepcidin and iron levels were measured 1 hour, 6 hours, 1, 2, 4, 8, 16 days post-dose (10 mg/kg, IV).

In FIG. 3 shows the dose-dependent effects of an anti-BMP6 antibody on serum biomarkers of iron metabolism. Top: Serum hIgG concentration versus time after a single IV injection of antibody 6 at the indicated doses. Bottom: The left panel is a quantitative analysis of serum hepcidin concentration after a single injection of antibody 6 or control human IgG, while the right panel is the serum iron concentration.

In FIG. 4 shows therapeutic treatment with an anti-BMP6 antibody in a mouse model of ESA-resistant inflammatory anemia. Top: Experimental design in a model of BA-induced ESA-resistant inflammatory anemia. Bottom: Erythropoiesis parameters 13 days after BA treatment. HGB: hemoglobin; HCT: hematocrit; RETA: reticulocyte count; RET-HE: reticulocyte hemoglobin equivalent. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to BA+EPO+hIgG1.

In FIG. 5 shows linear epitope mapping by HDxMS (hydrogen/deuterium exchange combined with mass spectrometry). The BMP6 epitope bound by antibody 7 is shown (residues 88-102 in human BMP6 (QTLVHLMNPEYVPKP (SEQ ID NO: 98))). Using HDxMS, antibody 676, a humanized version of a commercially available anti-BMP6 antibody, was found to bind to the human BMP6 epitope of residues 23-35 (VSSASDYNSSELK (SEQ ID NO: 99)).

In FIG. 6 shows the protocol for Part 1 of the clinical program to study the safety and efficacy of anti-BMP6 antibodies.

In FIG. 7 shows a dose adjustment decision tree for a clinical program to study the safety and efficacy of anti-BMP6 antibodies.

In FIG. 8 shows the protocol for Part 2 of the clinical program to study the safety and efficacy of anti-BMP6 antibodies.

In FIG. 9 shows single dose pharmacokinetic profiles of antibody 7 in male rats.

In FIG. 10 shows dose dependent effects of antibody 7 on serum biomarkers of iron metabolism in rats. A quantitative analysis of the concentration of hepcidin in the blood serum after a single injection of antibody 7 or control (carrier medium) at the indicated dose is shown. The left panel shows an expanded view of the effects in the first 24 hours after administration.

In FIG. 11 shows the dependent effects of antibody 7 on serum iron levels in rats. A quantitative analysis of the concentration of iron in the blood serum after a single injection of antibody 7 or control (carrier medium) at the indicated dose is shown. The left panel shows an expanded view of the effects in the first 24 hours after administration.

In FIG. 12 concentration versus time profile for IV injection of a single dose of antibody 7 (3 mg/kg) in cynomolgus monkeys. The graph shows the total concentration of antibody 7 (both unbound and bound to BMP6).

In FIG. 13 shows the serum concentrations of hepcidin and Fe in male cynomolgus monkeys after a single intravenous injection of antibody 7 at a dose of 3 mg/kg. Shown are data from three different macaques along with the average.

In FIG. 14 shows peak TSAT (% iron saturation) for hemodialysis patients before treatment (baseline) and within 3 days of receiving treatment with 0.01 mg/kg antibody 7 (Ab7) (3 days post-treatment). doses). All patients in Cohort 1 had pretreatment ferritin levels less than or equal to 500 ng/mL. All patients in cohort 2 had pretreatment ferritin levels ranging from 500 to 1000 ng/mL. The bold line shows the mean TSAT level for each group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating conditions associated with low iron levels, such as anemia, with BMP6 inhibitors.

BMP6 INHIBITORS

A wide range of BMP6 antagonists can be used in the methods of the present invention, such as, for example, antibodies, fusion proteins, adnectins, aptamers, anticalins, lipocalins, nucleic acids (for example, antisense molecules such as RNAi agents and ribozymes), immunoconjugates (eg, an antibody conjugated to a therapeutic agent), small molecules, fusion proteins, BMP6-derived peptide compounds, and receptor-based antagonists (eg, soluble domains of the BMP6 receptor).

Nucleic acids/antisense molecules

In another embodiment, the BMP6 antagonist used in the methods of the present invention is an antisense nucleic acid molecule that is complementary to a gene encoding BMP6, or a portion thereof, or a recombinant expression vector encoding an antisense nucleic acid molecule. As used herein, an "antisense" nucleic acid comprises a nucleotide sequence that is complementary to the "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence, or complementary to the coding strand of a gene. Accordingly, the antisense nucleic acid can form a hydrogen bond with the sense nucleic acid.

The use of antisense nucleic acids to down-modulate the expression of a particular protein in a cell is well known in the art (see, for example, Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986; Askart, F. K. and McDonnell, W. M. (1996) N. Eng. J. Med. 334:316-318; Bennett, M. R. and Schwartz, S. M. (1995) Circulation 92:1981-1993; Mercola, D and Cohen, J. S. (1995) Cancer Gene Ther 2:47-59; Rossi, J. J. (1995) Br. Med. Bull 51:217-225; Wagner, R. W. (1994) Nature 372:333-335). An antisense nucleic acid molecule contains a nucleotide sequence that is complementary to the coding strand of another nucleic acid molecule (eg, mRNA sequence) and, accordingly, can form hydrogen bonds with the coding strand of the other nucleic acid molecule. Antisense sequences complementary to mRNA sequences may be complementary to a sequence located in the mRNA coding region, the 5' or 3' untranslated region of the mRNA, or the region connecting the coding region and the untranslated region (for example, at the junction of the 5'-untranslated region and the coding areas). In addition, the antisense nucleic acid may be complementary in sequence to a region with regulators of the mRNA-encoding gene, such as a transcription initiation sequence or regulatory element. Preferably, the antisense nucleic acid is designed to be a complementary region upstream of the start codon or extending beyond the start codon in the coding strand/or in the 3'-untranslated region of the mRNA.

Antisense nucleic acids can be designed according to the rules of Watson-Crick base pairing. The antisense nucleic acid molecule may be complementary to the entire coding region of the BMP6 mRNA, but more preferably it is an oligonucleotide that is antisense to only a portion of the coding or non-coding region of the BMP6 mRNA. For example, the antisense oligonucleotide may be complementary to the region surrounding the translation start site in the BMP6 mRNA. The antisense oligonucleotide may be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. The antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, for example, phosphorothioate derivatives and acridine-substituted nucleotides can be used. Examples of modified nucleotides that can be used to create an antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-ioduracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2 -thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueuosine, inosine, N6-isopentenyladenine, methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6 -adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v ), vibutoxosin, pseudouracil, queuosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v) , 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w and 2,6-diami nopurine. Alternatively, an antisense nucleic acid can be produced biologically using an expression vector into which the nucleic acid has been subcloned in an antisense orientation (i.e., the RNA transcribed from the inserted nucleic acid will be present in an antisense orientation relative to the target nucleic acid of interest, further described in the next subsection).

Antisense nucleic acid molecules that can be used in the methods of the present invention are typically administered to a subject, or are generated in situ such that they hybridize or bind to cellular mRNA and/or genomic DNA encoding BMP6, thereby inhibiting expression by inhibiting transcription and/or translation. Hybridization can occur through conventional nucleotide complementarity to form a stable duplex or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An exemplary route of administration for antisense nucleic acid molecules includes direct injection into a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, in the case of systemic administration, antisense molecules can be modified so that they specifically bind to receptors or antigens expressed on the surface of selected cells, for example, by linking the antisense nucleic acid in the molecules to peptides or antibodies that bind to receptors or antigens on the surface of cells. . Antisense nucleic acid molecules can also be delivered to cells using vectors well known in the art and described, for example, in US20070111230, the full content of which is incorporated herein. In order to achieve sufficient intracellular concentrations of antisense molecules, vector constructs are preferred in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter.

In yet another embodiment, the antisense nucleic acid molecule used in the methods of the present invention may include an α-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, unlike conventional β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). Antisense nucleic acid molecules may also contain a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987) FEBS Lett. 215: 327-330).

In another embodiment, the antisense nucleic acid used in the methods of the present invention is a compound that mediates RNAi. RNA interference agents include, but are not limited to, nucleic acid molecules, including RNA molecules that are homologous to BMP6 or a fragment thereof, "short interfering RNA" (siRNA), "short hairpin" or "small hairpin RNA" (shRNA), and small molecules that prevent or inhibit the expression of the target gene through RNA interference (RNAi). RNA interference is a post-transcriptional targeted gene silencing technique in which a double-stranded RNA (dsRNA) is used to destroy a messenger RNA (mRNA) containing the same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287, 2431-2432 (2000 ); Zamore, P. D. et al. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197 (1999)). The process occurs when an endogenous ribonuclease cleaves a longer dsRNA into shorter RNAs of 21 or 22 nucleotides in length, called small interfering RNAs or siRNAs. Smaller segments of RNA then mediate the destruction of the target mRNA. RNAi synthesis kits are commercially available from, for example, New England Biolabs and Ambion. In one embodiment, one or more of the chemicals described above for use in antisense RNA can be used.

In another embodiment, the antisense nucleic acid is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, with which they have a complementary region. Thus, ribozymes (eg, hammerhead-type ribozymes (described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave BMP6 mRNA transcripts, thereby inhibiting BMP6 mRNA translation.

Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the BMP6 regulatory region (eg, promoter and/or enhancers) to form triple helical structures that prevent transcription of the BMP6 gene. In general, see Helene, C., 1991, Anticancer Drug Des. 6(6):569-84; Helene, C. et al., 1992, Ann. N.Y. Acad. sci. 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15.

Fusion proteins and BMP6-derived peptide compounds

In another embodiment, the BMP6 antagonist used in the methods of the present invention is a fusion protein or peptide compound derived from the BMP6 amino acid sequence. In particular, an inhibitory compound provides a fusion protein or portion of BMP6 (or a mimetic thereof) that mediates the interaction of BMP6 with a target molecule such that contact of BMP6 with the fusion protein or peptide compound competitively inhibits the interaction of BMP6 with the target molecule. Such fusion proteins and peptide compounds can be prepared using standard techniques known in the art. For example, peptide compounds can be prepared by chemical synthesis using standard peptide synthesis techniques and then introduced into cells using a number of means known in the art for introducing peptides into cells (eg, liposome, etc.).

The in vivo half-life of the fusion protein or peptide compounds of the present invention can be improved by making peptide modifications, such as adding N-linked glycosylation sites to a BMP6 peptide compound or conjugating a BMP6 peptide compound with polyethylene glycol (PEG; PEGylation), for example, by lysine monopegylation. Such techniques have proven to be useful in extending the half-life of protein-based therapeutic drugs. It is expected that pegylation of the BMP6 polypeptides of the present invention may result in similar pharmaceutical benefits.

In addition, pegylation can be achieved in any part of the polypeptide of the present invention by introducing a non-natural amino acid. Certain non-natural amino acids can be administered using the technology described in Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wang and Schultz, Science 301:964-967, 2003; Wang et al., Science 292:498-500, 2001; Zhang et al., Science 303:371-373, 2004; or US Pat. No. 7,083,970. Briefly, some of these expression systems provide for site-directed mutagenesis to introduce a nonsense codon, such as "amber" TAG, into an open reading frame encoding a polypeptide of the present invention. Such expression vectors are then introduced into a host that can use a tRNA specific for the introduced nonsense codon and loaded with a selected non-natural amino acid. Specific non-natural amino acids that are useful for conjugating fragments to the polypeptides of the present invention include amino acids with acetylene and azide containing side chains. Polypeptides from BMP6 containing these new amino acids can then be pegylated at these selected sites in the protein.

Receptor based antagonist

In another embodiment, the BMP6 antagonist used in the methods of the present invention is a receptor-based antagonist. Receptor-based antagonists include soluble BMP6 receptors that respectively bind BMP6 (or portions thereof) and interfere with BMP6 activity and/or function. In a specific embodiment, the receptor-based antagonist includes, without limitation, soluble hemojuvelin or a type I or type II BMP receptor. Variants of soluble hemojewelin include those described in US2010/0136015.

ANTIBODIES TO BMP6 AND THEIR ANTIGEN-BINDING FRAGMENTS

In the present invention, as inhibitors of BMP6 applicable in the methods and other embodiments and aspects of the present invention described herein, antibodies and antigen-binding fragments thereof that specifically bind to human BMP6 are provided.

BMP6, a secreted ligand of the BMP family of growth factors, has been identified as a critical endogenous regulator of hepatic expression of the hormone hepcidin involved in iron metabolism. Without wishing to be bound by any particular theory, the present invention provides an anti-BMP6 antagonist antibody as a therapy for lowering hepcidin levels, which is expected to benefit patients with iron deficiency anemia by overcoming resistance to an erythropoiesis-stimulating agent. (ESA), which makes a significant contribution to the incidence of underlying disease and is often a predictor of poor outcome.

Examples of such antibodies to human BMP6 are antibodies 3, 5, 6 and 7, the sequences of which are shown in Table 1, and antibodies to human BMP6, described in Table 14.

All of antibodies 5, 6 and 7 bind with high affinity to human BMP6 with high selectivity over human BMP7, human BMP5 and human BMP2 (see FIG. 1A). All of these antibodies also show a decrease in serum hepcidin and an increase in serum iron in rats (see FIG. 2).

Additional examples of antibodies to human BMP6 are LY3113593 (see, for example, clinical trial NCT02604160). Additional examples of antibodies to human BMP6 are described in PCT Application Publication Number WO2014/099391, the contents of which are incorporated herein by reference in their entirety. Specific antibodies to human BMP6 from WO2014/099391 include those described in Table 14.

Table 14 Examples of anti-human BMP6 antibodies

Sequence name Subsequence SEQID NO: Antibody 1 LCDR1 RSSENIYRNLA 1 LCDR2 ATNLAD 2 LCDR3 QGIWGTPLT 3 Light chain variable region (aa) DIQMTQSPSSLSASVGDRVTITCRSSENIYRNLAWYQQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGIWGTPLTFGGGTKVEIK 4 Light chain (aa) DIQMTQSPSSLSASVGDRVTITCRSSENIYRNLAWYQQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGIWGTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSVY 5 HCDR1 GYTFTSYAMH 6 HCDR2 YINPYNDGTKYNENFK 7 HDDR3 RPFGNAMDI 8 Heavy chain variable region (aa) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAMHWVRQAPGQGLEWMGYINPYNDGTKYNENFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARRPFGNAMDIWGQGTLVTVSS 92 Heavy chain (aa) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAMHWVRQAPGQGLEWMGYINPYNDGTKYNENFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARRPFGNAMDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 93 Antibody 2 LCDR1 RSSENIYRNLA 1 LCDR2 ATNLAD 2 LCDR3 QGIWGTPLT 3 Light chain variable region (aa) DIQMTQSPSSLSASVGDRVTITCRSSENIYRNLAWYQQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGIWGTPLTFGGGTKVEIK 4 Light chain (aa) DIQMTQSPSSLSASVGDRVTITCRSSENIYRNLAWYQQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGIWGTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSVY 5 HCDR1 GYTFTSYAMH 6 HCDR2 YINPYNRGTKYNENFK 94 HDDR3 RPFGNAMDI 8 Heavy chain variable region (aa) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAMHWVRQAPGQGLEWMGYINPYNRGTKYNENFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARRPFGNAMDIWGQGTLVTVSS 95 Heavy chain (aa) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAMHWVRQAPGQGLEWMGYINPYNRGTKYNENFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARRPFGNAMDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 96

To provide additional evidence that targeting this pathway can confer improved functional outcomes, we tested the ability of BMP6-specific antibodies, Antibodies 5-7, to modulate serum biomarkers of iron metabolism in normal mice and rats and reverse the course of resistant to ESA of anemia in a mouse model of anemia due to inflammation. The present inventors found that a single injection of anti-BMP6 antibody into animals resulted in a sustained increase in serum iron levels accompanied by a strong suppression of circulating hepcidin in the bloodstream. In addition, therapeutic treatment of mice exposed to inflammation-induced anemia significantly improved the parameters of erythropoiesis in response to simultaneous treatment with erythropoietin.

In the present invention, inhibition of BMP6 signaling in a mouse model of inflammatory anemia significantly improved iron-dependent erythrocyte parameters.

The BMP6 antagonist antibodies disclosed herein represent a novel therapeutic approach for the safe improvement of anemia with low erythropoietin responsiveness. Without wishing to be bound by any particular theory, the present invention suggests that this may be due to the mobilization and availability of iron stores on demand from the erythroid compartment.

In one embodiment, the present invention provides isolated antibodies, or antigen-binding fragments thereof, that bind to human BMP6 protein with an affinity that is 100-fold, 500-fold, or 1000-fold greater than that of either human BMP5 protein or human BMP7. Specificity for BMP6 without binding to BMP7 is important because BMP6 knockout is not lethal in mice. However, BMP7 knockout mice die after birth due to kidney, eye, and bone defects. Single knockouts of one of the genes do not alter cardiogenesis, but double knockouts of BMP6 and BMP7 demonstrated several defects and delays in heart development; embryos died due to heart failure. BMP7 is important in preventing the progression of chronic heart disease associated with fibrosis. Therefore, cross-reactivity of an anti-BMP6 antibody with BMP7 is not desirable. The antibodies provided herein are specific for BMP6, not BMP7; see, for example, Table 4A. In FIG. 1B also shows evidence of binding specificity to human BMP6 rather than human BMP2, BMP5 and BMP7 proteins. In contrast, a commercially available anti-BMP6 antibody from R&D Systems, for example, has been found to have strong cross-reactivity with BMP7 in the reporter gene assay and inhibit both BMP6 and BMP7.

The antibodies of the present invention include, without limitation, human monoclonal antibodies isolated as described in the examples (see section 6 below).

Examples of such antibodies to human BMP6 are antibodies 3, 5, 6 and 7, the sequences of which are shown in Table 1.

Mature antibody 7 is derived from NOV0442_VL(YGQ) with germline regression/PTM deletion, which is derived from primary IgG hit NOV0442 (VH3_3-15, Vl1_1e). Antibody 7 binds with high affinity to human BMP6 in an ELISA binding assay with over 500-fold selectivity over human BMP7 (i.e., affinity for human BMP6 is over 500-fold greater than for human BMP7) . This antibody also lacks detectable activity against human BMP2 or BMP5. The BMP6 peptide recognized by NOV0442 primary IgG and antibody 7 is shown in FIG. 5. The peptide contains the amino acids QTLVHLMNPEYVPKP (SEQ ID NO: 98) of human BMP6. Unlike IgG NOV0442 and antibody 7, humanized mAb507 (R&D Systems) binds to the sequence VSSASDYNSSELK (SEQ ID NO: 99) of human BMP6. Thus, the epitope recognized by IgG NOV0442 and antibody 7 is a novel BMP6 epitope. Antibody 7 also inhibits BMP6 binding to receptors in vitro. Binding of BMP6 to BMPR1A is maximally inhibited by 59%; binding to BMPR1B is maximally inhibited by 85% and binding to RGM-c is maximally inhibited by 72%. A single treatment with a dose of 10 mg/kg in rats results in a sustained suppression of circulating hepcidin in the bloodstream. The estimated minimum effective dose in mice is less than or equal to 0.1 mg/kg. After administration of a single dose of antibody 3 mg/kg to monkeys, an increase in serum iron levels was also observed and a decrease in hepcidin levels was observed. In mice treated with Brucella abortus antigen to mimic anemia, the effect of treatment with antibody 7 (2 mg/kg) was consistent with a clinically significant erythropoiesis response to continuous EPO therapy with a gradual increase in hemoglobin > 2.0 g/dl from baseline. level.

In antibody 7, a site of potential post-translational modification was removed by mutating N51Q in LCDR2 to increase the homogeneity of the subsequent product. The antibody derived from the VH3/lambda1 framework was designed to match the closest human germline sequence: in VH via the V40A mutation, in VL via the D1Q, I2S mutations and the introduction of amino acids Y49 and G50 to restore the framework in which these 2 residues were initially missing.

This work resulted in antibody 7 (=NOV0958=NOV0806_VH[V40A]_VL[D1Q, I2S, Y49, G50, N51Q]).

All of antibodies 3, 5, 6 and 7 show high specificity for human BMP6 protein compared to human BMP2, BMP5 or BMP7 protein. It was predicted that the epitope for all these antibodies is the same, since they are all derived from the same primary Fab before affinity maturation. Antibody 3, for example, shares the same Fab clone with both of antibodies 5 and 7 prior to HCDR2 affinity maturation. Antibody 5 is derived from NOV0442 (VH3_3-15, VI1_1e) → NOV0442_VL(YGQ) → (HCDR2 affinity maturation) → Antibody 5. Antibody 3 is derived from NOV0442 (VH3_3-15,VI1_1e) → NOV0442_VL(YGS) → (HCDR2 affinity maturation) → antibody 3. Additional details regarding the generation of the antibodies described herein are provided in the examples.

The present invention provides antibodies that specifically bind to BMP6 (e.g., the human BMP6 protein), wherein said antibodies comprise the VH domain shown in Table 1 and/or Table 14. The present invention also provides antibodies that specifically bind to the BMP6 protein , wherein said antibodies comprise a VH CDR having the amino acid sequence of any of the VH CDRs shown in Table 1 and/or Table 14. In particular, the present invention provides antibodies that specifically bind to the BMP6 protein, wherein said antibodies contain one, two, three, four, five or more VH CDRs (or alternatively consist of them) having the amino acid sequence of any of the VH CDRs shown in Table 1 and/or Table 14.

The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to BMP6, said antibodies or antigen-binding fragments thereof comprising (or alternatively consisting of) the VH amino acid sequence shown in Table 1 and/or Table 14, wherein no more than about 10 amino acids in the framework sequence (eg, a sequence that is not a CDR) have been mutated (where the mutation is, as various non-limiting examples, addition, substitution, or deletion). The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to BMP6, said antibodies or antigen-binding fragments thereof comprising (or alternatively consisting of) the VH amino acid sequence shown in Table 1 and/or Table 14, wherein no more than 10 amino acids in the framework sequence (eg, a sequence that is not a CDR) have been mutated (where the mutation is, as various non-limiting examples, addition, substitution, or deletion).

The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to BMP6, said antibodies or antigen-binding fragments thereof comprising (or alternatively consisting of) the VH amino acid sequence shown in Table 1 and/or Table 14, wherein no more than about 20 amino acids in the framework sequence (eg, a sequence that is not a CDR) have been mutated (where the mutation is, as various non-limiting examples, addition, substitution, or deletion). The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to BMP6, said antibodies or antigen-binding fragments thereof comprising (or alternatively consisting of) the VH amino acid sequence shown in Table 1 and/or Table 14, wherein no more than 20 amino acids in the framework sequence (eg, a sequence that is not a CDR) have been mutated (where the mutation is, as various non-limiting examples, an addition, a substitution, or a deletion).

The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to BMP6, said antibodies or antigen-binding fragments thereof comprising (or alternatively consisting of) the VL amino acid sequence shown in Table 1 and/or Table 14, wherein no more than about 10 amino acids in the framework sequence (eg, a sequence that is not a CDR) have been mutated (where the mutation is, as various non-limiting examples, addition, substitution, or deletion). The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to BMP6, said antibodies or antigen-binding fragments thereof comprising (or alternatively consisting of) the VL amino acid sequence shown in Table 1 and/or Table 14, wherein no more than 10 amino acids in the framework sequence (eg, a sequence that is not a CDR) have been mutated (where the mutation is, as various non-limiting examples, addition, substitution, or deletion).

The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to BMP6, said antibodies or antigen-binding fragments thereof comprising (or alternatively consisting of) the VL amino acid sequence shown in Table 1 and/or Table 14, wherein no more than about 20 amino acids in the framework sequence (eg, a sequence that is not a CDR) have been mutated (where the mutation is, as various non-limiting examples, addition, substitution, or deletion). The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to BMP6, said antibodies or antigen-binding fragments thereof comprising (or alternatively consisting of) the VL amino acid sequence shown in Table 1 and/or Table 14, wherein no more than 20 amino acids in the framework sequence (eg, a sequence that is not a CDR) have been mutated (where the mutation is, as various non-limiting examples, an addition, a substitution, or a deletion).

The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to the BMP6 protein, wherein said antibodies or antigen-binding fragments thereof comprise the VL domain shown in Table 1 and/or Table 14. The present invention also provides antibodies and antigen-binding fragments thereof, that specifically bind to the BMP6 protein, wherein said antibodies or antigen-binding fragments thereof comprise VL CDRs having the amino acid sequence of any of the VL CDRs shown in Table 1 and/or Table 14. In particular, the present invention provides antibodies and antigen-binding fragments thereof, that specifically bind to the BMP6 protein, wherein said antibodies or antigen-binding fragments thereof contain one, two, three or more VL CDRs (or alternatively consist of them) having the amino acid sequence of any of the VL CDRs shown in Table 1 and/or table 14.

Other antibodies and antigen-binding fragments of the present invention include amino acids that have been mutated and are characterized by at least 60, 70, 80, 90, or 95 percent identity of the CDR regions with the CDR regions shown in the sequences described in the table. 1 and/or Table 14. In one embodiment, they include mutant amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the CDR regions compared to the CDR regions shown in the sequence described in table 1 and/or table 14.

The present invention also provides nucleic acid sequences that encode the VH, VL, full length heavy chain and full length light chain of antibodies and antigen-binding fragments thereof that specifically bind to the BMP6 protein. Such nucleic acid sequences can be optimized for expression in mammalian cells (eg, Table 1 shows illustrative nucleic acid sequences for the heavy chain and light chain of antibodies 3, 5, 6 and 7).

TABLE 1. Examples of anti-BMP6 antibodies of the present invention

ANTIBODY 3 SEQID NO: Kabat HCDR1 SYVVH 9 Kabat HCDR2 RIKDHKQGYTTAYAASVKG 10 Kabat HDDR3 VERSKSGFDN eleven Chothia HCDR1 GFTFSSY 12 Chothia HCDR2 KDHKQGYT 13 Chothia HDDR3 VERSKSGFDN 14 vh QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYVVHWVRQAPGKGLEWVGRIKDHKQGYTTAYAASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSS 15 DNA VH caggtgcaattggtggaaagcggcggtggcctggtgaaaccaggcggcagcctgcgcctgagctgcgccgcctccggattcaccttttcttcttacgttgttcattgggtgcgccaggccccgggcaaaggtctcgagtgggtgggccgtatcaaagaccacaaacagggctacactactgcttatgccgcctctgtgaaaggccgctttaccattagccgcgatgattcgaaaaacaccctgtatctgcaaatgaacagcctgaaaaccgaagatacggccgtgtattattgcgcgcgtgttgaacgttctaaatctggtttcgataactggggccaaggcaccctggtgactgttagctca 16 heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYVVHWVRQAPGKGLEWVGRIKDHKQGYTTAYAASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 17 heavy chain DNA caggtgcaattggtggaaagcggcggtggcctggtgaaaccaggcggcagcctgcgcctgagctgcgccgcctccggattcaccttttcttcttacgttgttcattgggtgcgccaggccccgggcaaaggtctcgagtgggtgggccgtatcaaagaccacaaacagggctacactactgcttatgccgcctctgtgaaaggccgctttaccattagccgcgatgattcgaaaaacaccctgtatctgcaaatgaacagcctgaaaaccgaagatacggccgtgtattattgcgcgcgtgttgaacgttctaaatctggtttcgataactggggccaaggcaccctggtgactgttagctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 18 Kabat LCDR1 TGSSSNIGAGYSVH 19 Kabat LCDR2 GSSERPs 20 Kabat LCDR3 QSWDSSQTLVV 21 Chothia LCDR1 SSSNIGAGYS 22 Chothia LCDR2 GSS 23 Chothia LCDR3 WDSSQTLV 24 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWYQQLPGTAPKLLIYGSSERPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVL 25 DNA VL CAGAGCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACCGGGCCAGCGCGTGACCATTAGCTGTACCGGCAGCAGCAGCAACATTGGTGCTGGTTACTCTGTGCATTGGTACCAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTATGGTAGCTCTGAACGCCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTACTGCCAGTCTTGGGACTCTTCTCAGACTCTGGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA 26 light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWYQQLPGTAPKLLIYGSSERPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCAPTE 27 light chain DNA CAGAGCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACCGGGCCAGCGCGTGACCATTAGCTGTACCGGCAGCAGCAGCAACATTGGTGCTGGTTACTCTGTGCATTGGTACCAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTATGGTAGCTCTGAACGCCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTACTGCCAGTCTTGGGACTCTTCTCAGACTCTGGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 28 ANTIBODY 5 Kabat HCDR1 SYVVH 29 Kabat HCDR2 RIKRESSSYTTMYAAPVKG thirty Kabat HDDR3 VERSKSGFDN 31 Chothia HCDR1 GFTFSSY 32 Chothia HCDR2 KRESSSYT 33 Chothia HDDR3 VERSKSGFDN 34 vh QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYVVHWVRQAPGKGLEWVGRIKRESSSYTTMYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSS 35 DNA VH CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGATTAAGAGAGAGTCCTCTAGCTACACTACTATGTACGCCGCTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGC 36 heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYVVHWVRQAPGKGLEWVGRIKRESSSYTTMYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 37 heavy chain DNA CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGATTAAGAGAGAGTCCTCTAGCTACACTACTATGTACGCCGCTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAG CCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG 38 Kabat LCDR1 TGSSSNIGAGYSVH 39 Kabat LCDR2 GQSERPS 40 Kabat LCDR3 QSWDSSQTLVV 41 Chothia LCDR1 SSSNIGAGYS 42 Chothia LCDR2 GQS 43 Chothia LCDR3 WDSSQTLV 44 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVL 45 DNA VL CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTG 46 light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCVTAP 47 light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGCAGC 48 ANTIBODY 6 Kabat HCDR1 SYVVH 49 Kabat HCDR2 RTHRSDMGYATSYAAPVKG 50 Kabat HDDR3 VERSKSGFDN 51 Chothia HCDR1 GFTFSSY 52 Chothia HCDR2 RHSDMYA 53 Chothia HDDR3 VERSKSGFDN 54 vh QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYVVHWVRQAPGKGLEWVGRTRHSDMGYATSYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSS 55 DNA VH CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGACTAGACACTCAGATATGGGCTACGCTACTAGCTACGCCGCTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGC 56 heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYVVHWVRQAPGKGLEWVGRTRHSDMGYATSYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 57 heavy chain DNA CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGACTAGACACTCAGATATGGGCTACGCTACTAGCTACGCCGCTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAG CCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG 58 Kabat LCDR1 TGSSSNIGAGYSVH 59 Kabat LCDR2 GQSERPS 60 Kabat LCDR3 QSWDSSQTLVV 61 Chothia LCDR1 SSSNIGAGYS 62 Chothia LCDR2 GQS 63 Chothia LCDR3 WDSSQTLV 64 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVL 65 DNA VL CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTG 66 light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCVTAP 67 light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGCAGC 68 ANTIBODY 7 Kabat HCDR1 SYVVH 69 Kabat HCDR2 RIRLETHGYAAEYAASVKG 70 Kabat HDDR3 VERSKSGFDN 71 Chothia HCDR1 GFTFSSY 72 Chothia HCDR2 RLETHGYA 73 Chothia HDDR3 VERSKSGFDN 74 vh QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYVVHWVRQAPGKGLEWVGRIRLETHGYAAEYAASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSS 75 DNA VH CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGATTAGACTGGAAACTCACGGCTACGCCGCCGAGTACGCCGCTAGTGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGC 76 heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYVVHWVRQAPGKGLEWVGRIRLETHGYAAEYAASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 77 heavy chain DNA CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGATTAGACTGGAAACTCACGGCTACGCCGCCGAGTACGCCGCTAGTGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAG CCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG 78 Kabat LCDR1 TGSSSNIGAGYSVH 79 Kabat LCDR2 GQSERPS 80 Kabat LCDR3 QSWDSSQTLVV 81 Chothia LCDR1 SSSNIGAGYS 82 Chothia LCDR2 GQS 83 Chothia LCDR3 WDSSQTLV 84 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVL 85 DNA VL CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTG 86 light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCVTAP 87 light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGCAGC 88

Other antibodies and antigen-binding fragments thereof of the present invention include those in which the amino acids or nucleic acids encoding amino acids have been mutated and are at least 60%, 70%, 80%, 90%, or 95% identical to the sequences described in Table 1 and/or Table 14. In one embodiment, they include mutant amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the variable regions compared to the variable regions shown in the sequence described in table 1 and/or table 14, while maintaining almost the same therapeutic activity.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human BMP6 and contains the sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 of SEQ ID NOs. : 19, 20 and 21, respectively.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human BMP6 and contains the sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 12, 13 and 14, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 of SEQ ID NOs. : 22, 23 and 24, respectively.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human BMP6 and contains the sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 29, 30 and 31, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 of SEQ ID NO : 39, 40 and 41, respectively.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human BMP6 and contains the sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 32, 33 and 34, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 of SEQ ID NO : 42, 43 and 44, respectively.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human BMP6 and contains the sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 49, 50 and 51, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 of SEQ ID NO : 59, 60 and 61, respectively.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human BMP6 and contains the sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 52, 53 and 54, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 of SEQ ID NO : 62, 63 and 64, respectively.

In another specific embodiment, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to human BMP6 and contains the sequences HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 69, 70, and 71, respectively, and the sequences of LCDR1, LCDR2, and LCDR3, of SEQ ID NOs. : 79, 80 and 81, respectively.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human BMP6 and contains the sequences HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 72, 73 and 74, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 of SEQ ID NOs. : 82, 83 and 84, respectively.

Since each of these antibodies can bind to BMP6 sequences (amino acid sequences and nucleotide sequences encoding amino acid sequences), VH, VL, full length light chain and full length heavy chain can be "mixed and matched" to create other BMP6 binding antibodies and their antigen binding fragments of the present invention. Such "mix and match" BMP6 binding antibodies can be tested using binding assays known in the art (eg, ELISAs and other assays described in the Examples section). If these strands are mixed and matched, the VH sequence from the particular VH/VL pair should be replaced with a structurally similar VH sequence. Similarly, a full length heavy chain sequence from a particular full length heavy chain/full length light chain pair should be replaced with a structurally similar full length heavy chain sequence. Similarly, the VL sequence from a particular VH/VL pair should be replaced with a structurally similar VL sequence. Similarly, a full length light chain sequence from a particular full length heavy chain/full length light chain pair should be replaced with a structurally similar full length light chain sequence.

In another aspect, the present invention provides BMP6-binding antibodies that comprise the heavy chain and light chain CDR1, CDR2 and CDR3 described in Table 1 and/or Table 14, or combinations thereof. CDR regions are determined using the Kabat system (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) or using the Chothia system [Chothia et al. 1987 J. Mol. Biol. 196:901-917; and Al-Lazikani et al. 1997 J. Mol. Biol. 273: 927-948].

Given that each of these antibodies can bind to BMP6, and that antigen-binding specificity is provided predominantly by the CDR1, 2 and 3 regions, the CDR1, 2 and 3 VH sequences and the CDR1, 2 and 3 VL sequences can be "mixed and matched". pairs" (i.e., CDRs from different antibodies can be mixed and matched, although each antibody must contain CDR1, 2 and 3 VH and CDR1, 2 and 3 VL to create other BMP6 binding molecules of the present invention. Such "mix and match" BMP6 binding antibodies can be tested using the binding assays known in the art and the assays described in the examples (e.g., ELISA variants) If VH CDR sequences are mixed and matched, a CDR1, CDR2 and/or CDR3 sequence from a particular VH sequence should be replaced with structurally similar CDR sequence(s). To avoid mixing and matching, the CDR1, CDR2 and/or CDR3 sequence from a particular VL sequence should be replaced with a structurally similar CDR sequence(s). One of ordinary skill in the art will appreciate that new VH and VL sequences can be generated by mutating one or more VH and/or VL CDR region sequences with structurally similar sequences from the CDR sequences provided herein for monoclonal antibodies at the present invention.

Accordingly, the present invention provides an isolated monoclonal antibody or antigen-binding region thereof, comprising a heavy chain variable region CDR1 comprising an amino acid sequence selected from any of SEQ ID NOs: 29, 49, 69, 12, 32, 52, 72, or 9; a heavy chain variable region CDR2 comprising an amino acid sequence selected from any of SEQ ID NOs: 10, 30, 50, 70, 13, 33, 53, or 73; a heavy chain variable region CDR3 comprising an amino acid sequence selected from any of SEQ ID NOs: 11, 31, 51, 71, 14, 34, 54, or 74; A light chain variable region CDR1 comprising an amino acid sequence selected from any of SEQ ID NOs: 19, 39, 59, 79, 22, 42, 62, or 82; A light chain variable region CDR2 comprising an amino acid sequence selected from any of SEQ ID NOs: 20, 40, 60, 80, 23, 43, 63, or 83; and a light chain variable region CDR3 comprising an amino acid sequence selected from any of SEQ ID NOs: 21, 41, 61, 81, 24, 44, 64, or 84; where the antibody specifically binds to BMP6.

In one embodiment, the antibody that specifically binds to BMP6 is an antibody as described in Table 1 and/or Table 14.

As used herein, a human antibody comprises heavy or light chain variable regions or full-length heavy or light chains that are "the product of" or "derived from" a particular germline sequence if the antibody variable regions or full-length chains are derived from a system in which human germline immunoglobulin genes are used. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest, or screening a phage-displayed human immunoglobulin gene library with an antigen of interest. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e. has the highest % identity) to a human antibody sequence. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences from the germline sequence due to, for example, natural somatic mutations or deliberate introduction of site-directed mutations. However, in the VH or VL framework regions, the selected human antibody is typically at least 90% amino acid identical to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human. compared to germline immunoglobulin amino acid sequences from other species (eg, mouse germline sequences). In certain instances, the human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical. by the amino acid sequence of the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a recombinant human antibody will show no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene in the VH or VL framework regions. In certain instances, a human antibody may differ by no more than 5, or even no more than 4, 3, 2 or 1 amino acids from the amino acid sequence encoded by the germline immunoglobulin gene.

REPRESENTATIVES OF THE BMP FAMILY AND HEPSIDIN

In one embodiment, the present invention provides an antibody, or a binding fragment thereof, that specifically binds to BMP6, wherein it is an antibody. In one embodiment, the antibody or binding fragment thereof is described in Table 1 and/or Table 14.

In one embodiment, the antibody or binding fragment thereof specifically binds to BMP6 but not to other BMP proteins (such as BMP2, BMP5, or BMP7).

BMP6, a secreted ligand of the BMP family of growth factors, in its mature active form is a 30 kDa disulfide-linked homodimer. The protein is a member of the TGF-beta superfamily. Bone morphogenetic proteins are known for their ability to induce bone and cartilage growth. BMP6 is able to induce all osteogenic markers in mesenchymal stem cells.

Bone morphogenetic proteins (BMPs) belong to a family of secreted signaling molecules that can induce ectopic bone growth. BMPs are part of the transforming growth factor beta (TGF-beta) superfamily. BMP was originally identified based on the ability of a demineralized bone extract to induce endochondral osteogenesis in vivo in the extraskeletal region. Based on its expression in the early stages of embryogenesis, the BMP encoded by this gene plays a putative role in the early stages of development. In addition, the fact that this BMP is closely related to BMP5 and BMP7 led to the hypothesis of a possible activity in inducing bone development. An additional function of BMP6 has been identified as described in Nature Genetics April; 41[4]:386-8.

BMP6 knockout mice are viable and fertile and show normal bone and cartilage development.

BMP6 is a key regulator of hepcidin, a small peptide secreted by the liver that is the main regulator of iron metabolism in mammals. Hepcidin controls both the amount of dietary iron absorbed in the duodenum and the iron released by reticuloendothelial cells. Hepcidin expression is upregulated by a range of stimuli, including inflammation and iron overload, and downregulated in anemia, hypoxia, and iron deficiency.

Without wishing to be bound by any particular theory, the present invention suggests that an anti-BMP6 antagonist antibody as a therapy for lowering hepcidin levels is expected to benefit patients with iron deficiency anemia by overcoming resistance to the agent, stimulating erythropoiesis (ESA), which contributes significantly to the incidence of underlying disease and is often a predictor of poor outcome. When interacting with BMPR1 and BMPR2 receptors, it induces receptor dimerization and hepcidin transcription. BMP6 also binds to the HJV co-receptor in liver and muscle cells.

Thus, BMP6 is known to upregulate hepcidin expression. Hepcidin is known to be a key hormone involved in iron homeostasis. High levels of hepcidin are associated with iron-deficient erythropoiesis in ACD.

WO 2010/056981 discloses that administration of anti-BMP6 antibody to mice reduced hepcidin levels and increased iron levels.

BMP6 is further described in the prior art, for example, Hahn et al. 1992 Genomics 14: 759-62; Sauermann et al. 1993 J. Neurosci. Res. 33:142; Celeste et al. 1991 Proc. Natl. Acad. sci. USA 87: 9843; Schluesener et al. 1995 Atherosclerosis 113: 153; Gitelman et al. 1994 J. Cell Biol. 126:1595; Barnes et al. 1997 W. J. Urol. 13:337; and Hamdy et al. 1997 Cancer Res. 57:4427.

BMP2, like other bone morphogenetic proteins, plays an important role in bone and cartilage development. It is involved in the hedgehog pathway, the TGF-beta signaling pathway, and in the cytokine-cytokine receptor interaction. It is also involved in cardiac cell differentiation and the epithelial-mesenchymal transition. BMP2 plays many important roles, as stated in Kishimoto et al. 1997dev. 124:4457; Ma et al. 2005 dev. 132:5601; Wang et al. Bone 48: 524; and Rosen 2009 Cyt. Growth Fact. Rev. 20:475. Thus, it is preferred that the anti-BMP6 antibody does not bind to BMP2.

BMP2 is further described in, among others, Sampath et al. 1990 J Biol. Chem. 265:13198; Chen et al. 2004 Growth Factors 22:233; Marie et al. 2002 Histol. Histopath. 17:877; Nickel et al. 2001 J. Bone Joint Surg. 83-A Supp. 1: S7-14; Kirsch et al. 2000 FEBS Lett. 468:215; Kirsch et al. 2000 EMBO J. 19: 3314; Gilboa et al. 2000 Mol. Biol. Cell 11: 1023.

BMP5 is also a member of the TGF-beta superfamily. Like other BMPs, it is known for its ability to induce bone and cartilage development. BMP5 is expressed in the trabecular meshwork and optic nerve head and may play a role in development and normal function. It is also expressed in the lung and liver.

Additional information about BMP5 is known in the art, for example, Hahn et al. 1992 Genomics 14:759; Beck et al. 2003 BMC Neurosci. 2:12; Celeste et al. 1991 Proc. Natl. Acad. sci. USA 87: 9843; and Sakaue et al. 1996 Biochem. Biophys. Res. Comm. 221:768.

BMP7 is also a member of the TGF-beta superfamily. Like other members of the BMP protein family, it plays a key role in the transformation of mesenchymal cells into bone and cartilage. It induces the phosphorylation of SMAD1 and SMAD5, which in turn induce the transcription of numerous genes involved in osteogenesis.

As stated above, BMP6 knockout mice are viable and fertile, and have normal bone and cartilage development. However, BMP7 knockout mice die after birth due to kidney, eye, and bone defects. Single knockouts of one of the genes do not alter cardiogenesis, but double knockout of BMP6 and BMP7 demonstrated several defects and delays in heart development; embryos died due to heart failure. BMP7 is important in preventing the progression of chronic heart disease associated with fibrosis. Therefore, cross-reactivity of an anti-BMP6 antibody with BMP7 is not desirable.

Additional information related to BMP7 is provided in the prior art, for example, Hahn et al. 1992 Genomics 14:759; Chen et al. 2004 Growth Factors 22:233; Itoh et al. 2001 EMBO J. 20: 4132; Zeisberg et al. 2003 am. J Physiol. Renal physiol. 285: F1060; Kallui et al. 2009 J. Clin. Invest. 119:1420; and Wang et al. 2001 J. Am. soc. Neph. 12:2392.

Hepcidin is a peptide hormone, also known as HAMP (hepcidin antimicrobial protein or peptide).

A recent mouse evolutionary event of gene duplication resulted in the presence of two similar hepcidin genes in mice, Hepcidin1 and Hepcidin2. Ilyin et al. 2003 FEBS Lett. 542:22-26. Mouse hepcidin2 lacks several conserved residues found in mammalian hepcidins. Lou et al. 2004 Blood 103: 2816-2821.

The hepcidin gene product is involved in the maintenance of iron homeostasis and is required for the regulation of macrophage iron storage and intestinal iron absorption. These peptides exhibit antimicrobial activity.

Preproprotein (or preprohormone or preprohepcidin) (84 aa) and proprotein (or prohormone or prohepcidin) (60 aa) are processed into mature peptides of 20, 22 and 25 amino acids. The 25aa peptide is secreted predominantly by the liver and is considered the "master regulator" of iron metabolism. Metabolites from 20 and 22 aa are present in the urine. The N-terminal region of hepcidin is required for function; deletion of the 5 N-terminal amino acids results in loss of function.

The active hepcidin peptides have a high content of cysteines, which form intramolecular bonds that stabilize their beta sheet structures.

Hepcidin is predominantly synthesized in the liver, while other tissues have been found to synthesize smaller amounts. Bekri et al. 2006 Gastroent. 131:788-96.

The hepcidin peptide from 25aa is secreted predominantly by the liver and is considered the "master regulator" of iron metabolism. Hepcidin inhibits iron transport by binding to the iron export channel, ferroportin, which is located on the basolateral surface of intestinal enterocytes and the plasma membrane of reticuloendothelial cells (macrophages). By inhibiting ferroportin, hepcidin prevents intestinal enterocytes from secreting iron into the hepatic portal system, thereby functionally reducing iron absorption. The release of iron from macrophages is also prevented by inhibition of ferroportin; therefore, hepcidin maintains iron homeostasis. Hepcidin activity is also partly responsible for the iron sequestration seen in anemia due to chronic inflammation such as inflammatory bowel disease, chronic heart failure, carcinomas, rheumatoid arthritis, and renal failure.

Mutations in the hepcidin gene cause hemochromatosis type 2B, also known as juvenile hemochromatosis, a disease caused by severe iron overload that results in cardiomyopathy, cirrhosis, and endocrine insufficiency. Most cases of juvenile hemochromatosis are due to mutations in hemojuvelin, a regulator of hepcidin production.

Genetically engineered mice genetically engineered to overexpress hepcidin die shortly after birth due to severe iron deficiency, suggesting a central and non-redundant role in iron regulation. The first evidence linking hepcidin to inflammatory anemia came when researchers studied tissues from two liver tumor patients with severe microcytic anemia who did not respond to iron supplements. The tumor tissue overproduced hepcidin, and surgical removal of the tumors cured the anemia.

There are many diseases in which the inability to absorb enough iron contributes to the development of iron deficiency and iron deficiency anemia. Treatment will depend on hepcidin levels, as treatment with oral medications is likely to be ineffective if hepcidin blocks intestinal absorption.

In one embodiment, administration of an anti-BMP6 antibody or binding fragment thereof reduces the activity and/or level of hepcidin and is therefore useful in the treatment of anemia. In one embodiment, the present invention provides a method for reducing hepcidin activity or levels in a patient in need thereof, the method comprising the step of administering an anti-BMP6 antibody or antigen-binding fragment thereof to the patient. In one embodiment, the activity or level of hepcidin is reduced by at least 50%.

Hepcidin inhibitors, such as anti-BMP6 antibodies, can be used to treat hepcidin-associated disease. It includes any disease associated with hepcidin and/or mutation and/or overexpression of wild-type hepcidin and/or mutant hepcidin and/or diseases in which disease progression is enhanced or prognosis worsened by the presence of hepcidin and/or mutation and/or overexpression wild-type and/or mutant hepcidin and/or reduced urinary excretion of hepcidin by the kidneys. Non-limiting examples of hepcidin related diseases include anemia, iron deficiency erythropoiesis, hypoferremia, impaired dietary iron absorption, iron sequestration, inflammatory anemia (AI), atherosclerosis, diabetes, and many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Friedrich's ataxia. heart failure, chronic kidney disease, cardiorenal anemic syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, growth retardation and/or hair loss. In one embodiment, the subject is a patient on dialysis. In one embodiment, the hepcidin-associated disease is anemia and the subject is a dialysis patient. The prevalence of iron and ESA refractory anemia in the chronic hemodialysis population is high.

Anemia includes, but is not limited to, anemia due to chronic disease (ACD), anemia due to chronic kidney disease (CKD), anemia due to cancer, anemia resistant to erythropoiesis stimulating agents (ESA), and/or anemia due to iron deficiency. .

Anemia due to CKD is a common and early complication of chronic kidney disease. Cancer anemia is caused by hematologic malignancies and certain solid tumors. As defined herein, the term also includes chemotherapy-induced anemia, which is anemia caused by chemotherapeutic agents. Anemia in chronic kidney disease can worsen diabetic neuropathy, cardiovascular disease, retinopathy, and other problems. Cancer-related anemia is associated with an increased risk of death.

Some chronic diseases such as cancer, kidney disease, and autoimmune disorders can lead to anemia. Overactive inflammatory cytokines can cause dysregulation of iron homeostasis, reduced erythropoiesis, and reduced erythrocyte lifespan. Some treatments for anemia include administration of ESA, erythropoietin, iron (as a dietary supplement), or blood transfusion.

Hepcidin is a key hormone involved in iron homeostasis. High hepcidin levels have been associated with iron-deficient erythropoiesis in ACD. BMP6 is known to upregulate hepcidin expression.

Various types of anti-BMP6 antibodies and their antigen-binding fragments are described below.

HOMOLOGUE ANTIBODIES

In another embodiment, the present invention provides an antibody or antigen-binding fragment containing amino acid sequences that are homologous to the sequences described in table 1 and/or table 14, and the specified antibody binds to BMP6 and retains the desired functional properties of the antibodies described in table 1 and/or table 14.

For example, the present invention provides an isolated monoclonal antibody (or a functional antigen-binding fragment thereof) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains an amino acid sequence that is at least 80%, at least 90% or at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 16; 36; 56 or 76; the light chain variable region contains an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 26; 46; 66 or 86; the antibody specifically binds to the BMP6 protein, and the antibody can inhibit erythrocyte lysis in a hemolysis assay, the hemolysis assay being known in the art. In a specific example, such antibodies have an IC ₅₀ value of 20-200 pM in a hemolysis assay using human BMP6-depleted serum that is reconstituted with 100 pM human BMP6.

In one embodiment, the VH and/or VL amino acid sequences may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in Table 1 and/or Table 14. In one embodiment, the amino acid sequences of VH and/or VL may be identical except for amino acid substitutions at no more than 1, 2, 3, 4, or 5 amino acid positions. An antibody having VH and VL regions that have high (i.e., 80% or greater) identity with the VH and VL regions of the antibodies described in Table 1 and/or Table 14 can be generated by mutagenesis (e.g., site-directed or PCR). -mediated mutagenesis) nucleic acid molecules encoding respectively SEQ ID NO: 16; 36; 56 or 76 and 26; 46; 66 or 86, followed by testing the encoded altered antibody for retained function using the functional assays described herein.

In one embodiment, the amino acid sequences of the full length heavy chain and/or full length light chain may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identical, set forth in Table 1 and/or Table 14. An antibody having a full-length heavy chain and a full-length light chain characterized by high (ie, 80% or greater) identity to the full-length heavy chains of any of SEQ ID NO: 18; 38; 58 or 78 and full-length light chains under any of SEQ ID NO: 28; 48; 68 or 88, respectively, can be generated by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding such polypeptides, respectively, followed by testing the encoded altered antibody for retained function using the functional assays described herein. .

In one embodiment, the nucleotide sequences of the full length heavy chain and/or full length light chain may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in Table 1 and/or table 14.

In one embodiment, the heavy chain and/or light chain variable region nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in Table 1 and/or table 14.

As used herein, the percent identity of two sequences depends on the number of identical positions shared by the sequences (i.e., % identity equals the number of identical positions / total number of positions X 100), taking into account the number of gaps and the length of each gap that are needed. enter for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using the mathematical algorithm described in the non-limiting examples below.

In addition or alternatively, the protein sequences of the present invention can also be used as a "query sequence" for searches in public databases, for example, to identify related sequences. For example, such searches can be performed using the BLAST program (version 2.0) Altschul, et al., 1990 J. Mol. Biol. 215:403-10.

Antibodies with conservative modifications

In one embodiment, an antibody of the present invention has a heavy chain variable region comprising the sequences CDR1, CDR2, and CDR3, and a light chain variable region comprising the sequences CDR1, CDR2, and CDR3, wherein one or more of these CDR sequences have specific amino acid sequences, based on the antibodies described herein, or conservative modifications thereof, while the antibodies retain the desired functional properties of the BMP6-binding antibodies and antigen-binding fragments of the present invention. Accordingly, the present invention provides an isolated monoclonal antibody or a functional antigen-binding fragment thereof, consisting of a heavy chain variable region containing the sequences CDR1, CDR2 and CDR3, and a light chain variable region containing the sequences CDR1, CDR2 and CDR3, wherein the CDR1 of the heavy chain variable region the chain contains an amino acid sequence selected from any of SEQ ID NOs: 29, 49, 69, 12, 32, 52, 72, or 9 or conservative variants thereof; The heavy chain variable region CDR2 comprises an amino acid sequence selected from any of SEQ ID NOs: 10, 30, 50, 70, 13, 33, 53, or 73 or conservative variants thereof; The heavy chain variable region CDR3 comprises an amino acid sequence selected from any of SEQ ID NOs: 11, 31, 51, 71, 14, 34, 54, or 74, or conservative variants thereof; The light chain variable region CDR1 comprises an amino acid sequence selected from any of SEQ ID NOs: 19, 39, 59, 79, 22, 42, 62, or 82, or conservative variants thereof; The light chain variable region CDR2 comprises an amino acid sequence selected from any of SEQ ID NOs: 20, 40, 60, 80, 23, 43, 63, or 83, or conservative variants thereof; and the light chain variable region CDR3 comprises an amino acid sequence selected from any of SEQ ID NOs: 21, 41, 61, 81, 24, 44, 64, or 84, or conservative variants thereof; the antibody, or antigen-binding fragment thereof, specifically binds to BMP6 and inhibits erythrocyte lysis in a hemolysis assay.

In one embodiment, an antibody of the invention optimized for expression in a mammalian cell has a full length heavy chain sequence and a full length light chain sequence, one or more of these sequences having specific amino acid sequences based on the antibodies described herein, or their conservative modifications, while the antibodies retain the desired functional properties of the BMP6-binding antibodies and their antigen-binding fragments of the present invention. Accordingly, the present invention provides an isolated monoclonal antibody optimized for expression in a mammalian cell, consisting of a full-length heavy chain and a full-length light chain, where the full-length heavy chain has amino acid sequences selected from the group of SEQ ID NO: 18; 38; 58 or 78 and their conservative modifications; and the full-length light chain has amino acid sequences selected from the group of SEQ ID NO: 28; 48; 68 or 88 and their conservative modifications; the antibody specifically binds to BMP6; and the antibody inhibits erythrocyte lysis in the hemolysis assay described herein. In a specific embodiment, such antibodies have an IC ₅₀ value of 20-200 pM in a hemolysis assay using human BMP6-depleted serum that is reconstituted with 100 pM human BMP6.

ANTIBODIES THAT BIND TO THE SAME EPITOPE

The present invention provides antibodies that bind to the same epitope as the BMP6 binding antibodies shown in Table 1 and/or Table 14. The epitope bound by antibody 7 is shown in FIG. 5. Therefore, additional antibodies can be identified based on their ability to cross-compete (eg, competitively inhibit binding in a statistically significant manner) with other antibodies and their antigen-binding fragments of the present invention in BMP6 binding assays. The ability of a test antibody to inhibit the binding of the antibodies and antigen-binding fragments thereof of the present invention to the BMP6 protein demonstrates that the test antibody can compete with this antibody for binding to BMP6; such an antibody, in accordance with non-limiting theory, may bind to the same or related (eg, structurally similar or spatially similar) epitope on BMP6 as the antibody with which it competes. In a particular embodiment, an antibody that binds to the same epitope on BMP6 as the antibodies and antigen-binding fragments thereof of the present invention is a human monoclonal antibody. Such human monoclonal antibodies can be generated and isolated as described herein.

Once a desired epitope has been determined on an antigen, antibodies to that epitope can be generated, for example, using the techniques described in the present invention. Alternatively, during the development process, antibody generation and characterization can provide information on the required epitopes. Based on this information, antibodies can then be competitively screened for binding to the same epitope. An approach to achieve this is to conduct cross-competition studies to detect antibodies that compete with each other, such as antibodies that compete for binding to an antigen. A high throughput method for "sorting" antibodies based on their cross-competition is described in International Patent Application No. WO 2003/48731. As will be appreciated by one of skill in the art, substantially anything that an antibody can specifically bind to can be an epitope. The epitope may contain those residues to which the antibody binds.

In general, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

Regions of a given polypeptide that include an epitope can be identified using a variety of epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined, for example, by simultaneously synthesizing a large number of peptides on solid supports, peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and are described, for example, in US patent No. 4708871; Geysen et al., (1984) Proc. Natl. Acad. sci. USA 8:3998-4002; Geysen et al., (1985) Proc. Natl. Acad. sci. USA 82:78-182; Geysen et al., (1986) Mol. Immunol. 23:709-715. Similarly, conformational epitopes are easily identified by determining the spatial conformation of the amino acids in BMP6, such as by hydrogen/deuterium exchange, X-ray crystallography, and 2D nuclear magnetic resonance. See, for example, Epitope Mapping Protocols above. Antigenic regions of proteins can also be identified using standard antigenicity and hydrophobicity plots, such as those plotted using, for example, software from the Omiga software system version 1.0 available from the Oxford Molecular Group. This computer program uses the Hopp/Woods method, Hopp et al., (1981) Proc. Natl. Acad. Sci USA 78:3824-3828; for antigenicity profiling and the Kyte-Doolittle method, Kyte et al., (1982) J.MoI. Biol. 157:105-132; for hydrophobicity plots.

Engineered and modified antibodies

An antibody of the present invention can also be made using an antibody having one or more of the VH and/or VL sequences shown herein as a starting material for constructing a modified antibody, which modified antibody may have altered properties of the original antibody. An antibody can be constructed by modifying one or more residues within one or both of the variable regions (i.e., VH and/or VL), for example, within one or more CDR regions and/or within one or more framework regions. In addition or alternatively, the antibody can be engineered by modifying residues in the constant(s) region(s), for example, to change the effector(s) function(s) of the antibody.

One type of variable region construction that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six complementarity determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDR are more diverse in individual antibodies than those outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that contain CDR sequences from a specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, for example, Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. et al., 1989 Proc. Natl Acad., U.S.A. 86:10029-10033; U.S. Patent Nos. 5,225,539 to Winter and U.S. Patents 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

Such framework sequences can be obtained from public DNA databases or published sources that include germline antibody gene sequences. For example, the germline DNA sequences for the human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), and also in Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. J Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference.

An example of framework sequences for use in antibodies and their antigen-binding fragments of the present invention are sequences that are structurally similar to the framework sequences used in selected antibodies and antigen-binding fragments of the present invention, for example, consensus sequences and/or framework sequences used in monoclonal antibodies according to the present invention. The CDR1, 2 and 3 VH sequences and the CDR1, 2 and 3 VL sequences can be grafted onto framework regions that have a sequence identical to that found in the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations compared to germline sequences. For example, it has been found beneficial in certain cases to mutate residues within framework regions to maintain or increase the ability to bind an antigen by an antibody (see, for example, U.S. Patent Nos. 5,530,101; .

Another type of variable region modification is to mutate amino acid residues within the CDR1, CDR2, and/or CDR3 regions of the VH and/or VL, thereby improving one or more of the binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation". ". To introduce the mutation(s), site-directed mutagenesis or PCR-mediated mutagenesis can be performed and the effect on antibody binding or other functional property of interest can be assessed in the in vitro or in vivo assays described herein and presented in examples. You can enter conservative modifications (which are discussed above). Mutations can be substitutions, additions or deletions of amino acids. Moreover, as a rule, change no more than one, two, three, four or five residues within the CDR region.

GRAFTING ANTIGEN-BINDING DOMAIN ON ALTERNATIVE FRAMEWORK OR BACKGROUND

A wide variety of antibody/immunoglobulin scaffolds or backbones can be used, provided that the resulting polypeptide contains at least one binding region that specifically binds to BMP6. Such scaffolds or scaffolds include the 5 major human immunoglobulin idiotypes, their antigen-binding fragments, and include immunoglobulins from other animal species, preferably having humanized moieties. Single heavy chain antibodies, such as those identified in camelids, are of particular interest in this regard. Those skilled in the art continue to discover and develop new scaffolds, cores, and fragments.

In one aspect, the present invention relates to a method for producing antibodies based on proteins other than immunoglobulins using backbones other than immunoglobulins onto which the CDRs of the present invention can be grafted. Known or future scaffolds and backbones other than immunoglobulins may be used, provided they contain a binding region specific for the target BMP6 protein. Known scaffolds or scaffolds other than immunoglobulins include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv , Zwijnarde, Belgium), Lipocalin (Pieris Proteolab AG, Freising, Germany), Small Modular Immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), Maxitel (Avidia, Inc., Mountain View, CA), Protein A (Affibody AG, Sweden) and affilin (gamma crystallin or ubiquitin) (SciI Proteins GmbH, Halle, Germany).

Fibronectin scaffolds are based on a type III fibronectin domain (eg, the tenth type III fibronectin module (Fn3 domain 10)). The type III fibronectin domain has 7 or 8 beta strands that are distributed between two folded beta layers, which are themselves sandwiched between each other to form the core of the protein, and additionally contains loops (similar to CDRs) that connect the beta strands to each other and are solvent accessible. At least three such loops are present at each edge of the sandwich of pleated beta layers, where the edge is a protein boundary perpendicular to the direction of the folded beta layers (see US patent No. 6818418). These fibronectin-based backbones are not immunoglobulin, although the overall fold is very similar to that of the smallest functional antibody fragment, the heavy chain variable region, which contains the complete antigen recognition unit in camel and llama IgG. Due to this structure, an antibody other than an immunoglobulin mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These backbones can be used in an in vitro randomization and loop shuffling strategy that is similar to the in vivo antibody affinity maturation process. These fibronectin-based molecules can be used as scaffolds and the loop regions of the molecule can be replaced with the CDRs of the present invention using standard cloning techniques.

Ankyrin technology is based on the use of proteins with ankyrin repeat-derived modules as scaffolds for the transfer of variable regions that can be used to bind to various targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two antiparallel alpha helices and a beta fold. Binding of variable regions is generally optimized by the use of ribosome display.

Avimers are derived from a naturally occurring A-domain containing protein such as LRP-1. These domains are used inherently for protein-protein interactions, and in humans, more than 250 proteins have A-domains in their structure. Avimers are composed of a number of different "A-domain" monomers (2-10) linked via amino acid linkers. Avimers that can bind to a target antigen can be generated using the methodology described, for example, in US Patent Application Publication Nos. 20040175756; 20050053973; 20050048512 and 20060008844.

Affinity ligands, affiteles, are small, simple proteins composed of a three-stranded bundle based on a backbone from one of the IgG binding domains of protein A. Protein A is the surface protein of the bacterium Staphylococcus aureus. This backbone domain consists of 58 amino acids, 13 of which are randomized to generate affitel libraries with a large number of ligand variants (see, for example, US Pat. No. 5,831,012). Affitel molecules mimic antibodies, they have a molecular weight of 6 kD compared to the molecular weight of antibodies which is 150 kD. Despite its small size, the binding site in the affitel molecules is similar to that of an antibody.

Anticalins are products developed by Pieris ProteoLab AG. They are derived from lipocalins, a common group of small and stable proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several naturally occurring lipocalins are found in human tissues or body fluids. The structure of the protein resembles immunoglobulins with hypervariable loops on top of a rigid scaffold. However, in contrast to antibodies or their recombinant fragments, lipocalins consist of a single polypeptide chain with 160-180 amino acid residues, and are only slightly larger than a single immunoglobulin domain. The set of four loops that form the binding pocket exhibits marked structural plasticity and allows for a number of side chains. Thus, the binding site can be modified in a proprietary manner in order to recognize given target molecules of various shapes with high affinity and specificity. One lipocalin family protein, bilin-binding protein (BBP), from Pieris Brassicae was used to develop anticalins by performing four-loop set mutagenesis. One example of a patent application describing anticalins is PCT Publication No. WO 199916873.

Affilin molecules are small proteins, other than immunoglobulin, that are designed with specific affinities for proteins and small molecules. New affilin molecules can be very quickly selected from two libraries, each based on a different human-derived scaffold protein. Affilin molecules do not show any structural homology with immunoglobulin proteins. Two affilin backbones are currently in use, one of which is gamma-crystallin, a structural protein of the human eye lens, and the other is a protein of the "ubiquitin" superfamily. Both human backbones are very small, stable at high temperature, and most resistant to pH changes and denaturants. This high stability is mainly due to the unfolded beta sheet structure of proteins. Examples of proteins derived from gamma crystallin are described in WO200104144 and examples of "ubiquitin-like" proteins are described in WO2004106368.

Protein epitope mimetics (PEMs) are medium-sized cyclic peptide-like molecules (MW 1-2 kDa) that mimic beta-hairpin secondary structures of proteins, the main secondary structure involved in protein-protein interactions.

Human antibodies that bind BMP6 can be created using methods that are known in the art. For example, "human engineering" technology is used to convert non-human antibodies into engineered human antibodies. US Patent Application Publication No. 20050008625 describes an in vivo method for replacing a non-human antibody variable region with a human variable region in an antibody while maintaining the same or better binding characteristics than a non-human antibody. The method relies on epitope-defined replacement of the variable regions of a non-human reference antibody with those of a fully human antibody. The resulting human antibody is generally structurally unrelated to the non-human reference antibody, but binds to the same epitope on the same antigen as the reference antibody. Briefly, the sequential epitope-determined complementary substitution approach is achieved by creating cellular competition between a "competitor" and a library of diverse hybrids of a reference antibody ("test antibodies") for binding to an antigen present in limited amounts in the presence of a reporter system that is responsive to binding of the test antibody to the antigen. The competitor may be a reference antibody or a derivative thereof, such as a single chain Fv fragment. A competitor can also be a natural or artificial antigen ligand that binds to the same epitope as the reference antibody. The only requirements for a competitor are that it binds to the same epitope as the reference antibody and that it competes with the reference antibody for antigen binding. Test antibodies share one antigen-binding V region from a non-human reference antibody and another V region randomly selected from a different source, such as a human antibody repertory library. The overall V-region from the reference antibody serves as a guide, placing test antibodies on the same epitope on the antigen and in the same orientation in order to lean towards the highest quality of antigen binding relative to the reference antibody.

Many types of reporter systems can be used to detect the desired interactions between test antibodies and antigen. For example, matching reporter fragments can be linked to an antigen and a test antibody, respectively, such that reporter activation due to fragment matching occurs only when the test antibody binds to the antigen. If the fusion products from the test antibody-reporter fragment and the antigen-reporter fragment are co-expressed with a competitor, reporter activation becomes dependent on the ability of the test antibody to compete with the competitor, which is proportional to the affinity of the test antibody for the antigen. Other reporter systems that may be used include the self-inhibiting reporter reactivation (RAIR) system reactivator as disclosed in U.S. Patent Application Serial No. 10/208730 (Publication No. No. 10/076845 (Publication No. 20030157579).

When using a sequential epitope-determined complementary substitution system, a selection is made to identify cells expressing a single test antibody along with competitor, antigen, and reporter components. In these cells, each antibody tested competes individually with a competitor for binding to a limited amount of antigen. Reporter activity is proportional to the amount of antigen bound to the test antibody, which in turn is proportional to the affinity of the test antibody for the antigen and the stability of the test antibody. Test antibodies are initially selected based on their activity compared to that of a reference antibody when expressed as a test antibody. The result of the first round of selection is a set of "hybrid" antibodies, each of which consists of the same non-human V-region from the reference antibody and the human V-region from the library, and each of which binds to the same epitope on the antigen, the same as the reference antibody. One or more hybrid antibodies selected in the first round will have an affinity for the antigen comparable to or greater than that of the reference antibody.

In the second step of replacing the V region, the human V regions selected in the first step are used as a guide to select human replacement fragments for the rest of the V region of the non-human reference antibody using a diverse library of cognate human V regions. Hybrid antibodies selected in the first round can also be used as competitors for the second round of selection. The result of the second round of selection is a set of fully human antibodies that are structurally different from the reference antibody, but that compete with the reference antibody for binding to the same antigen. Some of the selected human antibodies bind to the same epitope on the same antigen as the reference antibody. Among these selected human antibodies, one or more bind to the same epitope with an affinity that is comparable to or greater than that of the reference antibody.

By using one of the above described murine or chimeric BMP6-binding antibodies as a reference antibody, this method can be easily used to generate human antibodies that bind to human BMP6 with the same binding specificity and the same or better binding affinity. In addition, such BMP6-binding human antibodies can also be obtained commercially from companies that manufacture custom human antibodies, such as KaloBios, Inc. (Mountain View, California).

CAMELID ANTIBODIES

Proteins that are antibodies derived from members of the family of camels and dromedaries (Camelus bactrianus and Calelus dromaderius), including members of New World species such as llama species (Lama paccos, Lama glama and Lama vicugna) , have been characterized in terms of size, structural complexity, and antigenicity in human subjects. Certain IgG antibodies of this family of mammals that are found naturally lack light chains and therefore differ in structure from the typical four chain quaternary structure characterized by two heavy and two light chains found in antibodies from other animals. See PCT/EP93/02214 (WO 94/04678 published March 3, 1994).

The camelid antibody region, which is a small single variable domain identified as VHH, can be genetically engineered to produce a small protein with high affinity for the target, resulting in a low molecular weight antibody-derived protein known as "camelid nanobody". See US patent No. 5759808, issued June 2, 1998; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available from, for example, Ablynx, Ghent, Belgium. As with other non-human antibodies and antigen-binding fragments thereof, the amino acid sequence of a camelid antibody can be altered recombinantly to produce a sequence that more closely resembles that of a human, i.e., the nanobody can be "humanized". In this way, the naturally occurring low antigenicity of camelid antibodies in humans can be further reduced.

The camelid nanobody has a molecular weight about one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigen sites that are functionally invisible to larger antibody proteins, i.e. camelid nanobodies are useful as reagents for detecting antigens that are otherwise hidden using classical immunological methods, and as possible therapeutic agents. Thus, another consequence of the small size is that the camelid nanobody can exert inhibition by binding to a specific site in the groove or narrow recess of the target protein and therefore can serve as a function that more closely resembles the function of a classic small molecule drug. than that of a classical antibody.

The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, resistant to pH extremes and to proteolytic degradation, and have low antigenicity. Another implication is that camelid nanobodies easily move from the circulatory system to tissues and even cross the blood-brain barrier and may provide treatment for disorders that affect neural tissue. Nanobodies can further facilitate drug transport across the blood-brain barrier. See U.S. Patent Application 20040161738 published August 19, 2004. These features, combined with low antigenicity in humans, point to great therapeutic potential. In addition, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins using bacteriophage and are functional.

Accordingly, a feature of the present invention is a camelid antibody or nanobody having high affinity for BMP6. In one embodiment, herein, a camelid antibody or nanobody is produced naturally in an animal of the camelid family, i.e., it is produced by camelids after immunization with BMP6 or a fragment of its peptide using the techniques described herein for other antibodies. Alternatively, a BMP6-binding camelid nanobody is constructed, i.e. obtained by selecting, for example, from a library of phages that display appropriately mutated camelid nanobody proteins using BMP6-targeted panning procedures as described. in the examples in this document. The engineered nanobodies can be further tailored to specific requirements by genetic engineering so that they have a half-life in the recipient subject from 45 minutes to two weeks. In a specific embodiment, a camelid antibody or nanobody is produced by grafting heavy or light chain CDR sequences of human antibodies of the present invention onto nanobody or single domain antibody framework sequences as described, for example, in PCT/EP93/02214.

BISPECIFIC MOLECULES AND MULTIVALENT ANTIBODIES

In another aspect, the present invention relates to bispecific or multispecific molecules containing an antibody that binds BMP6, or a fragment of the present invention. An antibody of the present invention, or its antigen binding regions, can be derivatized or linked to another functional molecule, such as another peptide or protein (for example, another antibody or receptor ligand) to produce a bispecific molecule that binds to at least two different binding sites or targets. molecules. The antibody of the present invention, in fact, can be derivatized or associated with more than one other functional molecule to obtain multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the present invention, an antibody of the present invention may be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent bonding, or otherwise) with one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, resulting in a bispecific molecule.

Accordingly, the present invention includes bispecific molecules containing at least one first element providing binding specificity for BMP6 and a second element providing binding specificity for a second target epitope. For example, the second target epitope is a different BMP6 epitope than the first target epitope.

In addition, in the case of the present invention in which the bispecific molecule is multispecific, the molecule may further include a third binding specificity element in addition to those for the first and second target epitopes.

In one embodiment, the bispecific molecules of the present invention comprise, as a binding specificity element, at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab')2, Fv, or single chain Fv . The antibody can also be a dimer of light chains or heavy chains, or any minimal fragment thereof, such as Fv or a single chain construct, as described in Ladner et al., US patent No. 4946778.

Diabodies are bivalent bispecific molecules in which the VH and VL domains are expressed on the same polypeptide chain connected by a linker that is too short to allow pairing between two domains on the same chain. The VH and VL domains pair with complementary domains of the other strand, thus creating two antigen binding sites (see, e.g., Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poijak et al. , 1994 Structure 2:1121-1123). Diabodies can be generated by expressing two polypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VL configuration) or VLA-VHB and VLB-VHA (VL-VH configuration) in the same cell. Most of them can be expressed in soluble form in bacteria. Single chain diabodies (scDb) are prepared by joining two diabody-forming polypeptide chains with a linker of about 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(3-4):128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36). scDb can be expressed in bacteria in a soluble active monomeric form (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45 (34): 128-30; Wu et al., 1996 Immunotechnology, 2 (1): 21-36; Pluckthun and Pack, 1997 Immunotechnology, 3 (2): 83-105; Ridgway et al., 1996 Protein Eng., 9 (7): 617-21). The diabody can be fused with Fc to form a "di-diabody" (see Lu et al., 2004 J. Biol. Chem., 279(4):2856-65).

Other antibodies that can be used in the bispecific molecules of the present invention are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be obtained by conjugating constituent elements that provide binding specificity using methods known in the art. For example, each binding specificity element in a bispecific molecule can be prepared separately and then conjugated to each other. When the elements providing binding specificity are proteins or peptides, a number of binding or crosslinking agents can be used for covalent conjugation. Examples of crosslinkers include protein A, carbodiimide, N-succinimidyl-5-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-( 2-pyridyldithio)propionate (SPDP) and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see e.g. Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. no. 78, 118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al., 1987 J. Immunol. 139: 2367-2375). The conjugants are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, Illinois).

If the binding specificity elements are antibodies, they can be conjugated by linking via the sulfhydryl group of the C-terminal hinge regions of the two heavy chains. In a particular embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, eg one, prior to conjugation.

Alternatively, both binding specificity elements can be encoded in the same vector and can be expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb X mAb, mAb X Fab, Fab X F(ab')2 fusion protein, or a Fab X ligand. The bispecific molecule of the present invention may be a single chain molecule containing one single chain antibody and a binding determinant, or a single chain bispecific molecule containing two binding determinants. Bispecific molecules may contain at least two single chain molecules. Methods for obtaining bispecific molecules are described, for example, in US patent No. 5260203; US patent No. 5455030; US Pat. No. 4,881,175; US patent No. 5132405; US Pat. No. 5,091,513; US patent No. 5476786; US patent No. 5013653; U.S. Patent No. 5,258,498 and U.S. Patent No. 5,482,858.

Binding of bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS assay, biological assay (eg, growth inhibition) or Western blot assay. Each of these assays typically detects the presence of protein-antibody complexes of interest by using a labeled reagent (eg, antibody) specific for the complex of interest.

In another aspect, the present invention provides multivalent compounds containing at least two identical or different antigen-binding portions of the antibodies and antigen-binding fragments of the present invention that bind to BMP6. The antigen-binding moieties may be linked together by protein fusion or covalent or non-covalent bonding. Alternatively, binding methods have been described for bispecific molecules. Tetravalent compounds can be obtained, for example, by cross-linking antibodies and antigen-binding fragments of the present invention with an antibody or antigen-binding fragment that binds to the constant regions of the antibodies and antigen-binding fragments of the present invention, for example, Fc or hinge region.

The trimerizing domain is described, for example, in EP 1012280B1 owned by Borean. Pentamerizing modules are described, for example, in PCT/EP97/05897.

ANTIBODIES WITH LONGER HALF-LIFE

The present invention provides antibodies that specifically bind to BMP6, which are characterized by an increased half-life in vivo.

Many factors can affect the in vivo half-life of a protein. For example, kidney filtration, liver metabolism, degradation by proteolytic enzymes (proteases), and immune responses (eg, protein neutralization by antibodies and uptake by macrophages and dendritic cells). A number of strategies can be used to increase the half-life of the antibodies and their antigen-binding fragments of the present invention. For example, chemical coupling to polyethylene glycol (PEG), reCODE PEG, antibody backbone, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate protection moieties; genetic fusion with serum protein binding proteins such as albumin, IgG, FcRn and transfer; coupling (genetic or chemical) to other binding moieties that bind to blood serum proteins, such as nanobodies, Fab molecules, DARPin molecules, avimers, affitels, and anticalins; genetic fusion with rPEG, albumin, albumin domain, albumin-binding proteins and Fc; or incorporation into nanocarriers, slow release formulations, or medical devices.

To prolong the circulation of antibodies in the blood serum in vivo, molecules of an inert polymer, such as high molecular weight PEG, can be attached to antibodies or their fragments with or without a multifunctional linker, through site-specific conjugation of PEG to the N- or C-terminus of antibodies, or through epsilon -amino groups present in lysine residues. To PEGylate an antibody, the antibody, its antigen-binding fragment, is typically reacted with a polyethylene glycol (PEG), such as a reactive ester or an aldehyde derivative of PEG, under conditions where one or more PEG moieties are attached to the antibody or antibody fragment. Pegylation can be carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10)aloxy or aryloxy polyethylene glycol or polyethylene glycol-maleimide. In one embodiment, the antibody to be pegylated is an aglycosylated antibody. Derivatization with a linear or branched polymer will be used which results in minimal loss of biological activity. The degree of conjugation can be carefully monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by size exclusion or ion exchange chromatography. PEG-derivatized antibodies can be tested for binding activity as well as in vivo potency using methods well known to those skilled in the art, such as the immunoassays described herein. Methods for PEGylating proteins are known in the art and can be applied to the antibodies and antigen-binding fragments thereof of the present invention. See, for example, EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

Other modified PEGylation technologies include directed engineering technology that provides chemical orthogonality using a reshaped biosynthetic system (ReCODE PEG), in which chemically specific side chains are incorporated into biosynthetic proteins via a redesigned system that includes tRNA and tRNA synthetase. This technology allows more than 30 new amino acids to be incorporated into biosynthetic proteins in E. coli, yeast and mammalian cells. The tRNA includes a standard amino acid at any location where a stop codon is located, converting the codon from a stop codon to one that signals the inclusion of a chemically specific amino acid.

Recombinant pegylation technology (rPEG) can also be used to increase serum half-life. This technology involves the genetic fusion of a protein tail of 300-600 amino acids of arbitrary structure with an existing pharmaceutical protein. Because the apparent molecular weight of such an arbitrary protein chain is about 15 times its actual molecular weight, the serum half-life of the protein is greatly increased. Unlike traditional PEGylation, which requires chemical conjugation and repurification, the manufacturing process is greatly simplified and the product is homogeneous.

Polysialylation is another technology that uses a natural polymer, polysialic acid (PSA), to extend the potency and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (sugar). When used to deliver a drug based protein and therapeutic peptide, polysialic acid provides a protective microenvironment upon conjugation. This increases the duration of the therapeutic protein in the bloodstream and prevents it from being recognized by the immune system. The polymer, which is PSA, is naturally found in the human body. It has been taken up by some bacteria that have evolved over millions of years to coat their walls with it. These naturally polysialylated bacteria were then able, through molecular mimicry, to counteract the body's defense system. PSA, which can be classified as nature's perfect stealth technology, can be easily obtained from such bacteria in large quantities and with desired physical characteristics. Bacterial PSA is completely non-immunogenic, even when bound to proteins, as it is chemically identical to PSA in the human body.

Another technology involves the use of derivatives of hydroxyethyl starch ("HES") associated with antibodies. HES is a modified natural polymer derived from waxy corn starch and can be metabolized through body enzymes. HES solutions are usually administered to replenish the missing blood volume and improve the rheological properties of the blood. Modification of an antibody with HES allows for an extension of the circulating half-life by increasing the stability of the molecule, as well as by decreasing renal clearance, resulting in increased biological activity. By changing various parameters, such as the molecular weight of the HES, a wide range of HES-antibody conjugates can be tailored to specific requirements.

Antibodies having an extended in vivo half-life can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) to the IgG constant domain or FcRn-binding fragment (preferably an Fc or hinge Fc domain fragment). See, for example, International Publication No. WO 98/23289; International Publication No. WO 97/34631 and US Patent No. 6277375.

Additionally, antibodies can be conjugated to albumin to make the antibody or antibody fragment more stable in vivo, or to have a longer in vivo half-life. The techniques are well known in the art, see for example International Publications Nos. WO 93/15199, WO 93/15200 and WO 01/77137 and European Patent No. EP 413622.

Strategies for increasing half-life are particularly applicable to nanobodies, fibronectin-based binding molecules, and other antibodies or proteins that require an extended in vivo half-life.

ANTIBODY CONJUGATES

The present invention provides antibodies, or antigen-binding fragments thereof, that specifically bind to BMP6, are recombinantly fused or chemically conjugated (including via both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or an antigen-binding fragment thereof, preferably with a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to give fusion proteins. In particular, the present invention provides fusion proteins comprising an antigen-binding fragment of an antibody described herein (e.g., Fab fragment, Fd fragment, Fv fragment, F(ab) ₂ fragment, VH domain, CDR VH, VL -domain or CDR VL) and a heterologous protein, polypeptide or peptide. Methods for fusion or conjugation of proteins, polypeptides or peptides with an antibody or antibody fragment are known in the art. See, for example, US Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; European Patents Nos. EP 307434 and EP 367166; International Publications Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Wil et al., 1992, Proc. Natl. Acad. sci. USA 89:11337-11341.

Additional fusion proteins can be generated by gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively referred to as "DNA shuffling") techniques. DNA shuffling can be used to change the activities of antibodies and antigen-binding fragments of the present invention (eg, antibodies and antigen-binding fragments with higher affinity values and lower dissociation rates). See, in general, US Pat. Nos. 5,605,793; 5,811,238; 5,830,721; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications are incorporated herein by reference in their entirety). Antibodies and their antigen-binding fragments, or encoded antibodies and their antigen-binding fragments, can be altered by subjecting them to random mutagenesis by error-correction PCR, random insertion of nucleotides, or other methods prior to recombination. A polynucleotide encoding an antibody, an antigen-binding fragment thereof that specifically binds to BMP6, can be recombined with one or more components, motifs, sections, moieties, domains, fragments, etc. of one or more heterologous molecules.

Moreover, antibodies and their antigen-binding fragments can be fused to marker sequences, such as a peptide, to facilitate purification. In one embodiment, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 97), such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311), among others, many of which are commercially available. For example, as described in Gentz et al., 1989, Proc. Natl. Acad. sci. USA 86:821-824, hexahistidine (SEQ ID NO: 97) provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, a hemagglutinin ("HA") tag that corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and a "flag" tag.

In one embodiment, the antibodies and antigen-binding fragments of the present invention are conjugated to a diagnostic or detectable agent. Such antibodies may be useful in monitoring or predicting the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure such as determining the efficacy of a particular therapy. Such diagnosis and detection can be accomplished by binding the antibody to detectable substances, including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholine transferase; prosthetic groups such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescenin, dansyl chloride, or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive materials such as but not limited to iodine (131I, 125I, 123I and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re , 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn and 117Tin; and positron-active metals used in various methods of positron emission tomography, and ions of non-radioactive paramagnetic metals.

The present invention further covers the ways of using antibodies and their antigen-binding fragments conjugated to a therapeutic fragment. The antibody, its antigen-binding fragment may be conjugated to a therapeutic moiety such as a cytotoxin, eg a cytostatic or cytocidal agent, a therapeutic agent, or a radioactive metal ion, eg alpha particle emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.

In addition, the antibody, its antigen-binding fragment can be conjugated to a therapeutic or drug fragment that modifies a given biological response. Therapeutic or drug moieties are not to be construed as being limited to classical chemical therapeutics. For example, a drug moiety may be a protein, peptide, or polypeptide having the desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, interferon alpha, interferon beta, nerve growth factor, platelet growth factor, tissue plasminogen activator, apoptotic agent, antiangiogenic agent or biological response modifier such as, for example, a lymphokine.

Moreover, the antibody can be conjugated to therapeutic moieties such as a radioactive metal ion, such as alpha particle emitters such as 213Bi, or to macrocyclic chelators suitable for conjugating radioactive metal ions, including but not limited to 131In, 131LU, 131Y , 131Ho, 131Sm, with polypeptides. In one embodiment, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are well known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference in its entirety.

Techniques for conjugating therapeutic fragments to antibodies are well known, see, for example, Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62:119-58.

Antibodies can also be attached to solid supports, which are particularly useful for immunoassays or target antigen purification. Such solid substrates include, without limitation, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

METHODS FOR PRODUCING ANTIBODIES OF THE PRESENT INVENTION

Nucleic acids encoding antibodies

The present invention provides substantially purified nucleic acid molecules that encode polypeptides containing the above-described segments or chain domains of an antibody that binds BMP6. Some of the nucleic acids of the present invention contain a nucleotide sequence encoding a heavy chain variable region shown in any of SEQ ID NO: 16; 36; 56 or 76 and/or a nucleotide sequence encoding a light chain variable region shown in any of SEQ ID NOs: 26; 46; 66 or 86. In a specific embodiment, the nucleic acid molecules are those identified in Table 1. Some other nucleic acid molecules of the present invention contain nucleotide sequences that are substantially identical (e.g., at least 65%, 80%, 95% or 99%) of the nucleotide sequences identified in Table 1. When expressed from appropriate expression vectors, the polypeptides encoded by these polynucleotides are capable of exhibiting the ability to bind the BMP6 antigen.

The present invention also provides polynucleotides that encode at least one CDR region, and typically all three CDR regions, from the heavy or light chain of a BMP6-binding antibody set forth in Table 1 and/or Table 14. Certain other polynucleotides encode all or substantially all of the heavy chain and/or light chain sequence of the BMP6 binding antibody set forth in Table 1 and/or Table 14. Due to the degeneracy of the code, a number of nucleic acid sequences will code for each of the immunoglobulin amino acid sequences.

The nucleic acid molecules of the present invention may encode both the variable region and the constant region of an antibody. Some of the nucleic acid sequences of the present invention comprise nucleotides encoding a mature heavy chain variable region sequence that is substantially identical (e.g., at least 80%, 90%, or 99%) to the mature heavy chain variable region sequence set forth in any of SEQ ID NO: 16; 36; 56 or 76. Certain other nucleic acid sequences comprising a nucleotide encoding a mature light chain variable region sequence that is substantially identical (e.g., at least 80%, 90%, or 99%) to the mature light chain variable region sequence set forth in any of SEQ ID NO: 26; 46; 66 or 86.

Polynucleotide sequences can be obtained by de novo solid phase DNA synthesis or by PCR mutagenesis of an existing sequence (eg, those described in the examples below) encoding a BMP6 binding antibody or binding fragment thereof. Direct chemical synthesis of nucleic acids can be carried out using methods known in the art, such as the phosphotriester method from Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method from Brown et al., Meth. Enzymol. 68:109, 1979; diethylphosphoramidite method from Beaucage et al., Tetra. Lett. 22:1859, 1981; and the solid support method of US Pat. No. 4,458,066. Introduction of mutations into a polynucleotide sequence by PCR can be performed as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, NY , N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

Also provided in the present invention are expression vectors and host cells for producing the above-described antibodies that bind BMP6. Various expression vectors can be used to express polynucleotides encoding BMP6-binding antibody chains or binding fragments. Both viral and non-viral expression vectors can be used to produce antibodies in a mammalian host cell. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for protein or RNA expression, and human artificial chromosomes (see, for example, Harrington et al., Nat Genet. 15:345, 1997). For example, non-viral vectors useful for expressing BMP6-binding polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen, San. Diego, Calif.), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40, human papillomavirus, Epstein-Barr virus HBP, vaccinia virus, and Semliki forest virus (SFV) vectors. See Brent et al., supra; Smith, Anna. Rev. microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (eg, enhancers) that are operably linked to polynucleotides encoding the BMP6-binding antibody chain, antigen-binding fragment. In one embodiment, an inducible promoter is used to prevent expression of the inserted sequences under conditions other than inducing. Inducible promoters include, for example, the arabinose, lacZ, metallothionein or heat shock protein promoter. Cultures of transformed organisms can be propagated under non-inducing conditions without affecting the population with coding sequences whose expression products are better tolerated by host cells. In addition to promoters, other regulatory elements may also be necessary or required for efficient expression of the BMP6-binding antibody chain, antigen-binding fragment. These elements typically include an ATG start codon and an adjacent ribosome binding site or other sequences. In addition, expression efficiency can be increased by including enhancers appropriate to the cell system used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153 :516, 1987). For example, an SV40 enhancer or a CMV enhancer can be used to increase expression in mammalian host cells.

Expression vectors can also position the secretion signal sequence to form a fusion protein with polypeptides encoded by the inserted BMP6-binding antibody sequences. More commonly, the inserted BMP6-binding antibody sequences are linked to signal sequences prior to inclusion in the vector. The vectors to be used to obtain sequences encoding the light and heavy chain variable domains of an antibody that binds BMP6 sometimes also encode constant regions or portions thereof. Such vectors allow the expression of variable regions as fusion proteins with constant regions, thus leading to the production of intact antibodies and their antigen-binding fragments. Typically, such constant regions are human.

Host cells for assembly and expression of BMP6 binding antibody chains can be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expression of the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis and other enterobacteria such as Salmonella, Serratia and various Pseudomonas species. For these prokaryotic hosts, expression vectors can also be created, which typically contain expression control sequences compatible with the host cell (eg, origin of replication). In addition, any number of various well known promoters will be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system or the lambda phage promoter system. Promoters typically drive expression, optionally with an operator sequence, and have ribosome binding site sequences and the like to initiate and complete transcription and translation. Other microbes such as yeast can also be used to express the BMP6 binding polypeptides of the present invention. It is also possible to use insect cells in combination with baculovirus vectors.

In one embodiment, mammalian host cells are used to express and produce the BMP6 binding polypeptides of the present invention. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes (e.g., myeloma hybridoma clone 1D6.C9 as described in the examples) or a mammalian cell line carrying an exogenous expression vector (e.g., SP2/0 myeloma cells, given as an example below). They include any normal mortal or normal or abnormal immortal animal or human cells. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue culture for the expression of polypeptides is generally discussed in, for example, Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells may include expression control sequences, such as an origin of replication , promoter and enhancer (see, for example, Queen, et al., Immunol. Rev. 89:49-68, 1986), and necessary information processing sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and terminator sequences transcription. These expression vectors typically contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell type specific, developmental stage specific, and/or modulated or regulated. Useful promoters include, but are not limited to, the metallothionein promoter, the adenovirus constitutive large late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the poIIII MRP promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human CMV immediate early promoter), the constitutive CMV promoter, and combinations of promoter- enhancer known in the art.

Methods for introducing expression vectors containing polynucleotide sequences of interest vary depending on the type of host cell. For example, calcium chloride transfection is typically used on prokaryotic cells, while calcium phosphate treatment or electroporation can be used on other host cells. (See, in general, Sambrook, et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, deproteinized DNA, artificial virions, fusion with the structural protein VP22 of the herpes virus (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced DNA uptake and ex vivo transduction. Long-term production of recombinant proteins in high yield will often require stable expression. For example, cell lines that stably express BMP6-binding antibody chains or binding fragments can be generated using expression vectors of the present invention that contain viral origins or endogenous expression elements and a selectable marker gene. After the introduction of the vector, the cells can be allowed to grow for 1-2 days in the enriched medium before they are transferred to the selective medium. The purpose of the selectable marker is to confer resistance to selection factors, and its presence allows the growth of cells that successfully express the introduced sequences in selective media. Proliferation of resistant stably transfected cells can be achieved using tissue culture techniques appropriate to the cell type.

PRODUCTION OF MONOCLONAL ANTIBODIES OF THE PRESENT INVENTION

Monoclonal antibodies (mAbs) can be generated by a number of techniques, including the conventional monoclonal antibody method, such as the standard somatic cell hybridization technique of Kohler and Milstein, 1975 Nature 256:495. oncogenic transformation of B-lymphocytes.

The animal system for producing hybridomas is a mouse system. Obtaining a hybridoma in a mouse is a well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg mouse myeloma cells) and fusion procedures are also known.

The chimeric or humanized antibodies and antigen-binding fragments thereof of the present invention can be generated based on a mouse monoclonal antibody sequence obtained as described above. DNA encoding immunoglobulin heavy and light chains can be obtained from a mouse hybridoma of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, mouse variable regions can be linked to human constant regions using methods known in the art (see, for example, US Pat. No. 4,816,567 by Cabilly et al.). To create a humanized antibody, mouse CDR regions can be inserted into a human scaffold using methods known in the art. See, for example, US Pat. No. 5,225,539 by Winter and US Pat. Nos. 5,530,101; 5585089; 5693762 and 6180370 by Queen et al.

In a specific embodiment, the antibodies of the present invention are human monoclonal antibodies. Such human monoclonal antibodies directed against BMP6 can be generated using transgenic or transchromosomal mice carrying portions of the human immune system instead of the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice".

The HuMAb® mouse (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human immunoglobulin heavy chain (mu and gamma) and kappa light chain sequences, together with targeted mutations that inactivate the endogenous mu and kappa loci. chains (see, for example, Lonberg, et al., 1994 Nature 368 (6474): 856-859). Accordingly, mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity monoclonal human IgG-kappa (Lonberg, N. et al., 1994 supra; review in Lonberg, N., 1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. , 1995 Ann. N. Y. Acad. Sci. 764:536-546). The production and use of HuMAb mice and the genomic modifications carried by such mice are further described in Taylor, L. et al., 1992 Nucleic Acids Research 20:6287-6295; Chen, J. et al., 1993 International Immunology 5: 647-656; Tuaillon et al., 1993 Proc. Natl. Acad. sci. USA 94:3720-3724; Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBO J. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor, L. et al., 1994 International Immunology 579-591; and Fishwild, D. et al., 1996 Nature Biotechnology 14: 845-851, all of which are expressly incorporated herein by reference in their entirety. See also U.S. Patent Nos. 5,545,806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299 and 5770429; all by Lonberg and Kay; US patent No. 5545807 by Surani et al.; PCT Publication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO 98/24884 and WO 99/45962, all by Lonberg and Kay; and PCT Publication No. WO 01/14424 by Korman et al.

In another embodiment, human antibodies of the present invention can be generated using a mouse that carries human immunoglobulin sequences on transgenes and transchromosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice", are described in detail in PCT publication WO 02/43478 by Ishida et al.

Moreover, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate BMP6-binding antibodies and antigen-binding fragments thereof of the present invention. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in US patent No. 5939598; 6075181; 6114598; 6150584 and 6162963 by Kucherlapati et al.

Moreover, alternative systems based on transchromosomal animals expressing human immunoglobulin genes are available in the art and can be used to generate the BMP6 binding antibodies of the present invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice" can be used; such mice are described in Tomizuka et al., 2000 Proc. Natl. Acad. sci. USA 97:722-727. In addition, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., 2002 Nature Biotechnology 20:889-894) and can be used to generate BMP6 binding antibodies of the present invention.

The human monoclonal antibodies of the present invention can also be generated using phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are defined in the art or described in the examples below. See, for example, US Pat. Nos. 5,223,409; 5403484 and 5571698 by Ladner et al; US Pat. Nos. 5,427,908 and 5,580,717 by Dower et al; US Pat. Nos. 5,969,108 and 6,172,197 by McCafferty et al. and US Patent Nos. 5,885,793; 6521404; 6544731; 6555313; 6582915 and 6593081 by Griffiths et al.

The human monoclonal antibodies of the present invention can also be prepared using SCID mice in which human immune cells have been transferred such that a human antibody response can be generated after immunization. Such mice are described, for example, in US patent No. 5476996 and 5698767 authored by Wilson et al.

FRAMEWORK OR Fc CONSTRUCTION

The engineered antibodies and antigen-binding fragments thereof of the present invention include those in which modifications have been made to the framework residues within the VH and/or VL, for example, to improve the properties of the antibody. Typically, such backbone modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "mutate back" one or more framework residues into the appropriate germline sequence. More specifically, an antibody that has undergone a somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequences of the antibody with the germline sequences from which the antibody is derived. To return framework region sequences to their germline configuration, somatic mutations can be introduced to "mutate back" into the germline sequence, for example, by site-directed mutagenesis. It is also contemplated that such "mutated to the original form" antibodies are covered by the present invention.

Another type of framework modification involves mutating one or more residues within a framework region, or even within one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in US Patent Application Publication No. 20030153043 by Carr et al.

As an addition or alternative to modifications made within the framework or CDR regions, the antibodies of the present invention can be designed to include modifications within the Fc region, typically to change one or more of the functional properties of the antibody, such as period serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, an antibody of the present invention may be chemically modified (for example, one or more chemical moieties may be attached to the antibody), or it may be modified to change its glycosylation pattern, again to alter one or more of the functional properties of the antibody. . Each of these embodiments is described in more detail below. Residues in the Fc region are numbered according to the Kabat EU index.

In one embodiment, the CH1 hinge is modified such that the number of cysteine residues in the hinge changes, eg increases or decreases. This approach is further described in US Pat. No. 5,677,425 to Bodmer et al. The number of cysteine residues in the CH1 hinge region is changed, for example, to facilitate the assembly of light and heavy chains, or to increase or decrease the stability of the antibody.

In another embodiment, the Fc-hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the region at the CH2-CH3 domain interface in the Fc-hinge fragment such that the antibody has impaired binding to Staphylococcus A protein (SpA) compared to the binding of the native Fc-hinge domain to SpA. This approach is described in more detail in US patent No. 6165745 by Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in Ward US Pat. No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be modified within the CH1 or CL region such that it contains a recycling receptor binding epitope taken from two loops of the CH2 domain of the IgG Fc region, as described in US Pat. No. 5,869,046 and 6121022 by Presta et al.

In one embodiment, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue in order to change the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for the effector ligand but retains the ability to bind the primary antibody antigen. The effector ligand for which the affinity is changed may be, for example, the Fc receptor or the C1 component of complement. This approach is described in more detail in US Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described in more detail in US patent No. 6194551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT publication WO 94/29351 by Bodmer et al.

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the Fc-gamma receptor by modifying one or more amino acids. This approach is further described in PCT publication WO 00/42072 by Presta. Moreover, binding sites on human IgG1 for Fc-gamma RI, Fc-gamma RII, Fc-gamma RIII and FcRn have been mapped and variants with improved binding have been described (See Shields, R. L. et al., 2001 J. Biol. Chen 276:6591-6604).

In yet another embodiment, the glycosylation pattern of the antibody is modified. For example, an aglycosylated antibody (i.e., an antibody without glycosylation) can be prepared. The pattern of glycosylation can be altered, for example, to increase the affinity of the antibody for the "antigen". Such modifications of carbohydrates can be carried out, for example, by changing one or more glycosylation sites within the sequence of the antibody. For example, one or more amino acid substitutions can be made that result in the exclusion of one or more glycosylation sites in the variable region framework, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is described in more detail in US patent No. 5714350 and 6350861 authored by Co et al.

Alternatively or alternatively, an antibody can be made that has an altered glycosylation pattern, such as a hypofucosylated antibody having reduced amounts of fucosyl residues, or an antibody having an increased content of structures with an additional GlcNac residue at the branch point. This altered glycosylation pattern has been shown to enhance the ability of antibodies to ADCC. Such carbohydrate modifications can be accomplished, for example, by expressing an antibody in a host cell with an altered glycosylation mechanism. Glycosylation-altered cells have been described in the art and can be used as host cells in which the recombinant antibodies of the present invention are expressed, thereby producing a glycosylation-altered antibody. For example, in EP 1176195 by Hang et al. describes a cell line with a functional disruption of the FUT8 gene, which encodes a fucosyltransferase, as a result of which the antibodies expressed in such a cell line exhibit hypofucosylation. PCT publication WO 03/035835 by Presta describes a variant of the CHO cell line, LecI3 cells, with reduced ability to attach fucose to Asn(297)-bound carbohydrates, which also leads to hypofucosylation of antibodies expressed in such a host cell (see also Shields, R. L. et al., 2002 J. Biol Chem 277:26733-26740). In PCT publication WO 99/54342 by Umana et al. describes cell lines designed to express glycoprotein-modifying glycosyltransferases (e.g., beta-(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), resulting in antibodies expressed in the engineered cell lines characterized by an increased content of structures with an additional GlcNac residue in branch point, resulting in increased ADCC activity in antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

METHODS FOR CONSTRUCTING MODIFIED ANTIBODIES

As discussed above, BMP6-binding antibodies having the VH and VL sequences or the full-length heavy and light chain sequences shown herein can be used to generate new BMP6-binding antibodies by modifying the full-length heavy chain and/or light chain sequences, sequences VH and/or VL or constant(s) region(s) attached(s) to them. Thus, in another aspect of the present invention, the structural features of the BMP6-binding antibody of the present invention are used to generate structurally related BMP6-binding antibodies that retain at least one functional property of the antibodies and antigen-binding fragments of the present invention, such as binding with human BMP6, as well as inhibition of one or more functional properties of BMP6 (for example, inhibition of erythrocyte lysis in a hemolysis assay).

For example, one or more CDR regions of antibodies and their antigen-binding fragments of the present invention, or mutant variants thereof, can be combined using recombination techniques with known framework regions and/or other CDRs to generate additional recombination-engineered BMP6-binding antibodies and their antigen-binding fragments of the present invention, as discussed above. Other types of modifications include those described in the previous section. The starting material for the design method is one or more of the VH and/or VL sequences provided herein, or one or more of their CDR regions. It is not necessary to actually generate (ie, express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein, or one or more of their CDR regions, in order to generate a engineered antibody. Rather, the information contained in the sequence(s) is used as input to create "second generation" sequence(s) derived from the original sequence(s) and then the sequence(s) " second generation" is obtained and expressed as a protein.

An altered antibody sequence can also be obtained by screening antibody libraries having fixed CDR3 sequences or minimum required binding determinants as described in US20050255552 and a variety of CDR1 and CDR2 sequences. Screening can be performed according to any screening technology suitable for screening antibodies from antibody libraries, such as phage display technology.

Standard molecular biology techniques can be used to generate and express an altered antibody sequence. An antibody encoded by an altered antibody sequence(s) is one that retains one, some, or all of the functional properties of the BMP6 binding antibodies described herein, the functional properties including, without limitation, specific binding to the human BMP6 protein. ; and the antibody inhibits erythrocyte lysis in the hemolysis assay.

The functional properties of altered antibodies can be assessed using standard assays available in the art and/or described herein, such as assays set forth in the examples (eg, ELISA variants).

In one embodiment of the methods for constructing antibodies and antigen-binding fragments thereof of the present invention, mutations can be introduced arbitrarily or selectively at all or part of the coding sequence of a BMP6-binding antibody, and the resulting modified BMP6-binding antibodies can be screened for binding activity and/or other functional properties as described in this document. Methods for introducing mutations have been described in the prior art. For example, PCT publication WO 02/092780 by Short describes methods for generating and screening antibody mutants using saturation mutagenesis, ligation assembly of synthesized fragments, or combinations thereof. Alternatively, in PCT publication WO 03/074679 by Lazar et al. describes how to use computer screening methods to optimize the physicochemical properties of antibodies.

CHARACTERIZATION OF ANTIBODIES OF THE PRESENT INVENTION

The characteristics of the antibodies and their antigen-binding fragments of the present invention can be determined using various functional assays. For example, they can be characterized by their ability to inhibit BMP6.

The ability of an antibody to bind to BMP6 can be detected by directly labeling the antibody of interest, or the antibody can be unlabeled and binding detected indirectly using various sandwich assay formats known in the art.

In one embodiment, the BMP6-binding antibodies and antigen-binding fragments thereof of the present invention block or compete with a BMP6-binding reference antibody for binding to a BMP6 polypeptide. They may be the fully human BMP6-binding antibodies described above. They can also be other murine, chimeric, or humanized BMP6-binding antibodies that bind to the same epitope as the reference antibody. The ability to block or compete with the reference antibody for binding indicates that the BMP6-binding antibody under test binds to the same or similar epitope as defined by the reference antibody, or to an epitope that is sufficiently close to the epitope bindable reference antibody that binds BMP6. It is particularly likely that such antibodies have the advantageous properties identified for the reference antibody. The ability to block or compete with a reference antibody can be determined, for example, using a competitive binding assay. In a competitive binding assay, a test antibody is evaluated for its ability to inhibit specific binding of a reference antibody to a common antigen, such as a BMP6 polypeptide. A test antibody competes with a reference antibody for specific binding to an antigen if an excess of test antibody provides significant inhibition of reference antibody binding. Significant inhibition means that the test antibody reduces the specific binding of the reference antibody, typically by at least 10%, 25%, 50%, 75%, or 90%.

There are a number of known competitive binding assays that can be used to evaluate competition between an antibody and a reference antibody for binding to a particular protein, in this case BMP6. These include, for example, solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), competitive sandwich assay (see Stahli et al., Methods in Enzymology 9:242-253, 1983); solid phase direct EIA using biotin-avidin (see Kirkland et al., J. Immunol. 137:3614-3619, 1986); direct labeled solid phase analysis, direct labeled sandwich solid phase analysis (see Harlow & Lane, supra); direct labeling solid phase RIA using the I-125 label (see Morel et al., Molec. Immunol. 25:7-15, 1988); solid phase direct EIA using biotin-avidin (Cheung et al., Virology 176:546-552, 1990) and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82, 1990). Typically, such an assay involves the use of a purified solid surface antigen or cells bearing either of these, an unlabeled test antibody that binds BMP6, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of the test antibody. Typically, the antibody to be tested is present in excess. Antibodies identified by competitive analysis (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope that is close enough to the epitope bound by the reference antibody that steric hindrance.

To determine if selected BMP6-binding monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (eg, reagents from Pierce, Rockford, IL). Competitive studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using BMP6 polypeptide-coated ELISA plates. Binding of biotinylated MAb can be detected using a streptavidin-alkaline phosphatase probe. To determine the isotype of a purified BMP6-binding antibody, isotype ELISAs can be performed. For example, wells of microtiter plates can be coated with 1 µg/ml anti-human IgG overnight at 4°C. purified isotype controls at ambient temperature for one to two hours. The contents of the wells can then be reacted with specific human IgG1 or human IgM alkaline phosphatase-conjugated probes. The plates are then developed and analyzed so that the isotype of the purified antibody can be determined.

Flow cytometry can be used to demonstrate binding of BMP6-binding monoclonal antibodies to living cells expressing a BMP6 polypeptide. Briefly, cell lines expressing BMP6 (grown under standard growth conditions) can be mixed with various concentrations of BMP6 binding antibody in PBS containing 0.1% BSA and 10% fetal calf serum and incubated at 37°C for 1 hours. After washing, the cells are reacted with fluorescein-labeled anti-human IgG under the same conditions as when stained with the primary antibody. Samples can be analyzed with the FACScan instrument using light scatter and side scatter properties to gate individual cells. An alternative analysis using fluorescence microscopy (in addition to or instead of) flow cytometry can be used. Cells can be stained exactly as described above and examined using fluorescence microscopy. This method allows visualization of individual cells, but may be characterized by reduced sensitivity depending on the density of the antigen.

The BMP6-binding antibodies and antigen-binding fragments thereof of the present invention can be further tested for reactivity with a BMP6 polypeptide or antigenic fragment by Western blotting. Briefly, purified BMP6 polypeptides or fusion proteins thereof or cell extracts from cells expressing BMP6 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, separated antigens are transferred to nitrocellulose membranes blocked with 10% fetal calf serum and examined with the test monoclonal antibodies. Human IgG binding can be detected using an anti-human IgG alkaline phosphatase antibody and visualized with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

Examples of functional assays are also described in the Examples section below.

PREVENTIVE AND THERAPEUTIC APPLICATIONS

The present invention provides methods for treating a disease or disorder associated with elevated BMP6 activity by administering to a subject in need thereof an effective amount of the antibodies and antigen-binding fragments thereof of the present invention. In a specific embodiment, the present invention provides a method for treating anemia by administering to a subject in need thereof an effective amount of the antibodies and antigen-binding fragments thereof of the present invention.

The antibodies and antigen-binding fragments thereof of the present invention can be used, among other things, to prevent the progression of anemia. They can also be used in combination with other therapies to treat patients with anemia.

In one embodiment, the present invention provides methods for treating a BMP6-associated disease or disorder by administering to a subject in need thereof an effective amount of the antibodies and antigen-binding fragments thereof of the present invention. Examples of known BMP6-associated diseases or disorders include anemia, including, but not limited to, anemia due to chronic disease (ACD), anemia due to (e.g., associated with) chronic kidney disease (CKD), anemia due to cancer, anemia, inflammatory, erythropoietic stimulant (ESA)-resistant anemia (eg, erythropoietin-resistant (EPO) anemia), ESA-hyporesponsive anemia (eg, EPO-hyporesponsive anemia), functional iron deficiency anemia, and/or anemia due to iron deficiency.

In a specific embodiment, the present invention provides methods for treating a BMP6-associated disease or disorder by administering to a subject in need thereof an effective amount of the antibodies and antigen-binding fragments thereof of the present invention, wherein said disease or disorder is anemia. In an embodiment, the anemia is anemia due to a chronic disease. In an embodiment, the chronic disease is chronic kidney disease. In an embodiment, the chronic disease is cancer. In an embodiment, the chronic disease is inflammation. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia resistant to ESAs (eg, EPO). In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia with ESA (eg, EPO) hyporeactivity. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia due to iron deficiency. In embodiments, including any of the above embodiments, the subject is a subject on chronic hemodialysis (HD). In embodiments, including any of the above embodiments, the subject has a kidney disease, such as end stage kidney disease.

In a specific embodiment, the present invention provides methods for treating a BMP6-associated disease or disorder by administering to a subject in need thereof an effective amount of an antibody and an antigen-binding fragment thereof of the present invention, wherein said disease or disorder is functional iron deficiency anemia. In an embodiment, the subject is a patient on chronic hemodialysis treated with an ESA (eg, EPO). In an embodiment, the subject is a chronic kidney disease patient on hemodialysis treated with an ESA (eg, EPO).

In a specific embodiment, the present invention provides methods for treating anemia by administering to a subject in need thereof an effective amount of a composition comprising an antibody of the present invention. In a specific embodiment, the present invention provides methods for treating anemia by administering to a subject in need thereof an effective amount of a composition comprising an antibody of the present invention. In an embodiment, the anemia is anemia due to chronic kidney disease. In an embodiment, the anemia is ESA (eg, EPO) resistant anemia. In an embodiment, the anemia is ESA (eg, EPO) hyporesponsive anemia. In an embodiment, the anemia is iron deficiency anemia. In an embodiment, the anemia is anemia associated with a kidney disease, such as chronic kidney disease. In embodiments, including any of the above embodiments, the subject is a subject on chronic hemodialysis (HD). In embodiments, including any of the above embodiments, the subject has a kidney disease, such as end stage kidney disease.

In a specific embodiment, the present invention provides methods for treating a BMP6-associated disease or disorder by administering to a subject in need thereof an effective amount of a composition comprising an antibody of the present invention, wherein said disease or disorder is functional iron deficiency anemia. In an embodiment, the subject is a patient on chronic hemodialysis treated with an ESA (eg, EPO). In an embodiment, the subject is a chronic kidney disease patient on hemodialysis treated with an ESA (eg, EPO).

In a specific embodiment, the present invention provides methods for treating anemia.

In a specific embodiment, the present invention provides a method for reducing ESA (eg, EPO) dose requirements in a subject by administering to the subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the present invention. In an embodiment, the anemia is anemia due to a chronic disease. In an embodiment, the chronic disease is chronic kidney disease. In an embodiment, the chronic disease is cancer. In an embodiment, the chronic disease is inflammation. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia resistant to ESAs (eg, EPO). In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia with ESA (eg, EPO) hyporeactivity. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia due to iron deficiency. In embodiments, including any of the above embodiments, the subject is a subject on chronic hemodialysis (HD). In embodiments, including any of the above embodiments, the subject has a kidney disease, such as end stage kidney disease.

In embodiments, the methods and uses of the present invention result in a reduction in the ESA Resistance Index (ERI) in patients.

In a specific embodiment, the present invention provides methods for reducing ESA (e.g., EPO) dose requirements in a subject by administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the present invention, wherein said subject has anemia due to functional iron deficiency. In an embodiment, the subject is a patient on chronic hemodialysis and receiving treatment with an ESA (eg, EPO). In an embodiment, the subject is a chronic kidney disease patient on hemodialysis treated with an ESA (eg, EPO).

In a specific embodiment, the present invention provides a method of reducing ESA (eg, EPO) dose requirements in a subject by administering to the subject in need thereof an effective amount of a composition comprising an antibody of the present invention. In an embodiment, the subject is anemic. In an embodiment, the anemia is anemia due to a chronic disease. In an embodiment, the chronic disease is chronic kidney disease. In an embodiment, the chronic disease is cancer. In an embodiment, the chronic disease is inflammation. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia resistant to ESAs (eg, EPO). In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia with ESA (eg, EPO) hyporeactivity. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia due to iron deficiency. In embodiments, including any of the above embodiments, the subject is a subject on chronic hemodialysis (HD). In embodiments, including any of the above embodiments, the subject has a kidney disease, such as end stage kidney disease.

In a specific embodiment, the present invention provides methods for reducing ESA (e.g., EPO) dose requirements in a subject by administering to a subject in need thereof an effective amount of a composition comprising an antibody of the present invention, wherein said subject has functional iron deficiency anemia. In an embodiment, the subject is a patient on chronic hemodialysis and receiving treatment with an ESA (eg, EPO). In an embodiment, the subject is a chronic kidney disease patient on hemodialysis treated with an ESA (eg, EPO).

In a specific embodiment, the present invention provides a method of reducing iron (eg, iron IV) dosage requirements in a subject by administering to the subject in need thereof an effective amount of the antibodies and antigen-binding fragments thereof of the present invention. In an embodiment, the subject is anemic. In an embodiment, the anemia is anemia due to a chronic disease. In an embodiment, the chronic disease is chronic kidney disease. In an embodiment, the chronic disease is cancer. In an embodiment, the chronic disease is inflammation. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia resistant to ESAs (eg, EPO). In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia with ESA (eg, EPO) hyporeactivity. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia due to iron deficiency. In embodiments, including any of the above embodiments, the subject is a subject on chronic hemodialysis (HD). In embodiments, including any of the above embodiments, the subject has a kidney disease, such as end stage kidney disease.

In a specific embodiment, the present invention provides methods for reducing iron (e.g., iron IV) dose requirements in a subject by administering to a subject in need thereof an effective amount of the antibodies and antigen-binding fragments thereof of the present invention, wherein said subject has anemia due to functional iron deficiency. . In an embodiment, the subject is a patient on chronic hemodialysis and receiving treatment with an ESA (eg, EPO). In an embodiment, the subject is a chronic kidney disease patient on hemodialysis treated with an ESA (eg, EPO).

In a specific embodiment, the present invention provides a method of reducing iron (eg, IV iron) dose requirements in a subject by administering to the subject in need thereof an effective amount of a composition comprising an antibody of the present invention. In an embodiment, the subject is anemic. In an embodiment, the anemia is anemia due to a chronic disease. In an embodiment, the chronic disease is chronic kidney disease. In an embodiment, the chronic disease is cancer. In an embodiment, the chronic disease is inflammation. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia resistant to ESAs (eg, EPO). In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia with ESA (eg, EPO) hyporeactivity. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia due to iron deficiency. In embodiments, including any of the above embodiments, the subject is a subject on chronic hemodialysis (HD). In embodiments, including any of the above embodiments, the subject has a kidney disease, such as end stage kidney disease.

In a specific embodiment, the present invention provides methods for reducing iron (e.g., IV iron) dose requirements in a subject by administering to a subject in need thereof an effective amount of a composition comprising an antibody of the present invention, wherein said subject has anemia due to functional iron deficiency. In an embodiment, the subject is a patient on chronic hemodialysis and receiving treatment with an ESA (eg, EPO). In an embodiment, the subject is a chronic kidney disease patient on hemodialysis treated with an ESA (eg, EPO).

In a particular embodiment, the present invention provides a method for reducing iron (e.g., iron IV) dose requirements in a subject and reducing ESA (e.g., EPO) dose requirements in a subject, comprising administering an antibody or antigen-binding fragment of the present invention, or a composition comprising said antibody. or an antigen-binding fragment. In an embodiment, the anemia is anemia due to a chronic disease. In an embodiment, the chronic disease is chronic kidney disease. In an embodiment, the chronic disease is cancer. In an embodiment, the subject is anemic. In an embodiment, the chronic disease is inflammation. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia resistant to ESAs (eg, EPO). In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia with ESA (eg, EPO) hyporeactivity. In an embodiment, the anemia (eg, anemia due to chronic disease) is anemia due to iron deficiency. In embodiments, including any of the above embodiments, the subject is a subject on chronic hemodialysis (HD). In embodiments, including any of the above embodiments, the subject has a kidney disease, such as end stage kidney disease.

In an embodiment, the present invention provides methods for mobilizing sequestered iron by administering to a subject in need thereof an effective amount of the antibodies and antigen-binding fragments thereof of the present invention.

In an embodiment, the present invention provides methods for mobilizing sequestered iron by administering to a subject in need thereof an effective amount of a composition comprising an antibody of the present invention.

In an embodiment, the present invention provides a method for improving (e.g., increasing) the hemoglobin level in an anemic subject while reducing the dose requirement of erythropoietin and/or iron (e.g., iron IV), said method comprising administering to the subject in need thereof an antibody or its antigennegative fragment of the present invention. In an embodiment, the anemia is anemia associated with a chronic disease. In an embodiment, improving the hemoglobin level involves improving the level to meet the requirements of a clinical practice guideline, such as Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidney inter., Suppl. 2012; 2: 279-335, the contents of which are incorporated herein by reference in their entirety. In an embodiment, improving the hemoglobin level involves improving the hemoglobin level to at least about 11.0 g/dl, for example, to a level from about 11.0 g/dl to about 12.5 g/dl. In an embodiment, improving the hemoglobin level involves improving the hemoglobin level to at least 11.0 g/dl, for example, to a level from 11.0 g/dl to 12.5 g/dl.

In an embodiment, the present invention provides a method for improving (e.g., increasing) the hemoglobin level in an anemic subject while reducing the dose requirement of erythropoietin and/or iron (e.g., iron IV), said method comprising administering to a subject in need thereof a composition containing the antibody of the present invention. In an embodiment, the anemia is anemia associated with a chronic disease. In an embodiment, improving the hemoglobin level involves improving the level to meet the requirements of a clinical practice guideline, such as Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidney inter., Suppl. 2012; 2: 279-335, the contents of which are incorporated herein by reference in their entirety. In an embodiment, improving the hemoglobin level involves improving the hemoglobin level to at least about 11.0 g/dl, for example, to a level from about 11.0 g/dl to about 12.5 g/dl. In an embodiment, improving the hemoglobin level involves improving the hemoglobin level to at least 11.0 g/dl, for example, to a level from 11.0 g/dl to 12.5 g/dl.

In an embodiment, the present invention provides a method for maintaining a hemoglobin level in a subject with anemia while reducing the need for a dose of erythropoietin and/or iron (e.g., iron IV), wherein said method comprises administering to a subject in need thereof an antibody or an antigen-binding fragment thereof according to the present invention. In an embodiment, the anemia is anemia associated with a chronic disease. In an embodiment, improving the hemoglobin level involves improving the level to a level that meets the requirements of a clinical practice guide, for example, Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidney inter., Suppl. 2012; 2: 279-335, the contents of which are incorporated herein by reference in their entirety. In an embodiment, improving the hemoglobin level includes improving the hemoglobin level to at least about 11.0 g/dl, for example, to a level from about 11.0 g/dl to about 12.5 g/dl. In an embodiment, improving the hemoglobin level involves improving the hemoglobin level to at least 11.0 g/dl, for example, to a level from 11.0 g/dl to 12.5 g/dl.

In an embodiment, the present invention provides a method for maintaining a hemoglobin level in an anemic subject while reducing the dose requirement of erythropoietin and/or iron (e.g., iron IV), said method comprising administering to a subject in need thereof a composition comprising an antibody of the present invention. invention. In an embodiment, the anemia is anemia associated with a chronic disease. In an embodiment, improving the hemoglobin level involves improving the level to meet the requirements of a clinical practice guideline, such as Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidney inter., Suppl. 2012; 2: 279-335, the contents of which are incorporated herein by reference in their entirety. In an embodiment, improving the hemoglobin level involves improving the hemoglobin level to at least about 11.0 g/dl, for example, to a level from about 11.0 g/dl to about 12.5 g/dl. In an embodiment, improving the hemoglobin level involves improving the hemoglobin level to at least 11.0 g/dl, for example, to a level from 11.0 g/dl to 12.5 g/dl.

In one embodiment, the isolated antibody or antigen-binding fragment thereof described in Table 1 and/or Table 14 may be administered to a patient in need thereof in combination with a therapeutic method or procedure such as those described herein or known in the art. Such method or procedure includes, but is not limited to, administration of a therapeutically effective amount of ESA (eg, EPO), erythropoietin, or iron, and blood transfusion. Treatment is generally continued at intervals over a period of a week, a month, three months, six months, or a year. In some patients, treatment is carried out for the rest of the patient's life.

If the therapeutic agents of the present invention are administered together with another agent, both of them can be administered sequentially in any order or simultaneously. In some aspects, an antibody of the present invention is administered to a subject who is also receiving therapy with a second agent or method (eg, ESA, erythropoietin, iron supplements, blood transfusion). In other aspects, the binding molecule is administered in conjunction with surgical treatments.

Suitable agents for combination treatment with BMP6-binding antibodies include agents known in the art that inhibit or reduce the expression, level, stability and/or activity of BMP6. Such agents include antibodies, siRNA and small molecules targeting BMP6.

Various anti-BMP6 antibodies are known in the art, including but not limited to those described in

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WO 2010/056981.

Additional antibodies to BMP6 are known in the art; many of them are commercially available.

Various BMP6-targeting siRNAs are known in the art, including but not limited to the siRNAs described in

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Additional BMP6 inhibitors are known. Any of these can be used in combination with any antibody or antigen-binding fragment disclosed herein.

The combination therapy regimen may be additive, or it may provide synergistic results (eg, effects in the form of a decrease in BMP6 activity that are greater than expected for the combined use of the two agents). In one embodiment, the present invention provides a combination therapy for the prevention and/or treatment of anemia or other BMP6-associated disease described above with a BMP6-binding antibody of the present invention and an antianemic agent or method, such as ESA, erythropoietin, iron supplements or blood transfusions.

DIAGNOSTIC WAYS OF APPLICATION

In one aspect, the present invention encompasses diagnostic assays for determining BMP6 and/or nucleic acid expression, as well as BMP6 function, in a biological sample (e.g., blood, serum, cells, tissue) or from an individual afflicted with a disease or disorder or at risk of developing a disorder. associated with anemia.

Diagnostic assays, such as competitive assays, rely on the ability of a labeled analog ("labeled substance") to compete with an analyte in a test sample for a limited number of binding sites on a common binding partner. The binding partner is usually rendered insoluble before or after competition, and then the labeled and analyte bound to the binding partner are separated from the unbound labeled and analyte. This separation is carried out by decanting (where the binding partner has previously been converted to an insoluble form) or by centrifugation (where the binding partner has precipitated after the competition reaction). The amount of analyte in the test sample is inversely proportional to the amount of bound labeled substance, which is measured by the amount of marker substance. Dose-response curves with known amounts of analyte are plotted and compared with test results to quantify the amount of analyte present in the test sample. These assays are called ELISA systems when enzymes are used as detectable markers. In this assay format, competitive binding between antibodies and BMP6-binding antibodies results in bound BMP6, preferably the BMP6 epitopes of the present invention, which are a measurable indication of the content of antibodies in a blood serum sample, most specifically neutralizing antibodies in a blood serum sample.

A significant advantage of the assay is the direct measurement of neutralizing antibodies (i.e., antibodies that interfere with the binding of BMP6, in particular epitopes). Such an assay, especially in the form of an ELISA assay, has wide application in the clinical setting and in routine blood screening.

In clinical diagnosis or monitoring of patients with disorders associated with anemia, the detection of BMP6 proteins when compared with levels in a corresponding biological sample from a normal subject indicates a patient with disorders associated with anemia.

In vivo diagnostic or imaging is described in US2006/0067935. Briefly, these methods generally involve administering or providing a patient with a diagnostically effective amount of a BMP6 binding molecule that is operably attached to a marker or label that can be detected by non-invasive methods. The antibody-marker conjugate is given sufficient time to be detected and bound to BMP6. The patient is then exposed to a detecting device to identify the detectable marker, thus obtaining an image of the location of BMP6 binding molecules in the patient's tissue. The presence of a BMP6-binding antibody or antigen-binding fragment thereof is detected by determining whether the marker antibody binds to a tissue component. The detection of an elevated level of BMP6 proteins or a protein combination compared to that of a normal individual without anemia indicates a predisposition to disorders associated with anemia, and/or the onset of their manifestation. These aspects of the present invention also relate to use in tissue imaging methods and combined diagnostic and therapeutic methods.

The present invention also relates to the field of predictive medicine, in which diagnostic assays, prognostic assays, pharmacogenomic studies, and monitoring in clinical trials are used for predictive (predictive) purposes to thereby provide prophylactic treatment to an individual.

The present invention also provides prognostic (or predictive) assays to determine whether an individual is at risk of developing a disorder associated with dysregulation of the BMP6 pathway. For example, mutations in the BMP6 gene can be analyzed in a biological sample. Such assays can be used to predict or predict, thereby prophylactically treating an individual before the onset of a disorder characterized by or associated with expression of a nucleic acid or activity of BMP6.

In another aspect of the present invention, methods are provided for determining BMP6 nucleic acid expression or BMP6 activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomic studies"). Pharmacogenomic studies provide the ability to select agents (eg, drugs) for therapeutic or prophylactic treatment of an individual based on the individual's genotype (eg, the genotype of the individual being assessed to determine whether the individual is likely to respond to a particular agent).

In yet another aspect of the present invention, a method is provided for monitoring the effect of agents (eg, drugs) on BMP6 expression or activity in clinical trials.

PHARMACEUTICAL COMPOSITIONS

The present invention provides pharmaceutical compositions comprising a BMP6-binding antibody, or binding fragment thereof, formulated with a pharmaceutically acceptable carrier. The compositions may further comprise one or more other therapeutic agents that are suitable for the treatment or prevention of a disease associated with BMP6 (eg anemia). Pharmaceutical carriers enhance or stabilize the composition or facilitate the preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible.

The pharmaceutical composition of the present invention can be administered using a number of methods known in the art. The route and/or route of administration will vary depending on the desired results. Administration may be intravenous, intramuscular, intraperitoneal or subcutaneous, or administration is carried out near the target site. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, vertebral, or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bispecific and multispecific molecule, may be coated with a material to protect the compound from acids and other natural conditions that may inactivate the compound.

The composition must be sterile and fluid. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases it is preferred to include isotonic agents, for example sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate or gelatin.

Pharmaceutical compositions of the present invention can be obtained in accordance with methods well known in the art and usually carried out in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured in accordance with the provisions of GMP. Generally, a therapeutically effective dose or effective dose of a BMP6 binding antibody is used in the pharmaceutical compositions of the present invention. BMP6 binding antibodies are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art. Dosing regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated according to the needs of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unit doses for subjects to be treated; wherein each unit contains a predetermined amount of active compound, calculated to obtain the desired therapeutic effect, in combination with the necessary pharmaceutical carrier.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, effective in terms of composition and route of administration, without being toxic to the patient. The selected dose level depends on a number of pharmacokinetic factors, including the activity of the specific compositions of the present invention used, or their presence in the form of an ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs. , compounds and/or materials used in combination with the specific compositions used, age, sex, weight, condition, general health and history of the patient to be treated, and similar factors.

A physician or veterinarian may begin administering doses of the antibodies and antigen-binding fragments of the present invention used in a pharmaceutical composition at levels below those needed to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. . In general, effective dosages of the compositions of the present invention for the treatment of the allergic inflammatory disease described herein will vary depending on many different factors, including routes of administration, target site, physiological state of the patient, whether the patient is a human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Doses for treatment should be adjusted to optimize safety and efficacy. In the case of systemic administration of the antibody, the dose is in the range of about 0.0001 to 100 mg/kg, and more typically 0.001-15 mg/kg of the host's body weight. An exemplary treatment regimen involves systemic administration (eg, intravenous or subcutaneous) once, or alternatively, more than once according to the dosing regimen (eg, with repeated dosing), for example, once every two weeks, once every three weeks , or once a month, or once every 3-6 months. In the case of intravitreal administration of the antibody, the dose is in the range of about 0.0001 to about 10 mg. An exemplary treatment regimen involves systemic administration once every two weeks, once every three weeks, or once a month, or once every 3-6 months.

The antibody is usually injected several times. The intervals between individual doses may be weeks, months or years. The intervals may also be irregular, as indicated by measurement of BMP6-binding antibody levels in the patient's blood. In some methods of systemic administration, the dose is adjusted to achieve a plasma concentration of the antibody of 1-1000 μg/ml, and in some methods 25-500 μg/ml. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the antibody in the patient. In general, humanized antibodies exhibit a longer half-life than chimeric and non-human antibodies. The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dose is administered at relatively large intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer a relatively high dose at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the patient experiences partial or complete relief of the symptoms of the disease. After that, the patient can be administered according to the prophylactic scheme.

In a particular embodiment, a composition comprising an antibody or antigen-binding fragment of the present invention is administered at a dose (of the antibody or antigen-binding fragment thereof) of 0.001 mg/kg to 0.1 mg/kg. In a specific embodiment, a composition comprising an antibody or antigen-binding fragment of the present invention is administered at a dose of about 0.001 mg/kg to about 0.1 mg/kg. In a specific embodiment, a composition containing an antibody or antigen-binding fragment of the present invention is administered at a dose of 0.001 mg/kg, 0.0016 mg/kg, 0.0025 mg/kg, 0.0040 mg/kg, 0.0063 mg /kg, 0.01 mg/kg, 0.016 mg/kg, 0.025 mg/kg, 0.040 mg/kg, 0.063 mg/kg or 0.1 mg/kg. In a particular embodiment, a composition comprising an antibody or antigen binding fragment of the present invention is administered at a dose of about 0.001 mg/kg, about 0.0016 mg/kg, about 0.0025 mg/kg, about 0.0040 mg/kg, about 0.0063 mg/kg, about 0.01 mg/kg, about 0.016 mg/kg, about 0.025 mg/kg, about 0.040 mg/kg, about 0.063 mg/kg, or about 0.1 mg/kg. In an embodiment, a composition comprising an antibody or antigen-binding fragment of the present invention is administered, including, for example, at any of the doses listed above, intravenously. In embodiments, the intravenous administration is an intravenous infusion. In embodiments, the infusion lasts for 30-60 minutes. In embodiments, the infusion lasts for about 30-60 minutes.

FERRITIN LEVEL

The disclosed methods may include determining the level of ferritin in a biological sample, such as blood or blood serum, and in embodiments, patients, such as patients having a disease described herein, such as anemia, are selected for treatment with a BMP6 antagonist (for example, which described herein, for example, antibodies to BMP6, for example, antibodies 7, for example, which are described in table 1), or they are predicted to respond to therapy with a BMP6 antagonist based on the level of ferritin.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is ≤ about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is ≤ 1500 ng/mL. In embodiments, the ferritin level is < 1500 ng/mL. In embodiments, the ferritin level is ≤ about 2000 ng/mL. In embodiments, the ferritin level is ≤ 2000 ng/mL. In embodiments, the ferritin level is < about 2000 ng/mL.

In embodiments, the ferritin level, e.g., the ferritin level predictive of response (e.g., increased response) to treatment with BMP6, is ≤ about 1450 ng/ml, e.g., ≤ about 1400 ng/ml, ≤ about 1350 ng/ml, ≤ about 1300 ng/mL, ≤ approximately 1250 ng/mL, ≤ approximately 1200 ng/mL, ≤ approximately 1150 ng/mL, ≤ approximately 1100 ng/mL, ≤ approximately 1050 ng/mL or ≤ approximately 1000 ng/mL, etc. .

In embodiments, the ferritin level, e.g., the ferritin level predictive of response (e.g., increased response) to treatment with BMP6, is ≥ about 200 ng/mL, e.g., ≥ about 250 ng/mL, ≥ about 300 ng/mL, ≥ about 350 ng/mL, ≥ approximately 400 ng/mL, ≥ approximately 450 ng/mL, ≥ approximately 500 ng/mL, ≥ approximately 550 ng/mL, ≥ approximately 600 ng/mL, ≥ approximately 650 ng/mL, ≥ approximately 700 ng/mL, ≥ about 750 ng/mL, ≥ about 800 ng/mL, ≥ about 850 ng/mL, ≥ about 900 ng/mL, ≥ about 950 ng/mL, ≥ about 1000 ng/mL or ≥ about 1500 ng /ml, etc. In embodiments, the ferritin level ranges from any of the levels listed above to about 2000 ng/ml.

In embodiments, the ferritin level, e.g., the ferritin level predictive of response (e.g., increased response) to treatment with BMP6, is ≥ about 500 ng/mL, ≥ about 550 ng/mL, ≥ about 600 ng/mL, ≥ about 650 ng /ml, ≥ approximately 700 ng/mL, ≥ approximately 750 ng/mL, ≥ approximately 800 ng/mL, ≥ approximately 850 ng/mL, ≥ approximately 900 ng/mL, ≥ approximately 950 ng/mL, ≥ approximately 1000 ng/mL ml or ≥ about 1500 ng/ml, etc. In embodiments, the ferritin level ranges from any of the levels listed above to about 2000 ng/ml.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is ≥ about 500 ng/mL. In embodiments, the ferritin level is from about 500 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level is from about 500 ng/mL to about 1000 ng/mL. In embodiments, the ferritin level is from about 500 ng/mL to about 1500 ng/mL.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 200 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 300 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 400 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 500 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 600 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 700 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 800 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 900 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1000 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1100 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1200 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1300 ng/mL to about 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1400 ng/mL to about 1500 ng/mL.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 200 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 300 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 400 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 500 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 600 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 700 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 800 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 900 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1000 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1100 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1200 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1300 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1400 ng/mL to about 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1500 ng/mL to about 1600 ng/mL.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 200 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 300 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 400 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 500 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 600 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 700 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 800 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 900 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1000 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1100 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1200 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1300 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1400 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1500 ng/mL to about 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1600 ng/mL to about 1700 ng/mL.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 200 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 300 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 400 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 500 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 600 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 700 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 800 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 900 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1000 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1100 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1200 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1300 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1400 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1500 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1600 ng/mL to about 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1700 ng/mL to about 1800 ng/mL.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 200 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 300 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 400 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 500 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 600 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 700 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 800 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 900 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1000 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1100 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1200 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1300 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1400 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1500 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1600 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1700 ng/mL to about 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1800 ng/mL to about 1900 ng/mL.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 200 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 300 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 400 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 500 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 600 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 700 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 800 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 900 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1000 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1100 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1200 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1300 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1400 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1500 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1600 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1700 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1800 ng/mL to about 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is from about 1900 ng/mL to about 2000 ng/mL.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 200 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 300 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 400 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 500 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 600 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 700 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 800 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 900 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1000 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1100 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1200 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1300 ng/mL and 1500 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1400 ng/mL and 1500 ng/mL. In preferred embodiments, all ranges include the listed extremes.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 200 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 300 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 400 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 500 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 600 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 700 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 800 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 900 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1000 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1100 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1200 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1300 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1400 ng/mL and 1600 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1500 ng/mL and 1600 ng/mL. In preferred embodiments, all ranges include the listed extremes.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 200 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 300 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 400 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 500 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 600 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 700 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 800 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 900 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1000 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1100 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1200 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1300 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1400 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1500 ng/mL and 1700 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1600 ng/mL and 1700 ng/mL. In preferred embodiments, all ranges include the listed extremes.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 200 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 300 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 400 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 500 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 600 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 700 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 800 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 900 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1000 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1100 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1200 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1300 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1400 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1500 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1600 ng/mL and 1800 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1700 ng/mL and 1800 ng/mL. In preferred embodiments, all ranges include the listed extremes.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 200 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 300 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 400 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 500 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 600 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 700 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 800 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 900 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1000 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1100 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1200 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1300 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1400 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1500 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1600 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1700 ng/mL and 1900 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1800 ng/mL and 1900 ng/mL. In preferred embodiments, all ranges include the listed extremes.

In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 200 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 300 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 400 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 500 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 600 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 700 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 800 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 900 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1000 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1100 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1200 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1300 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1400 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1500 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1600 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1700 ng/mL and 2000 ng/mL. In embodiments, the ferritin level, eg, the ferritin level predictive of response (eg, increased response) to treatment with BMP6, is between 1800 ng/mL and 2000 ng/mL. In preferred embodiments, all ranges include the listed extremes.

In embodiments, the ferritin levels described above are measured in a biological sample selected from blood and serum. In embodiments, the ferritin levels described above are measured in serum.

METHODS OF ANALYSIS, METHODS OF DIAGNOSTICS AND METHODS OF OBTAINING A FORM OF INFORMATION SUITABLE FOR TRANSMISSION

The disclosed methods are useful for treating, preventing, or alleviating the severity of diseases associated with low iron levels, for example, the diseases described herein, for example, anemia, as well as for predicting the likelihood of a patient with a disease to respond to treatment with a BMP6 antagonist, for example, antibody 7 (eg, as described in Table 1). These methods use, among other things, determining whether a patient has a particular ferritin level, such as that described herein, in a patient sample.

A biological sample from a patient can be analyzed for ferritin levels using any applicable conventional methods.

Numerous biological samples can be used to identify the presence of a marker, such as proteins, the level of expression of genes or proteins, and protein activity, for example, in blood, synovial fluid, buffy coat, serum, blood plasma, lymph, feces, urine, tears, saliva, cerebrospinal fluid, buccal swabs, sputum or tissue. In the context of a biological sample, various sources may be used in the disclosed methods, for example, a biological sample, such as blood or blood serum, from a patient may be analyzed for ferritin levels.

The authors of the present invention have determined that the level of ferritin can be a useful biomarker in determining or predicting response to therapy with a BMP9 antagonist. Accordingly, one skilled in the art will appreciate that it is possible to identify whether a subject has a target ferritin level by performing an analysis of a biological sample, eg, blood or serum, from the patient for ferritin levels. In preferred embodiments, the patient's serum is analyzed to determine if the subject has a specific ferritin level. In other preferred embodiments, the patient's blood is analyzed to determine if the subject has a particular ferritin level.

As described herein, the present invention is based in part on the finding that ferritin levels may be useful in predicting an improved response to BMP6 antagonism (e.g., antibody 7 as described in Table 1) in the presence of anemia or other disease as described in this document. Detection of ferritin can be performed using any method known in the art, including, without limitation, immunocytochemical staining, ELISA, flow cytometry, Western blotting, spectrophotometry, HPLC, and mass spectrometry. One method for detecting polypeptide products in a sample is with a probe, which is a binding protein capable of interacting specifically with a marker protein (eg, an antibody capable of binding the ferritin protein). Preferably, labeled antibodies, their binding portions, or other binding partners can be used. Antibodies may be monoclonal or polyclonal in origin, or they may be produced by biosynthesis. Binding partners can also be naturally occurring molecules or obtained by synthesis. The number of proteins in the complex is determined using standard protein detection techniques described in the prior art. A detailed review of immunoassay design, theory, and protocols can be found in numerous books in the prior art, including Practical Immunology, Butt, W. R., ed., Marcel Dekker, New York, 1984. A number of assays are available for protein detection using labeled antibodies. Labels for direct measurement include fluorescent or luminescent labels, metals, dyes, radionuclides, and the like attached to the antibody. Labels for indirect measurement include various enzymes well known in the art, such as alkaline phosphatase, hydrogen peroxide, etc. In a one-step assay, polypeptide products, if present, are immobilized and incubated with labeled antibody. The labeled antibody binds to the immobilized target molecule. After washing to remove unbound molecules, the sample is analyzed for a label.

The present invention also contemplates the use of immobilized antibodies specific for proteins or polypeptides. Antibodies can be immobilized on a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay site (such as microtiter plate wells), pieces of a solid material support (such as plastic, nylon, paper), etc. An assay strip can be prepared by coating an antibody or a plurality of antibodies in a row on a solid support. The strip can then be dipped into the test sample and then subjected to rapid processing through the washing and detection steps to form a measurable signal, such as a colored spot.

In a two-step assay, an immobilized marker (eg, ferritin) can be incubated with unlabeled antibody. The unlabeled antibody complex, if present, then binds to a second labeled antibody that is specific for the unlabeled antibody. The sample is washed and analyzed for the presence of the label. The choice of marker used to label antibodies will vary depending on the application. However, the choice of marker can be easily determined by one skilled in the art. Antibodies can be labeled with a radioactive atom, an enzyme, a chromophore or fluorescent fragment, or a colorimetric label. The choice of tag for tagging will also depend on the detection constraints required. Enzymatic assays (types of ELISA) typically provide the ability to detect a colored product resulting from the interaction of an enzyme-labeled complex with an enzyme substrate. Some examples of radioactive atoms include 32P, 125I, 3H, and 14P. Some examples of enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase. Some examples of chromophore moieties include fluorescein and rhodamine. Antibodies can be conjugated to these labels using methods known in the art. For example, enzymes and chromophore molecules can be conjugated to antibodies using binding agents such as dialdehydes, carbodiimides, dimaleimides, and the like. Alternatively, conjugation can be via a ligand-receptor pair. Some suitable ligand-receptor pairs include, for example, biotin-avidin or β-streptavidin and antibody-antigen.

In one aspect, the present invention contemplates the use of a sandwich technique for the detection of polypeptide products in biological samples. The technique requires two antibodies capable of binding to the protein of interest, for example, one immobilized on a solid support and one unbound, in solution but labeled with some easily detectable chemical compound. Examples of chemical labels that can be used for the second antibody include, but are not limited to, radioisotopes, fluorescent compounds, and enzymes or other molecules that form colored or electrochemically active products when exposed to an enzyme reagent or substrate. When samples containing polypeptide products are placed in this system, the polypeptide products bind to both the immobilized antibody and the labeled antibody. The result is an immune complex in the form of a "sandwich" on the surface of the substrate. The complexed protein is detected by washing off unbound sample components and excess labeled antibody and measuring the amount of labeled antibody complexed with the protein on the surface of the support. The immunological sandwich assay is highly specific and very sensitive, provided labels with proper detection limits are used.

Preferably, the presence of polypeptide products in the sample is detected by radioimmunoassays or enzyme immunoassays, competitive enzyme binding assays, dot blot, western blot, chromatography, preferably high performance liquid chromatography (HPLC), or other assays known in the art. the level of technology. Specific immune binding of an antibody to a protein or polypeptide can be detected directly or indirectly.

Dot blotting is usually practiced by a skilled person to detect the desired protein using an antibody as a probe (Promega Protocols and Applications Guide, Second Edition, 1991, Page 263, Promega Corporation). Samples are applied to the membrane using a dot blot device. The labeled probe is incubated with the membrane and the presence of protein is detected.

Western blotting analysis is well known to the skilled artisan (Sambrook et al., Molecular Cloning, A Laboratory Manual, 1989, Vol. 3, Chapter 18, Cold Spring Harbor Laboratory). In Western blotting, the sample is separated using SDS-PAGE. The gel is transferred to the membrane. The membrane is incubated with labeled antibody to detect the desired protein.

The assays described above include steps such as, but not limited to, immunoblotting, immunodiffusion, immunoelectrophoresis, or immunoprecipitation. In some embodiments, an automated analyzer is used to determine the presence and/or level of ferritin.

The level of ferritin activity can be analyzed using various methods disclosed in the prior art, for example, through the methods set forth in Kochan et al. (2011) Proc Natl Acad Sci U S A. 108(19):7745-50.

For the purpose of comparison, the expression level of ferritin, ferritin protein, or ferritin activity in a patient can be compared with the level of expression of ferritin, ferritin protein, or ferritin activity in a control. The control may be a reference level of ferritin expression, ferritin protein, or ferritin activity obtained from subjects (e.g., anemic patients) known to respond well to treatment with a BMP6 antagonist (e.g., antibody 7 described in Table 1), or subjects known to have poor response to treatment with a BMP6 antagonist (eg, antibody 7 described in Table 1), as appropriate. Control level expression can be obtained from biological samples from reference subjects (i.e., anemic patients who are known to respond well to treatment with a BMP6 antagonist (e.g., antibody 7 described in Table 1), or subjects who are known to have poor response to treatment with a BMP6 antagonist response to treatment with a BMP6 antagonist (e.g., antibody 7 described in Table 1), or it may simply be a numerical standard (e.g., mean, median, range, [+/- standard deviation]) previously obtained from reference subjects In some embodiments, the control is a reference level of ferritin, ferritin protein, or ferritin activity obtained from a subject known to have poor response to treatment with a BMP6 antagonist, and the level of ferritin, ferritin protein, or ferritin activity (depending on case) in the patient is compared to that control. is a reference level of ferritin expression, ferritin protein or ferritin activity obtained from a subject known to have a good response to treatment with a BMP6 antagonist, and the level of ferritin expression, ferritin protein or ferritin activity in the patient to be treated is compared with this control, while a similar (e.g., statistically similar) level of ferritin expression, ferritin protein, or ferritin activity in a patient (compared to a control) provides an indication that the patient will have an increased likelihood of responding to treatment with a BMP6 antagonist (e.g., antibody 7 described in table 7).

When performing any of the methods described herein that require determination of the presence or level of expression of ferritin, ferritin protein, or ferritin activity, such determination can be performed by accessing a data bank that contains sufficient information about the characteristics of the patient to determine whether patient marker (or marker level) of interest. Preferably, the database contains a list of markers and the level of the marker of interest in individuals. The data bank may include an individual's medical history, a medical record, a file (e.g., an ASCII file in a single-level database) accessible by a computer or other electronic or non-electronic medium on which relevant information or genetic data may be stored. In the context of this document, a medical data card is a portable storage device such as a magnetic data card, a smart card that has an embedded processing unit and is sold by vendors such as Siemens of Munich, Germany, or a flash memory card. If the databank is a file accessible by a computer; such files may be located on a variety of media, including a server, a client, a hard drive, a CD, a DVD, a personal digital assistant such as a smartphone, a Palm Pilot, a tape recorder, a zip drive, the internal ROM (Read Only Memory) of a computer, or the Internet, or the World Wide Web. Other storage media available with a computer will be apparent to those skilled in the art.

Typically, once ferritin expression levels, ferritin protein, or ferritin activity have been determined, clinicians or consultant geneticists or patients or other investigators can be informed of the result. In particular, the result can be implemented as information in a transferable form that can be communicated or shared with other researchers, or clinicians, or consultant geneticists, or patients. This form is subject to change and may be tangible or intangible. The result of the tested individual can be implemented in the form of descriptive conclusions, graphs, photographs, diagrams, images, or any other visual form. For example, images of gel electrophoresis results or capture assays may be used in explaining the results. Conclusions regarding ferritin expression levels, ferritin protein, or ferritin activity are also applicable when reporting test results. These conclusions and visual forms may be recorded on tangible media such as paper forms, machine-readable media such as floppy disks, CDs, etc., or intangible media such as electronic media in the form of an email message or website on the Internet or an internal electronic network. In addition, the results can also be recorded in audio form and can be transmitted using any suitable communication system, such as analog or digital cable lines, fiber optic cables, etc., telephone, facsimile, wireless mobile phone, telephone with access to the Internet, etc. All such forms (tangible and intangible) will constitute a "transferable form of information". Thus, information and data about the result of the study can be obtained anywhere in the world and transferred to another place. The test result in a transferable form can thus be imported into the U.S. Accordingly, the present invention also encompasses a method for obtaining information in a transferable form in terms of ferritin expression levels, ferritin protein or ferritin activity in an individual. This form of information is useful for predicting the susceptibility of a patient having a disease described herein, such as anemia, to a BMP6 antagonist, for selecting a course of treatment based on this information, and for selectively treating a patient based on this information.

Disclosed herein are methods for predicting the likelihood that a patient with ferritin protein levels will respond (e.g., respond with increased efficacy, e.g., compared to the general population of patients suffering from the same or similar disease) to treatment with a BMP6 antagonist, comprising: detecting a ferritin level in a biological sample from a patient where the ferritin level described herein, for example, ≤ 1500 ng/mL, indicates an increased likelihood that the patient will respond to treatment with a BMP6 antagonist.

In some embodiments, the method further comprises the step of obtaining a biological sample from the patient, wherein the step of obtaining is performed prior to the step of performing the analysis.

In some embodiments, the biological sample is selected from the group consisting of synovial fluid, blood, blood serum, feces, blood plasma, urine, tears, saliva, cerebrospinal fluid, leukocyte sample, and tissue sample. In some embodiments, the biological sample is blood or blood serum.

In some embodiments, the presence and/or level of expression of ferritin, ferritin protein, or ferritin activity is detected using a technique selected from the group consisting of immunoassays, immunohistochemistry, ELISA, flow cytometry, Western blotting, HPLC, and mass spectrometry.

In some embodiments of the disclosed methods and uses, the BMP6 antagonist is a BMP6 binding molecule or a BMP6 receptor binding molecule. In some embodiments, the BMP6 binding molecule or the BMP6 receptor binding molecule is a BMP6 binding molecule. In some embodiments, the BMP6 binding molecule is an anti-BMP6 antibody, or an antigen-binding portion thereof.

In some embodiments of the disclosed methods and uses, the anti-BMP6 antibody is Antibody 7, such as those described in Table 1.

TREATMENT METHODS AND ROUTES OF APPLICATION OF BMP6 ANTAGONISTS

The disclosed methods allow clinicians to provide personalized therapy for patients suffering from the disease described herein, for example, patients with anemia, i.e., they provide the ability to determine whether to prescribe a patient selective treatment with a BMP6 antagonist (for example, antibody 7 , for example, which is described in table 1). Thus, the clinician can maximize the beneficial effects and minimize the risk of BMP6 antagonism in an entire population of patients affected by the disease described herein, such as anemia. It will be understood that BMP6 antagonists, e.g., BMP6 binding molecules (e.g., an anti-BMP6 antibody or antigen-binding portion thereof, e.g., antibody 7, e.g., which is described in Table 1) or BMP6 receptor binding molecules (e.g., an anti-BMP6 receptor antibody or an antigen-binding portion thereof) are useful for treating, preventing, or alleviating the severity of the disease described herein, such as anemia (e.g., signs and symptoms, etc.) as disclosed herein, in particular in patients which have the ferritin level described in this document.

EXAMPLES

The following examples are provided to further illustrate the present invention and not to limit its scope. Other embodiments of the present invention will be fully apparent to those skilled in the art and are covered by the appended claims.

EXAMPLE 1 In vitro and in vivo activity and PK/PD of anti-BMP6 antibodies

materials

Test compounds were antibodies 5, 6, and 7 (Table 8) at ~8 mg/mL in 50 mM citrate buffer, pH 7.0, 150 mM NaCl, and diluted in PBS prior to administration to the animal. Male C56BL/6 mice or Sprague Dawley rats were used (Table 9).

Table 8. Properties of anti-BMP6 antagonist antibodies

Antibody ID frame KD (nM) BMP6 IC50 (µg/mL) of BMP6 reporter ANTIBODY 5 VH3_15, Vl1 0.1 0.06 ANTIBODY 6 VH3_15, Vl1 <0.1 0.08 ANTIBODY 7 VH3_15, Vl1 0.1 0.07

Table 9. Characteristics of animals

View Breed Category Provider Floor Age Mouse (Mus musculus) C57BL/6 wild type Jackson Laboratory, Bar Harbor, Maine Male 8-9 weeks Rat (Rattus norvegicu) Sprague Dawley wild type Charles River Laboratory, Wilmington, Massachusetts Male 8-12 weeks

For BMP reporter gene assays, a lentiviral vector was constructed containing the BMP sensitive element BRE in the promoter [Korchynskyi et al. 2002. J. Biol. Chem. 277: 4882-91] controlling firefly luciferase derived from pGL4-BRE2-Luc2. The lentiviral virus was used to stably transfect the HEP3B hepatoma cell line. The cell line was maintained in EMEM using 10% fetal bovine serum, 1% penicillin/streptomycin, and 5 μg/ml blasticidin. Recombinant human BMP proteins were purchased from R&D Systems.

Brucella abortus ring test antigen (strain 1119-3) in 60 ml flasks was purchased from USDA, Animal and Plant Health Services, National Veterinary Laboratories, Ames, Iowa. Brucella antigen for the ring test contains a suspension of destroyed stained cells of B. abortus strain 1119-3 in a phenolized buffer. The concentration in each 60 ml flask is approximately 10 ⁹ particles/ml. The original solution 5×10 ⁹ was washed and received as follows. First, the 60 ml flasks were removed from the refrigerator and thoroughly mixed. Then 500 ml of BA was transferred into a 500 ml centrifuge flask. They were then centrifuged at 10,000 rpm. within 15 minutes using an ultracentrifuge. The supernatant was removed and resuspended in 100 ml PBS, resulting in a stock solution of 5×10 ⁹ particles/ml, which was aliquoted and frozen at -80°C.

Animal conditions

Animals were housed together in microisolated hard bottom cages during acclimatization and study periods. Animals were maintained under a standard light cycle as follows: 12 hours dark, 12 hours light (lights on at 6:30 am, lights off at 6:30 pm) at room temperature 21-23°C and humidity 30-70%. During acclimatization and study periods, animals were provided ad lib access to rodent diet and water.

Experimental conditions

Determination of antibody activity in the BMP reporter gene assay

In a typical assay, 0.6×10 ⁴ BRE-Luc2 HEP3B cells were seeded in 384-well plates in 25 μl of basic culture medium, in addition to serum being restored to 2%. The next day, antibodies diluted in PBS were added, after which BMP6 was added to a final concentration of 10 ng/ml. The volume was adjusted to 50 μl with EMEM medium without any serum, resulting in a final serum concentration of 1%. As opposite assays, BMP2/4/7 activation was performed in parallel. A BrightGlo assay (Promega) was performed 24 hours after antibody addition according to the manufacturer's instructions using an Envision plate reader (PerkinElmer). Data were calculated as the percentage inhibition of each antibody compared to total reporter activation by the control antibody.

Pharmacokinetic study of a single dose of an antibody in the rat

The rat triage PK study did not involve the determination of classical PK parameters with definite statistical significance, but instead an estimate of the serum half-life of the antibody under study. 3 animals received one IV dose of the antibody.

In the case of the PK/PD dose-response study in mice, the animals were equally divided into 2 separate cohorts. Each cohort included both vehicle-treated mice and compound-treated mice. One cohort was analyzed on day 2.4, while the second cohort was analyzed on day 6.8 after antibody injection. The reason for dividing the cohorts was to reduce the need for periodic blood sampling, so that the effect on serum iron parameters was kept to a minimum. Animal groups are presented in table 10.

Table 10. Design, placement of animals and doses of study drugs

Experiment Group Quantity Dose
(mg/
kg, IV) Frequency PK/PD in mice Control hIgG 5 0.5 once 0.05 mg/kg ANTIBODY 6 5 0.05 once 0.1 mg/kg ANTIBODY 6 5 0.1 once 0.5 mg/kg ANTIBODY 6 5 0.5 once Anemia in mice treated with BA Imitation (no BA) 6 0 0 BA, EPO+ control hIgG 6 2 once BA, EPO+ ANTIBODY 5 6 2 once BA, EPO+ ANTIBODY 6 7 2 once BA, EPO+ ANTIBODY 7 6 2 once

Occurrence of anemia due to inflammation in mice and therapeutic treatment

5×10 ⁸ BA particles for injection were prepared as follows (example in case of 10 mice). The initial concentration should be 2.5×10 ⁹ particles/ml, since 200 μl/mouse was administered. The stock solution was diluted 2-fold with PBS. For example, for 10 mice multiplied by 0.200 µl, 2 ml + 20% of the allowable excess = 2.2 ml of 2.5×10 ⁹ particles/ml was required. 1.1 ml BA stock solution + 1.1 ml PBS. It has been shown that administration of BA 1-8 days prior to ESA treatment resulted in an attenuated HGB response after 6-7 days.

C57BL/6 mice were injected with BA (3×10 ⁸ particles/mouse) and serum IL6 levels were measured 5 hours after ELISA (KMC0061, Life Technologies) to determine the inflammatory response. Animals with an IL6 concentration below the 95% confidence interval of the mean for BA-treated animals were excluded from the study, resulting in fewer than 5-6 mice in some groups. This exclusion process was performed to reduce the likelihood of false positive results resulting from the inclusion of animals that did not have sufficient inflammation to attenuate the ESA response. Following the exclusion process, mice were given IV antibodies as indicated on day 6 and EPO (100 g/kg sc, darbepoetin alfa, Amgen) was administered at 100 mg/kg on day 7 relative to BA treatment. The response to ESA as well as the results of antibody therapy were measured after 6 days.

Pharmacokinetic, pharmacodynamic and efficacy endpoint analyzes

For PK/PD studies in mice and rats, serum samples were collected at the indicated time points after antibody injection. Serum aliquots were used to determine the concentration of circulating antibodies using a high throughput automated immunoassay system (Gyros) with a biotinylated anti-IgG antibody as the primary capture antibody. A second aliquot of serum from each sample was used for quantitative colorimetric analysis of iron (Quuntichrom, DIFE-250, Bioassay Systems). A third aliquot was processed for quantification by LC-MS of the rat or mouse hepcidin-25 peptide according to the modified procedure described previously. Li et al. 2009. J. Pharm. Tox. Meth. 59:171-80.

In the case of BA-induced anemia and the study with antibody treatment, the final blood draw in EDTA-coated BD Microtainer tubes was performed at the end of the study by cardiac puncture. In the case of extensive blood tests on the XT-2000iV hematology analyzer, whole blood was used. Efficacy endpoints included HGB, HCT, RETA, and RET-HE.

Statistical analyzes

One-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test was performed to analyze group differences (with p<0.05 considered significant) in hematological parameters. Data are presented as means ± SEM.

results

Biological activity of anti-BMP6 antagonistic antibodies in cellular BMP-dependent transcription assays

All three anti-BMP6 antagonist antibodies 5, 6 and 7 completely suppressed the biological activity of the human BMP6-induced BMP reporter (BRE-luc) in the human hepatoma cell line Hep3B (IC50=0.4 nM vs. 0.3 nM rhBMP6) and, thus were active at an Ag/mAb molar ratio of 1:1 or higher. The antibodies showed good selectivity for the relevant BMP family proteins, including BMP2, 5 and 7, with a window of 500 or more. See fig. 1.

Simultaneous pharmacokinetic and pharmacodynamic profiles of anti-BMP6 antagonistic antibodies in the rat

A single dose pharmacokinetic study with sorting in Sprague Dawley rats was performed for anti-BMP6 antibodies 5, 6 and 7 by IV injection via jugular vein catheter at 10 mg/kg body weight. Comparison of the relationship of total antibody concentration and time (in particular, t _1/2 , MRT) in serum in the case of three antibodies with a standard profile indicated characteristics comparable to typical human IgG (see Fig. 2 and table 11). There was no evidence of target-mediated drug distribution. At this dose, all anti-BMP6 antibodies suppressed serum hepcidin to levels below the detection limit on day 1 post-injection. The sustained strong downregulation of hepcidin expression was still evident at day 16, indicating a long duration of activity. Accordingly, a transient increase in peak circulating iron concentration was observed on day 2 following antibody injection, and levels remained elevated until day 16.

The serum antibody concentration was measured over time after a single antibody injection. Samples were collected 1 hour, 6 hours, 1, 2, 4, 8, 16, 28 days post dose (10 mg/kg, IV).

Table 11. Key parameters of the single dose PK study with triage in rats

Options ANTIBODY 5 ANTIBODY 6 ANTIBODY 7 T _1/2 (days) 9.1 7.8 9.2 _Cmax (µg/ml) 140.7 189.0 146.2 Average contact time (days) 8.6 7.0 6.9

In addition, the total concentration of antibody 7 (both free and BMP6-bound) in serum was measured in rats and cynomolgus monkeys after a single IV injection of antibody 7 (in rats at doses of 10, 3, 1, 0.3, 0, 1 and 0.03 mg/kg; in macaques at 3 mg/kg) at the indicated times by ELISA using LLOQ 46 ng/mL (dashed line) in rats and LLOQ 0.2 µg/mL (dashed line) in macaques . The results are shown in FIG. 9 (rat) and 12 (monkey).

Dose-dependent response to serum iron parameters following anti-BMP6 antibody treatment in mice and cynomolgus monkeys

To further determine the dose-dependent response of iron metabolism to anti-BMP6 antibody treatment, C57BL/6 naive mice were dosed incrementally with antibody 6 ranging from 0.02 to 0.5 mg/kg as indicated. Antibody 6 was chosen as representative of 3 antibodies because it has a similar backbone, rodent PK profile and in vitro activity. A single dose of 0.5 or 0.1 mg/kg significantly suppressed the serum level of hepcidin and correspondingly increased the serum iron concentration 2 days after treatment. However, only at 0.5 mg/kg was a marked sustained effect on iron metabolism up to 8 days post-injection observed. See fig. 3. These results indicate that dose-dependent saturable neutralization of the target can be easily achieved with potent BMP6 antagonist antibodies.

See fig. 3, dose-dependent effects of anti-BMP6 antibody on serum biomarkers of iron metabolism, top: serum hIgG concentration versus time after a single IV injection of antibody 6 at indicated doses, bottom: left panel is a quantitative analysis of serum hepcidin concentration after single injection of antibody 6 or control human IgG, while the right panel is the serum iron concentration.

Similar experiments were performed with antibody 7. Dose- and time-dependent suppression of circulating hepcidin in the blood serum by antibody 7 was studied in male Sprague-Dawley rats. Serum samples were collected at 0.25, 1, 2, 6 hours and 1, 2, 4, 7 and 14 days after administration of a single dose of antibody 7 by IV administration at a dose ranging from 0.03 mg/kg to 10 mg/kg. Serum hepcidin levels were measured by LC/MS using LLOQ=9 ng/mL. Serum iron levels were also measured in the same animals. The results are shown in FIG. eleven.

These results indicate that the anti-BMP6 antibodies of the present invention are capable of inducing a dose-dependent increase in serum iron levels. The effects were stable and stable for at least 2 weeks after administration of the antibody.

Effects on serum iron parameters in response to anti-BMP6 antibody were also investigated in the cynomolgus monkey. Male cynomolgus monkeys received a single intravenous injection of antibody 7 at a dose of 3 mg/kg. On indicated days after injection, serum samples were collected and analyzed for total serum iron (Fe) and hepcidin concentrations. The results are shown in FIG. 13. Data from 3 individual animals are shown (noted on the graph relative to pre-dose baseline levels). Mean values are indicated with an "x" line. An increase in serum iron levels and a suppression of serum hepcidin levels were observed 24 hours after antibody administration, and the effects persisted (relative to pre-dose levels) until the end of the 28-day study. These results indicate that the anti-BMP6 antibodies of the present invention significantly induce hepcidin expression and reduce circulating iron concentration in non-human primates.

Effect of anti-BMP6 antibodies on erythrocyte parameters in inflammation-induced ESA-resistant anemia in mice

Experiments were performed to evaluate the therapeutic utility of anti-BMP6 antibodies in a mouse model of anemia due to inflammation. See fig. 4. Mice treated with Brucella abortus antigen (BA) developed anemia after 6 days. Animals with anemia were treated with anti-BMP6 antibody together with recombinant erythropoietin (EPO), which was started one day later, and the effect of antibody therapy on the progression of anemia was monitored on day 13 relative to BA administration. HGB and HCT values decreased between the start of treatment and day 13, which was stable when treated with EPO alone. The combined anti-BMP6 and EPO antibody treatment effectively restored the EPO response and significantly increased HGB and HCT levels. This effect was associated with a concomitant stimulation of erythropoietin activity, which was reflected by a steady increase in RETA, as well as a restored reticulocyte hemoglobin content, indicating a correction of heme synthesis due to functional iron deficiency in the erythropoiesis compartment.

See fig. 4, therapeutic treatment with anti-BMP6 antibody in a mouse model of ESA-resistant inflammatory anemia. Top: Experimental design in a model of BA-induced ESA-resistant inflammatory anemia. Bottom: Erythropoiesis parameters 13 days after BA treatment. HGB: hemoglobin; HCT: hematocrit; RETA: reticulocyte count; RET-HE: reticulocyte hemoglobin equivalent.

* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to BA+EPO+hIgG1.

EXAMPLE 2 Human Clinical Design for Anti-BMP6 Antibodies

Clinical study plan. Evaluation of therapy using antibodies or antigen-binding fragments thereof that bind human BMP6.

Patients with end-stage kidney disease (ESRD) produce little or no erythropoietin (EPO) production and generally require intermittent exogenous EPO and intravenous (IV) iron infusions to provide EPO-induced Hgb synthesis. Up to a third of patients on chronic hemodialysis (HD) do not respond appropriately to EPO due to predominantly intracellular iron sequestration. Hepcidin is predominantly excreted by the kidneys, and removal by dialysis is ineffective. Thus, patients on chronic HD tend to have significantly elevated levels of hepcidin, which blocks iron mobilization for erythropoiesis. IV iron therapy is no longer effective or recommended when the body's iron stores reach a critical level (as evidenced by high serum ferritin levels). Current guidelines do not recommend IV iron administration to anemic patients on dialysis with high ferritin levels, and thus these patients may receive even higher doses of EPO with the potential associated risk of anemia with decreased EPO sensitivity (Kidney Disease Improving Global Outcomes (KDIGO) Anemia Work Group 2012). Anemia with reduced sensitivity to EPO causes a significantly increased risk of mortality from any cause associated with both anemia and a higher dose of EPO in patients on hemodialysis (Kilpatrick et al 2008, Lopez-Gomez et al 2008, Fukuma et al 2012 ), and patients on peritoneal dialysis (Suttorp et al 2013). Isolated antibodies or antigen-binding fragments thereof that bind human BMP6 of the present invention may benefit chronic kidney disease patients with iron deficiency anemia by increasing hemoglobin (Hgb) levels while reducing the need for EPO and IV iron. A lower EPO resistance index (the ratio of EPO dose to Hgb level) correlates with a lower risk of mortality.

In general, the goal of therapy using isolated antibodies or antigen-binding fragments thereof that bind human BMP6 of the present invention is to mobilize sequestered iron, which may then reduce EPO and iron dose requirements and improve Hgb levels, all of which are expected to improve patient outcomes. This is the first single-dose human study using isolated antibodies or antigen-binding fragments that bind human BMP6. This study will evaluate safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy in a population of patients on chronic hemodialysis. The aim of the present study is to evaluate whether therapy using isolated antibodies or antigen-binding fragments thereof that bind human BMP6 warrants further clinical investigation of anemia associated with chronic kidney disease.

Study plan

Study Design

This is a first human, two-part, non-confirmation study using a single dose of an isolated antibody or antigen-binding fragment thereof that binds BMP6 to evaluate safety, tolerability, PK, PD, and efficacy in the chronic hemodialysis patient population. Part 1 is the first open-label, single-dose titrated study in humans. Part 2 is a randomized, double-blind, placebo-controlled, single dose study that will compare two dose levels of an isolated antibody or antigen-binding fragment thereof that binds human BMP6.

Safety assessments will include physical examination, ECG, vital signs, routine clinical laboratory evaluations (CBC, chemistry, serum iron), monitoring for adverse events and serious adverse events.

Part 1

The objectives of Part 1 are (a) to evaluate single dose safety, PK, PD, and tolerability, and (b) to determine the minimum PAD of an isolated antibody or antigen-binding fragment thereof that binds human BMP6, defined as the minimum dose investigated in Part 1, which results in to an increase in Hgb levels (median change from baseline ~ 0.5 g/dL) 29 days post-dose.

During Part 1, a screening visit will take place to determine the patient's eligibility for participation in the study (Figure 6). Patients eligible for study participation will be identified at the study site and re-evaluated against eligibility criteria at the baseline visit. All baseline safety assessment results should be available and reviewed prior to dosing.

Figure 6 is a summary of the study design for Part 1. Patients will be asked to report to the study site on Day 1, immediately following their standard dialysis visit. Patients will then receive an infusion of an antibody or antigen-binding fragment thereof that binds human BMP6 (the exact dose will depend on the cohort). When possible, dosing should preferably occur on the day of dialysis before the two-day interval between dialysis (eg Friday or Saturday) and will occur after the dialysis session on that day. However, if this is not possible, then dosing may occur on the day of dialysis and not precede the two-day interval between dialysis. Following dosing, the first two patients in Part 1 will be hospitalized for at least 48 hours for safety and PK/PD evaluation. Patients will return to the study site on days 4 and 6 for PK/PD assessment, and then weekly for a total of 29 days for PK assessment, and a total of 12 weeks for safety assessment, with the end-of-study visit occurring approximately on day 85. Study visits, including all laboratory tests except for post-dialysis PK assessment, should take place prior to the scheduled patient visit for dialysis.

Part 1 will start with a dose that is not expected to be pharmacologically active. Dose will be adjusted for each subsequent Part 1 cohort based on the median change in Hgb levels for each cohort following a single dose of isolated antibody or antigen-binding fragment thereof that binds human BMP6 and transferrin saturation level (TSAT) as described in the Statistical Evaluation section and shown 7. The goal of this decision tree is to identify the lowest possible dose that induces iron mobilization (as evidenced by transferrin saturation levels (TSAT) >50% observed in at least 4 patients in a cohort of 6 patients one week post-dose ) and increased Hgb levels. If iron is mobilized but Hgb levels do not rise by at least 0.5 g/dl by 29 days post-dose, then clinical data will be analyzed to determine potential confounding factors (eg, blood loss due to excess non-study phlebotomy) . Appropriate investigators and sponsor representative(s) will study adverse events in each cohort and will define these events in the context of (a) known medical problems associated with chronic renal failure and (b) preclinical toxicological findings. Subsequent cohorts will not be dosed until the investigator and sponsor indicate that it is safe to continue the study.

Figure 7 shows the dose adjustment algorithm in Part 1. Blood testing, including Hgb measurements, will be performed prior to dialysis. The initial dose will be 0.01 mg/kg. In Part 1, patients will be assigned to one of up to 6 open dose titration cohorts of up to 6 patients each. The minimum PAD of an isolated antibody or antigen-binding fragment thereof that binds human BMP6 as defined above will be included in the lower dose group for Part 2. The dose for each subsequent cohort can be adjusted higher or lower as shown in Figure 7. If the lowest possible dose (0.001 mg/kg) results in a mean increase in Hgb levels ≥ 0.5 g/dl, then this will be the minimum PAD and the lower dose selected for part 2, and the next maximum dose estimated in part 1, will be included in the higher dose group for Part 2. If the maximum dose (0.1 mg/kg) estimated in Part 1 represents the minimum PAD, then Part 2 will continue with only 2 groups: placebo and minimum PAD . In the event that additional dose cohorts are needed in Part 1, these cohorts will be added as described in this document.

Each Part 1 cohort will include 6 patients. The first 2 patients in the first cohort of Part 1 will be dosed at least 7 days apart. Timing of subsequent doses for patients in the Part 1 cohorts will occur as the respective research site schedule and support resources allow. All Part 1 patients will be followed up for 12 weeks post-dose.

Part 2

The objectives of Part 2 are (a) to assess safety, PK, PD, and tolerability, and (b) to determine efficacy based on changes in Hgb levels in response to a single dose of an antibody that binds BMP6 compared to placebo. Part 2 will include no more than three groups: no more than two Ab dose groups and one placebo group (Figure 8). Two Ab dose groups will be generated from the data obtained in Part 1. Part 2 will include approximately 60 patients randomized into three groups 1:1:1. If in Part 1 the minimum PAD is also the maximum dose assessed (0.1 mg/kg), then in Part 2 there will be only two groups: minimum PAD and placebo. In this case, 40 patients will be randomized into two groups with a randomization ratio of 1:1. The sample size of Part 2 can be adjusted based on the variability in change from baseline Hgb in Part 1.

Figure 8 shows the study design for Part 2. During Part 2, there will be a screening visit to determine the patient's eligibility for participation in the study. Eligible patients will be re-evaluated for eligibility at the baseline visit. All baseline safety assessment results should be available and reviewed prior to dosing.

Patients will be asked to report to the Study Center on Day 1, immediately following their standard dialysis visit. Patients will then receive either an Ab infusion or placebo as determined by allocation according to randomization. When possible, dosing should preferably occur on the day of dialysis before the two-day interval between dialysis (eg Friday or Saturday) and will occur after the dialysis session on that day. However, if this is not possible, subsequent dosing may occur on the day of dialysis and not precede the two-day interval between dialysis. Patients will return to the study center on days 4 and 6, then weekly for follow-up evaluations. During follow-up visits, patients will undergo standard safety assessments and PK data will be collected at day 85. Study visits may take place after the patient's scheduled dialysis-related visit to fit the patient's dialysis schedule. All patients included in Part 2 will be followed up for 12 weeks post dose Ab (or placebo).

EPO dose control (both parts)

Adjustment of individual doses of EPO during both parts will be monitored according to the standard treatment protocol of each dialysis research center. Study site protocols will be reviewed as part of the clinical site evaluation and will be reviewed for compliance with treatment standards (KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease Anemia Work Group 2012). Patients who achieve Hgb levels ≥ 13 g/dl at any time during the study may be monitored with therapeutic phlebotomy, at the discretion of the investigator, in addition to site-specific guidelines for controlling Hgb values above target levels.

Control of intravenous iron (both parts)

Patients receiving IV loading doses of iron (100 mg/week) will be excluded from the study. Patients receiving weekly IV iron maintenance (<100 mg/week) may be included in this study. The weekly IV iron maintenance dose will be maintained at the start of the 1st week of Ab dosing. Iron values will be monitored during the first week after the administration of Ab doses and iron backup therapy and IV iron control will follow the standard of care in accordance with the protocol of the hemodialysis control unit. Study site protocols will be reviewed as part of the clinical site evaluation and will be reviewed for compliance with treatment standards (KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease Anemia Work Group 2012).

Study Design Rationale

Rationale for the design of the two-part study

The rationale for having two parts in the same patient population is to identify the safety and efficacy of minimal PAD aimed at minimizing the number of patients and cohorts exposed to potentially subtherapeutic doses. Part 2 will determine the efficacy of the minimum PAD and one dose level above the minimum PAD (as defined in Part 1) compared to the placebo group.

Part 1 was designed to evaluate safety, tolerability, single dose PK/PD, and minimal PAD Ab in an open study. The minimum PAD will be determined based on the median change in each Hgb dose cohort 29 days after the Ab dose. The rationale for defining PAD criteria is that it may take up to 4 weeks for clinically significant responses to EPO after changing the dose of EPO. If Ab mobilizes iron in the target population, then this may help the patient's current dose of EPO have a more sustained erythropoietic effect. The 29-day Hgb ranges given in defining the PAD criteria are based on clinically relevant and safe values for the rate of increase in Hgb levels in response to an EPO dose (~0.5 g/dL over 29 days). The rationale for looking for a minimum PAD rather than a maximum effect is that an overly sustained Hgb response is a safety risk in this patient population, as reflected by the Hgb target ranges in the current standard of care (Kidney Disease Improving Global Outcomes (KDIGO) Anemia Work Group). 2012). The objectives of the safety and tolerability assessments in Part 1 are (a) to identify safety signals, and (b) to communicate dose adjustment decisions so that the doses selected for Part 2 (lowest PAD+1 higher dose) are appropriate for further evaluation. both safety and efficacy relative to placebo. While Part 1 is not power enough to provide an unbiased estimate of safety, the placebo group and larger sample sizes in Part 2 will provide an unbiased estimate of safety at the lowest PAD and one higher dose. In addition to safety, tolerability, and PK/PD, Part 2 was designed to evaluate efficacy versus placebo in a double-blind study. Efficacy evaluation will be based primarily on Hgb along with the EPO resistance index (ERI=weekly weight-adjusted dose of EPO divided by Hgb) as a key secondary evaluation criterion. The ERI provides a quantitative measure of the amount of EPO required to achieve a given Hgb value and thus provides clinically important information in addition to Hgb alone.

Rationale for FIH in Dialysis Patients

This first-in-human (FIH) study will be conducted in patients on chronic hemodialysis (HD) rather than healthy volunteers (HV). Evaluation of the safety, tolerability and PK/PD response to Ab to human BMP6 in HV does not appear to be transferable to chronic HD patients for several reasons.

In contrast to HV, chronic HD patients with anemia, high serum ferritin, and low TSAT have chronically accumulated intracellular iron stores. Thus, the safety, tolerability, and pharmacological effects associated with iron mobilization in response to low doses of Ab are most appropriately assessed in patients with chronic HD. In HV patients with normal renal function, hepcidin (of which BMP6 is the main regulator) is filtered by the kidneys and efficiently excreted in the urine, resulting in low circulating levels. In contrast, dialysis filters hepcidin less efficiently and transiently, resulting in higher circulating levels in chronic HD patients (Zaritsky et al 2010). In addition, normal kidneys will dynamically adjust endogenous EPO levels and changes in Hgb levels may not be apparent in response to Ab. Thus, the safety and tolerability associated with the modulation of hepcidin by the BMP6 pathway and the effect of Ab on Hgb are most appropriately assessed in patients with chronic HD.

Rationale for the target patient population

This study was designed to evaluate Ab to human BMP6 in an iron-deficient anemic setting with reduced sensitivity to EPO. Common clinical practice guidelines (KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease Anemia Work Group 2012) define EPO deficient sensitivity as a requirement for two dose increases of EPO, 50% above the stable dose, to maintain a stable Hgb concentration. The proposed eligibility criteria are designed to select patients on stable chronic HD with anemia and clinical symptoms of iron deficiency: elevated ferritin and low TSAT (TSAT=serum iron/total iron-binding capacity; TSAT correlates very strongly with serum iron ). In addition, when adjusting EPO and IV iron doses, strict treatment standards will be adhered to in the case of Hgb, TSAT and ferritin targets. This design reduces the risk of deviation from the required Hgb targets, as changes in iron parameters and hematological parameters will continue to be monitored according to standard of care. In addition, patients receiving loading doses of IV iron within 1 week prior to baseline will be excluded. Patients receiving maintenance doses of IV iron may be included (if all other eligibility criteria are met). The rationale for including these patients is that the current standard of care in the US dictates that Hgb and TSAT be maintained within narrow limits, and thus completely withholding iron maintenance therapy to meet eligibility criteria at a lower TSAT would put patients at risk for TSAT. below 25%, necessitating a course of loading IV doses of iron supplements in accordance with the standard of care. However, study eligible patients receiving IV iron maintenance therapy will have their weekly IV iron dose maintained at the start of the week of Ab dosing and will resume IV iron maintenance therapy only as determined by the study center standard of care protocol. , based on iron values.

Justification of the dose / regimen, duration of treatment

Justification of the initial dose

The maximum recommended starting dose (MRSD) was calculated based on the No Adverse Event Level (NOAEL) based on 13 weeks (14 doses) GLP toxicology studies conducted in rats and cynomolgus monkeys. Animals received weekly IV bolus doses of 0.1, 1, 10, and 100 mg/kg. The 1 and 100 mg/kg (only) dose groups were subsequently followed for 16 weeks in the case of rats or 24 weeks in the case of cynomolgus monkeys after the last dose of isolated antibody or antigen-binding fragment thereof that binds human BMP6. MRSD was assessed first by calculating human equivalent dose (HED) for NOAEL from these studies (0.1 mg/kg), an approach considered appropriate for drugs with a molecular weight >100 kDa, and then using a safety factor of 10 , to explain differences between species in preclinical studies and patients, such as the amount of stored iron and the need for erythropoiesis. The PK parameters for preclinical species were derived from toxicokinetic (TK) data collected during IND-providing toxicology studies. The corresponding PK parameters in patients were then assessed by allometric scaling and these parameters were used to predict the concentration of free or isolated antibody or antigen-binding fragment thereof that bind human BMP6 over time in patients at a given dose. Comparison of TK data from toxicology studies with PK Ab based on a patient model indicated that a dose 10 times lower than NOAEL/HED would presumably result in a (minimum) 10-fold limit based on Ab concentration.

Peak serum iron levels observed in response to Ab in animal studies may underestimate the predicted human iron iron response to Ab because patients on HD who are on IV iron therapy have higher iron stores in tissues than healthy animals. However, unlike healthy animals without anemia, HD patients are expected to use released serum iron for erythropoiesis; thus, animal models may underestimate the duration of iron elevations. The liver pathology observed in the 13-week studies was not observed in the 4-week studies, indicating that the toxicity is a consequence of cumulative serum iron exposure and not a response to acute iron release.

MRSD was also predicted based on analysis using a model of serum iron concentrations to explain expected differences in stored iron between species in preclinical studies and patients. The cumulative serum iron exposure that resulted in toxicological results was presented as the area under the iron curve (Fe AUC). In this approach, Fe AUC calculated for a dose of NOAEL (0.1 mg/kg) was considered to be reasonably safe. Fe AUC was then predicted in proposed MRSD in patients and compared with Fe AUC in preclinical species in NOAEL. Because the model predicted serum iron exposure in MRSD in patients was >10-fold less than in NOAEL in preclinical species, a reduction in NOAEL/HED by a safety factor of 10 was considered sufficient to assess MRSD. Thus, the estimated MRSD was 0.01 mg/kg.

Rationale for Dose Adjustment

In the case of this study, the maximum study dose ( xmax ) will be the HED corresponding to the NOAEL in each of the 2 IND-providing toxicological studies: 0.1 mg/kg. The minimum possible investigational dose (xmin) is the lowest technically feasible dose based on compatibility studies: 0.001 mg/kg. MRSD (x0) will be evaluated according to safety criteria, TSAT and Hgb (figure 7). If x0 results in a median change in Hgb < 0.5 g/dl from predose, then the choice of x+ ( Figure 7) will be controlled by linear extrapolation on a natural log base scale (expecting a sigmoidal dose-response relationship) from x0 to xmax . Provisional doses for this dose escalation are presented above. These preliminary doses can be adjusted based on data review during informal interim analyzes between each cohort. This approach will continue until PAD is detected or xmax is reached. If xmax results in a median change in Hgb < 0.5 g/dl, then the safety of that dose will be assessed and a decision will be made as to whether the protocol needs to be modified to add additional cohorts at doses greater than xmax based on safety data, PK and PD. If the maximum study dose in Part 1 results in a mean increase in Hgb < 0.5 g/dL, does not increase TSAT above 50%, and is below xmax , then the protocol may be modified to add additional cohorts.

If x0 instead results in a median change in Hgb ≥ 0.5 g/dl from pre-dose, then the dose for the next cohort will be adjusted to xmin . If xmin also results in a median Hgb change ≥ 0.5 g/dL, then xmin will be considered a minimal PAD and xmin and x0 will be assessed in Part 2. If xmin results in a median Hgb change < 0.5 g/dL and TSAT ≤ 50% (figure 7), then doses will increase by linear extrapolation in the interval ( xmin , x0 ) on a base scale of natural logarithms until either the minimum PAD is detected or 6 doses (cohorts) are evaluated . Preliminary doses in the interval ( xmin , x0 ) are presented above. These preliminary doses can be adjusted based on data review during informal interim analyzes between each cohort. The minimum PAD will be defined as the lowest study dose that results in a median change in Hgb levels ≥ 0.5 g/dl from pre-dose status.

Ab will be administered as a single dose IV infusion in order to provide less serum iron exposure (Fe AUC) than that associated with adverse outcomes in preclinical toxicology studies. Ab solution will be infused immediately after the hemodialysis session on day 1, minimizing the potential effect of dialysis on PK or iron bioavailability immediately after dosing. An additional approximately 30 minutes of dosing after dialysis (on the day of dosing only) is not expected to create any significant risk or discomfort to patients.

Rationale for choosing a comparator drug

Placebo is used as a comparator in Part 2 to provide an unbiased assessment of clinical outcomes.

Purpose and timing of interim reviews/design adaptations

In Part 1, after each cohort of 6 patients completes evaluation at week 4 post-dose, an informal interim review will be conducted to decide on a dose adjustment for the next cohort. Safety markers and PDs will be reviewed by all members of the dose adjustment team, including the appropriate investigators and sponsor representative(s). New cohorts will only be formed if safety and tolerability is confirmed and if the PD conditions are met, as described in Figure 7. No more than 5 informal interim analyzes will be conducted in Part 1. An informal interim review is scheduled after all patients in the final Part 1 cohort complete evaluation at week 4 post-dose to assess the clinical effects of study doses and potentially initiate additional non-clinical studies, and may report on subsequent clinical studies. Body temperature, blood pressure, pulse rate, ECG score, blood chemistry, iron hematology, EPO resistance index, and adverse events obtained by day 29 of the last part 1 cohort will be included.

The minimum PAD and a dose one level higher than the minimum PAD will be selected for Part 2. If the lowest possible investigational dose induces an increase in Hgb ≥ 0.5 g/dL, then the two lowest investigational doses will be selected for Part 2.

Risks and Benefits

Potential benefit for patients in this study may include reduced EPO and IV iron requirements and improved Hgb levels during treatment and for some period thereafter.

Risk to patients in this study will be minimized by adhering to eligibility criteria and careful clinical observation of all patients (and hospitalization of the first two patients in Part 1) within the first 48 hours after Ab administration.

Potential risks associated with iron mobilization include (a) redistribution of iron to tissues and organs such as the spleen, liver, heart, pancreas, and pituitary gland, and (b) a slight increase in susceptibility to bacterial infection, particularly in patients with implanted vascular catheters. Several of the eligibility criteria reduce the risk of complications. Elevated levels of liver function tests may be seen in association with iron redistribution. Liver function can be monitored in parallel with hematological and iron parameters. Deviation from standard treatment of Hgb targets can lead to polycythemia. May monitor Hgb, EPO and iron therapy.

HD patients treated with IV iron may have higher tissue stores of iron than healthy animals; thus, peak serum iron levels observed in patients treated with isolated antibody or antigen-binding fragment thereof that bind human BMP6 may exceed those observed in animal studies. However, unlike healthy animals, HD patients are expected to use released serum iron for erythropoiesis; thus, animal models may underestimate the duration of iron elevations. The model-predicted Ab exposure (eg, Cmax, AUC) in MRSD is expected to be 10-fold less than that observed in NOAEL in preclinical studies. Such exposure is not expected to result in serum iron (AUC) exposure levels associated with elevated liver transaminases and single cell necrosis in the liver observed in preclinical studies. Dose escalation of Ab relative to NOAEL will occur after safety evaluations. Clinical experience in patients with chronic iron overload, as well as in those receiving parenteral iron, is probably not necessarily predictive of the effects that may occur due to the Ab-induced acute elevation of intracellular iron. Thus, the potential risk of Ab-induced acute iron toxicity is likely to be low. Acute iron toxicity can affect the heart, liver and/or pancreas. Clinical manifestations of acute iron toxicity may include cardiac conduction disturbances, elevated liver transaminases, and impaired glucose tolerance/hyperglycemia. Severe acute iron toxicity may also include metabolic acidosis, electrolyte disturbances, and neurological manifestations. When acute iron toxicity occurs, patients can be promptly treated with iron chelators such as deferoxamine in combination with hemodialysis. A maximum of 134 ml (Part 1) and 172 ml (Part 2) of blood is planned to be collected over a period of 115 days from each patient as part of the study. Additional samples to observe any safety results will be collected in addition to this. This is not considered a risk to this population.

To date, reproductive toxicity studies using antibodies to human BMP6 have not been performed. Potential effects on the male or female genital organs were determined using a rigorous standard histopathological examination of the ovaries and testes and accessory genital organs in a 13-week toxicity study in cynomolgus monkeys. No treatment-related effects were observed. BMP6 knockout mice were characterized by delayed sternum ossification and iron overload (Meynard et al 2009).

Significant intrauterine and maternal morbidity and mortality are associated with chronic hemodialysis. In one retrospective cohort study comparing women on chronic hemodialysis (267 births) with women receiving a kidney transplant (264 births), women on hemodialysis had higher rates of placental abruption, blood transfusion, neonatal malnutrition and maternal mortality (Saliem et al 2015). Thus, women of childbearing potential should use highly effective contraception to prevent pregnancy during administration of an isolated antibody or antigen-binding fragment that binds human BMP6 and for 125 days after the last dose.

population

The study population will consist of patients with end-stage kidney disease who require chronic hemodialysis therapy at least twice a week and who have clinical evidence of anemia due to functional iron deficiency, defined as anemia in the presence of apparently adequate iron stores, determined by the levels of ferritin and transferrin saturation. Part 1 includes a plan to evaluate up to 36 patients initially in 6 cohorts (6 patients/cohort). If no TSAT and Hgb effects are seen after 6 cohorts and there are no safety concerns (as determined by the respective investigators and sponsor representative(s), then no more than 2 additional cohorts of 6 patients can be added (total of 48 patients in Part 1) . Part 2 consists of 3 groups (2 dose levels selected for additional evaluation from Part 1 and a placebo group) with no more than about 20 patients per group (60 patients in Part 2 in total). Thus, a total of approximately 96 patients (up to a maximum of 108) are planned to be enrolled, of which approximately 60 will be randomized in Part 2. Approximately 60 patients (12 in Part 1, 48 in Part 2) are expected to complete the study. The investigator must ensure that all patients considered for participation in the study meet the following eligibility criteria. No additional criteria should be used by the investigator to ensure that the study population is representative of all patients eligible to participate in the study.

Patient selection is done by testing all eligibility criteria at screening and first baseline. An appropriate record (eg checklist) of all suitability criteria should be kept with the primary documentation at the study site.

Deviation from any inclusion criterion excludes the patient from inclusion in the study.

Inclusion Criteria (both parts)

Patients eligible to participate in this study must meet all of the following criteria.

Written informed consent must be obtained before any evaluation can be carried out. If consent cannot be expressed in writing, it should be formally documented and witnessed, ideally by an independent trusted witness.

Age at screening ≥ 18 years.

Dependence on hemodialysis for at least 2 months prior to screening.

Receiving appropriate hemodialysis at least 2 times a week in case of end-stage kidney disease; compliant is defined as Kt/V ≥ 1.2 at the most recent monthly assessment before screening.

Receiving chronic erythropoietin (EPO) therapy in accordance with the dialysis research center's anemia management protocol. The EPO dose did not increase by 50% or more within 14 days to baseline. EPO therapy should only include a fast-acting formulation (not darbopoetin) and be administered IV (not SC).

Hgb ≥ 8.5, including Hgb ≥ 8.5 and < 11.5 g/dL, and not increased by ≥ 0.5 g/dL at baseline compared to the previous 14 days.

Ferritin <1500 ng/mL (inclusive) for at least 28 days before baseline (may include screening). Alternatively, ferritin > 500 ng/mL and ≤ 1000 ng/mL at screening.

TSAT ≤ 30% at at least one time point during the 90-day period to baseline and TSAT ≤ 30% at baseline.

Exclusion criteria (both parts)

Patients meeting some of the following criteria are not eligible for inclusion in this study. No additional criteria may be used by the investigator to ensure that the study population is representative of all patients eligible to participate in the study.

1. Use of other investigational medicinal products within 5 half-lives of entry into the study, or until the expected pharmacodynamic effect returns to baseline, whichever is longer.

2. History of hypersensitivity to study drug or therapeutic antibodies.

3. Confirmed diagnosis of hemochromatosis.

4. Confirmed malignant tumor of the bone marrow, malignant tumor of the lymphatic system or myelodysplastic syndrome.

5. History of dialysis with AV fistula thrombosis within 2 months prior to screening or 2 or more episodes of AV fistula thrombosis within 6 months prior to screening.

6. Severe comorbid disease/liver dysfunction (Child-Pugh score ≥ 6) or previous liver transplant 7. Heart failure (functional class III or IV according to the New York Heart Association (NYHA)).

8. Gastrointestinal bleeding requiring intervention within the last 2 months prior to screening. Patients with hepatitis C virus (HCV) infection may be included if all other eligibility criteria for hepatic function are met.

9. ALT, AST or bilirubin ≥ 1.5x ULN within 4 weeks to baseline.

10. Uncontrolled nephrogenic osteodystrophy defined as PTH ≥ 750 pg/mL at screening.

11. Conditions that predispose to an increased risk of serious infection, such as an implanted vascular catheter (central venous catheter or hemodialysis catheter) or an active infection requiring antibiotic therapy at any time within 2 weeks prior to screening.

12. Blood transfusion given within 4 weeks to baseline.

13. Receive a loading dose (100 mg/week) of IV iron for 1 week to baseline.

14. History of drug and alcohol abuse within 12 months prior to dosing, or symptoms of such abuse as evidenced by laboratory tests performed at screening.

15. Positive test result for hepatitis A surface antigen.

16. History of immunodeficiency diseases, including a positive HIV test result (ELISA and Western blotting).

17. Women of childbearing potential may be included in this study if highly effective contraception is used for at least 125 days after a dose of an antibody or antigen-binding fragment that binds human BMP6. Highly effective contraception is defined as one of the following: a. total abstinence (in case it is in accordance with the patient's preferred or usual lifestyle; intermittent abstinence (eg, calendar, ovulation, symptomatic, post-ovulation methods) and withdrawal are not acceptable methods of contraception); b. sterilization of a man/woman; With. use of oral, injectable or implantable hormonal methods of contraception or insertion of an intrauterine device (IUD) or intrauterine system (IUS) or other forms of hormonal contraception that are comparable in effectiveness (<1% failure rate), such as a hormonal vaginal ring or transdermal hormonal contraception.

Treatment

Research treatment

The investigational therapy in this study is an antibody or antigen-binding fragment that binds human BMP6, eg anti-BMP6 IgG1, a fully human antibody. The antibody is provided in a liquid solution. The initial concentration will be diluted at the research center according to the dose to be administered. The infusion time will be kept relatively constant across all cohorts at about 30 minutes. Part 1 will be a single dose open label study and Part 2 will be a double blind single dose study versus a matched placebo (vehicle control). Anti-human BMP6 Ab and placebo will be supplied as liquid in vials. The excipients in the active substance and placebo are identical.

Treatment groups

In Part 1, patients will be assigned to one of up to 6 dose titration cohorts of 6 patients each. Part 1 is an open treatment. The starting dose, maximum dose, and rationale for dose adjustments are described above. Preliminary doses for Part 1 are shown in Table 12 (Hgb < 0.5 g/dL for MRSD) and Table 13 (Hgb ≥ 0.5 g/dL for MRSD).

Table 12 Predose Levels for Part 1

If Hgb is less than 0.5 g/dl at the MRSD dose level Predose Increase in previous dose 1 (MRSD) 0.010 mg/kg Initial dose 2 0.016 mg/kg 60% ↑ 3 0.025 mg/kg 60% ↑ 4 0.040 mg/kg 60% ↑ 5 0.063 mg/kg 60% ↑ 6 (NOAEL) 0.100 mg/kg 60% ↑

This table is provided as an example of dose adjustment in Part 1 for guidance only. Intermediate or higher dose levels may be used, and some dose levels may be omitted based on data evaluation during informal interim analysis between each cohort. Actual dosage levels may be confirmed in writing by Novartis and made available to all participating study sites prior to treatment of patients in a new cohort.

Table 13 . Predose Levels for Part 1

If Hgb is 0.5 g/dl or greater at the MRSD dose level Predose Increase in previous dose 1 (MRSD) 0.0100 mg/kg Initial dose 2 0.0010 mg/kg 90% ↓ 3 0.0016 mg/kg 60% ↑ 4 0.0025 mg/kg 60% ↑ 5 0.0040 mg/kg 60% ↑ 6 0.0063 mg/kg 60% ↑

This table is provided as an example of dose adjustment in Part 1 for guidance only. Intermediate or higher dose levels may be used, and some dose levels may be omitted based on data evaluation during informal interim analysis between each cohort.

Types of research treatment are defined as follows:

A: single dose placebo.

B: Single dose Ab to human BMP6 at minimum PAD as defined in Part 1.

C: Single dose Ab to human BMP6 at one dose level above the minimum PAD defined in Part 1.

Concomitant treatment

All prescription drugs, over-the-counter drugs, and significant non-drug therapies (including physical therapy and blood transfusions) administered or taken during the period of time specified in the pre-study and during-study enrollment criteria must be recorded See the appropriate section of the CRF "Concomitant Drugs/Significant Non-Medicinal Therapies". Drug administration should be specific in terms of brand, single dose and form, frequency and route of administration, date of initiation and discontinuation, and reason for treatment.

Efficacy/pharmacodynamics

Performance ratings are defined below. Samples for performance evaluations will be collected at various time points. Hematological laboratory parameters will be determined. Hgb and Fe will be examined during each informal inter-cohort interim analysis as part of the dose adjustment assessment during Part 1 of the study. If the sampling time points initially established are found to be suboptimal for understanding the relationship between iron and PK, then the sampling time points may be changed in subsequent cohorts in Part 1.

Iron Dashboard

Ab to human BMP6 is hypothesized to mobilize Fe derived from body stores, leading to changes in serum Fe parameters including: serum Fe levels, transferrin saturation (TSAT), unbound Fe binding capacity (UIBC), total Fe binding capacity (TIBC), ferritin level and hemoglobin content in reticulocytes (CHr). They will be measured in serum using validated assays.

Safety

The safety ratings are defined below.

Physical examination

A complete physical examination will include general appearance, skin, neck (including thyroid), eyes, ears, nose, throat, lungs, heart, abdomen, back, lymph nodes, limbs, vascular system, and neurological system. If necessary, based on medical history and/or symptoms, examinations of the rectum, vulva, breast, and/or pelvis may be performed.

Important findings that are present prior to the start of the study should be included in the "Relevant Medical History/Current Medical Conditions" screening in the patient's eCRF. Significant findings after initiation of the investigational medicinal product that meet the criteria for an "adverse event" should be recorded in the "Adverse Events" screening in the patient's eCRF.

Vital Signs

Body temperature

Blood pressure (BP)

Pulse

Height and weight

Height

Body weight

The body mass index (BMI) will be calculated as (body weight (kg)/[height (m)]2).

Assessments of laboratory indicators

Clinically significant laboratory test abnormalities will be evaluated against the criteria for defining an adverse event and reported as such if they meet the criteria. Reassessments are mandatory until the result(s) normalize or until the change is no longer clinically significant.

General blood analysis

Hemoglobin content, hematocrit, red blood cell count, white blood cell count and platelet count will be measured. Iron levels will be monitored.

Clinical biochemical analysis of blood

Sodium, potassium, creatinine, urea, chloride, albumin, calcium, alkaline phosphatase, total bilirubin, LDH, GGT, AST and ALT will be monitored. If the concentration of total bilirubin rises above 1.5 times the upper limit of normal, then it is necessary to differentiate between direct and indirect bilirubin.

Electrocardiogram (ECG)

PR interval, QRS duration, heart rate, RR, QT, QTc will be measured.

For clinical decision making, the Friederick formula for QT correction (QTcF) must be used.

Pregnancy and fertility assessments

Pregnancy tests are required for all female patients regardless of reported reproductive/menopausal status.

In this study, pregnancy tests based on blood serum will be performed. In case of a positive result, the patient must terminate the study ahead of schedule.

When performed at screening or at baseline, the result of this test must be obtained before the patient can receive a dose.

Pharmacokinetics

PK samples will be collected. PK data will be reviewed during each informal inter-cohort interim analysis as part of the dose adjustment assessment during Part 1 of the study. If the time points for collection of analyzes initially established are considered insufficient or inappropriate for understanding the characteristics of the PK profile, then the time points for collection of samples can be changed in subsequent cohorts. The number of blood draws and the total amount of blood collected will not exceed those specified in the protocol.

PK samples will be collected and evaluated at all dose levels in all patients.

The concentration of free Ab to BMP6 will be determined using ELISA analysis. The estimated lower limit of quantification (LLOQ) is 10 pg/mL.

Untreated (placebo) samples will not be analyzed.

The concentrations of free Ab to BMP6 will be expressed in μg/ml. All concentrations below the LLOQ or missing data will be flagged as such in the concentration data listings. Concentrations below the LLOQ will be treated as a null value in the aggregated statistical analysis in the case of concentration data only. They will not be taken into account when calculating the PK parameters.

PK samples remaining after determination of free Ab to human BMP6 can be used for research evaluations or other bioanalytical purposes (eg, cross-validation between different research centers, stability assessment).

The following pharmacokinetic parameters will be determined (when possible) using non-compartmental method(s) using Phoenix WinNonlin (version 6.2 or higher): Cmax, tmax, AUC(0-t), AUC(0-tlast), Cmax / D and AUC / D based on data on the dependence of concentration in blood serum from time to time. For AUC calculations, the linear trapezoidal rule will be used. The final half-life of an antibody or antigen-binding fragment that binds human BMP6 (t1/2) will also be estimated based on these data, if possible.

Other ratings

Immunogenicity

ELISA will be used to detect antibodies to BMP6. IG samples remaining after immunogenicity analysis may be used for exploratory evaluation or other bioanalytical purposes (eg, cross-validation between different research sites).

Research estimates

Biomarkers are objectively measured and evaluated indicators of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention (Biomarkers Definitions Working Group 2001).

The BMP6 hepcidin pathway is next. BMP6 signaling in hepatocytes is required for induced hepcidin expression, inhibition of iron uptake by enterocytes, and iron export by macrophages. A BMP6 neutralizing antibody as a hepcidin lowering therapy should be effective in patients with iron deficiency anemia by reducing the EPO requirement and increasing the number of patients who achieve the target Hgb level.

Based on the biological mechanism described above, research assessments of biomarkers include, without limitation, the content of hepcidin (measurement using LC-MS analysis).

Additional exploratory evaluations may explore the potential roles of bone demineralization markers as well as responding to inflammation as a contributing factor in the mechanism of action.

The research objectives are as follows:

determination of links between hepcidin levels and several key indicators, such as ERI and iron indicators;

studying the dynamics between primary and secondary endpoints and research biomarkers over time;

definition of pharmacogenetics;

determination of immunogenicity.

Sample(s) will be collected at various time points.

Additional details regarding the collection, numbering, processing and shipping of samples will be provided in the central laboratory manual.

DNA

DNA research is planned as part of this study to identify genetic factors that may (1) be associated with erythropoietin-treated patients on chronic hemodialysis with functional iron deficiency anemia, (2) predict response to Ab to BMP6 human or (3) predict genetic predisposition to side effects.

In addition, recent advances in genotyping technologies have made genome-wide approaches possible. Whole genome approaches can also be implemented within the limited scope of research data, as described above.

Soluble biomarkers

Hepcidin will be quantified in plasma as a potential PD/biomarker.

Detailed descriptions of the assays will be included in bioanalytical data reports.

Other biomarkers

No hypothesis platforms can be used to understand disease heterogeneity, mode of action, and/or potentially identify markers of stratification. Samples for studying immunogenicity (IG) will be collected at various time points. The immunogenicity of the Ab to human BMP6 will be determined by measuring antibodies recognizing the anti-human BMP6 antibody.

Bibliography

Fukuma S, Yamaguchi T, Hashimoto S et al (2012) Erythropoiesis-stimulating agent responsiveness and mortality in hemodialysis patients: results from a cohort study from the dialysis registry in Japan. Am J Kidney Dis; 59(1): p. 108-16.

Kilpatrick RD, Critchlow CW, Fishbane S et al (2008) Greater epoetin alfa responsiveness is associated with improved survival in hemodialysis patients. Clin J Am Soc Nephrol; 2008. 3(4):1077-83.

Lopez-Gomez JM, Portoles JM, and Aljama P (2008), Factors that condition the response to erythropoietin in patients on hemodialysis and their relation to mortality. Kidney Int Suppl; (111):S75-81.

Meynard D, Kautz L, Darnaud V et al (2009) Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat Genet; 41(4):478-81.

Saliem S, Patenaude V, Abenheim HA (2015) Pregnancy outcomes among renal transplant recipients and patients with end-stage renal disease on dialysis. J Perinat Med (electronic publication ahead of print) http://www.ncbi.nlm.nih.gov/pubmed/25719292.

Suttorp MM, Hoekstra T, Rotmans JI et al (2013) Erythropoiesis-stimulating agent resistance and mortality in hemodialysis and peritoneal dialysis patients . BMC Nephrol; 14(1):200.

Zaritsky J, Young B, Gales B, et al (2010) Reduction of serum hepcidin by hemodialysis in pediatric and adult patients. Clin J Am Soc Nephrol; 5(6):1010-14.

EXAMPLE 3 TSAT levels for patients treated with 0.01 mg/kg antibody 7

Data including TSAT levels (iron saturation, %) were evaluated for the first 10 patients treated according to the clinical protocol described in Example 2, with each patient receiving a single infusion of 0.01 mg/kg of antibody 7. None of the patients did not show any hepatic safety signals, which defined a no observed adverse event level (NOAEL) of 0.1 mg/kg/week. Cohort 1 included 5 anemic patients on hemodialysis with low ferritin levels of 500 ng/mL or less, while cohort 2 included 5 anemic patients on hemodialysis with higher ferritin levels (500 to 1000 ng/ml).

In five patients in cohort 1 (low ferritin) who received 0.01 mg/kg of antibody 7, TSAT levels increased by only 9.8% on average after dosing (mean 38.6% post-dose compared with 24, 8% prior to dosing). In contrast, post-dose TSAT levels increased by a median of 17.6% (mean 48.4% post-dose versus 30.8% pre-dose) in the five patients in cohort 2 (high ferritin) who received 0.01 mg/kg of antibody 7. The data are presented in figure 14, which shows the peak levels of TSAT before administration of antibody 7 compared with 72 hours after administration of antibody 7 in 2 cohorts. Surprisingly, unlike the patients in Cohort 1, the anemic patients in Cohort 2 showed a distinct increase in TSAT from baseline. These data indicate that patients with ferritin levels of 500 ng/mL or greater were suitable candidates for responding to anti-BMP6 antibody therapy and that a ferritin level, such as a ferritin level of 500 ng/mL or greater, may be indicative of a response.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains.

Unless otherwise indicated, all methods, steps, techniques and manipulations that are not specifically described in detail can be and have been carried out in a manner known per se, as will be clear to a person skilled in the art. For example, reference is repeatedly made to the standard manuals and the general art referred to in this document, and to additional sources of literature cited in this document. Unless otherwise indicated, each of the literature sources cited in this document is incorporated in its entirety by reference.

The claims are non-limiting and are presented below.

While certain aspects and claims have been detailed herein, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims or the scope of the subject matter of the claims, with any appropriate further application. In particular, the authors of the present invention assume that various substitutions, changes and modifications can be made in relation to the present invention without deviating from the essence and scope of the present invention defined by the claims. It is believed that the choice of nucleic acid starting material, clone of interest, or type of library is a familiar matter for a person skilled in the art with knowledge of the aspects described herein. It is believed that other aspects, advantages and modifications are within the scope of the following claims. Those skilled in the art will recognize many equivalents to the specific aspects of the present invention described herein, or through routine experimentation alone, will be able to ascertain such equivalents. Such equivalents are intended to be embraced by the following claims. Changes in the scope of a claim in later filed related applications may be due to limitations in the patent laws of various countries and should not be construed as a waiver of the subject matter of the claims.

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<120> METHODS OF TREATMENT OF DISEASE USING BONE BONE INHIBITORS

MORPHOGENETIC PROTEIN 6 (BMP6)

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atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acgcagaaga gcctctccct gtctccggggt aaa 1353

<210> 19

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 19

Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Ser Val His

1 5 10

<210> 20

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 20

Gly Ser Ser Glu Arg Pro Ser

15

<210> 21

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 21

Gln Ser Trp Asp Ser Ser Gln Thr Leu Val Val

1 5 10

<210> 22

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 22

Ser Ser Ser Asn Ile Gly Ala Gly Tyr Ser

1 5 10

<210> 23

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 23

Gly Ser Ser

1

<210> 24

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 24

Trp Asp Ser Ser Gln Thr Leu Val

15

<210> 25

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 25

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Ser Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Ser Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ser

85 90 95

Gln Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 26

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 26

cagagcgtgc tgacccagcc gccgagcgtg agcggtgcac cgggccagcg cgtgaccatt 60

agctgtaccg gcagcagcag caacattggt gctggttact ctgtgcattg gtaccagcag 120

ctgccgggca cggcgccgaa actgctgatc tatggtagct ctgaacgccc gagcggcgtg 180

ccggatcgct ttagcggatc caaaagcggc accagcgcca gcctggcgat taccggcctg 240

caagcagaag acgaagcgga ttattactgc cagtcttggg actcttctca gactctggtt 300

gtgtttggcg gcggcacgaa gttaaccgtc cta 333

<210> 27

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 27

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Ser Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Ser Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ser

85 90 95

Gln Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 28

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 28

cagagcgtgc tgacccagcc gccgagcgtg agcggtgcac cgggccagcg cgtgaccatt 60

agctgtaccg gcagcagcag caacattggt gctggttact ctgtgcattg gtaccagcag 120

ctgccgggca cggcgccgaa actgctgatc tatggtagct ctgaacgccc gagcggcgtg 180

ccggatcgct ttagcggatc caaaagcggc accagcgcca gcctggcgat taccggcctg 240

caagcagaag acgaagcgga ttattactgc cagtcttggg actcttctca gactctggtt 300

gtgtttggcg gcggcacgaa gttaaccgtc ctaggtcagc ccaaggctgc cccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc a 651

<210> 29

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 29

Ser Tyr Val Val His

15

<210> 30

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 30

Arg Ile Lys Arg Glu Ser Ser Ser Tyr Thr Thr Met Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 31

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 31

Val Glu Arg Ser Lys Ser Gly Phe Asp Asn

1 5 10

<210> 32

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 32

Gly Phe Thr Phe Ser Ser Tyr

15

<210> 33

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 33

Lys Arg Glu Ser Ser Ser Tyr Thr

15

<210> 34

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 34

Val Glu Arg Ser Lys Ser Gly Phe Asp Asn

1 5 10

<210> 35

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 35

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Gly Arg Ile Lys Arg Glu Ser Ser Ser Tyr Thr Thr Met Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Glu Arg Ser Lys Ser Gly Phe Asp Asn Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 36

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 36

caggtgcagc tggtggaatc aggcggcgga ctggtcaagc ctggcggtag cctgagactg 60

agctgcgctg ctagtggctt caccttctct agctacgtgg tgcactgggt cagacaggcc 120

cctggtaaag gcctggagtg ggtcggacgg attaagagag agtcctctag ctacactact 180

atgtacgccg ctcccgtgaa gggccggttc actatctcta gggacgactc taagaacacc 240

ctgtacctgc agatgaatag cctgaaaacc gaggacaccg ccgtctacta ctgcgctaga 300

gtggaacggt ctaagtcagg cttcgataac tggggtcagg gcaccctggt caccgtgtct 360

agc 363

<210> 37

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 37

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Gly Arg Ile Lys Arg Glu Ser Ser Ser Tyr Thr Thr Met Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Glu Arg Ser Lys Ser Gly Phe Asp Asn Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 38

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 38

caggtgcagc tggtggaatc aggcggcgga ctggtcaagc ctggcggtag cctgagactg 60

agctgcgctg ctagtggctt caccttctct agctacgtgg tgcactgggt cagacaggcc 120

cctggtaaag gcctggagtg ggtcggacgg attaagagag agtcctctag ctacactact 180

atgtacgccg ctcccgtgaa gggccggttc actatctcta gggacgactc taagaacacc 240

ctgtacctgc agatgaatag cctgaaaacc gaggacaccg ccgtctacta ctgcgctaga 300

gtggaacggt ctaagtcagg cttcgataac tggggtcagg gcaccctggt caccgtgtct 360

agcgctagca ctaagggccc aagtgtgttt cccctggccc ccagcagcaa gtctacttcc 420

ggcggaactg ctgccctgggg ttgcctggtg aaggactact tccccgagcc cgtgacagtg 480

tcctggaact ctggggctct gacttccggc gtgcacacct tccccgccgt gctgcagagc 540

agcggcctgt acagcctgag cagcgtggtg acagtgccct ccagctctct gggaacccag 600

acctatatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gagagtggag 660

cccaagagct gcgacaagac ccacacctgc cccccctgcc cagctccaga actgctggga 720

gggccttccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc 780

cccgaggtga cctgcgtggt ggtggacgtg tcccacgagg acccagaggt gaagttcaac 840

tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccagaga ggagcagtac 900

aacagcacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960

aaagaataca agtgcaaagt ctccaacaag gccctgccag ccccaatcga aaagacaatc 1020

1080

gagatgacca agaaccaggt gtccctgacc tgtctggtga agggcttcta ccccagcgat 1140

atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccca 1200

1260

tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320

acccagaagt ccctgagcct gagccccggc aag 1353

<210> 39

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 39

Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Ser Val His

1 5 10

<210> 40

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 40

Gly Gln Ser Glu Arg Pro Ser

15

<210> 41

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

<400> 41

Gln Ser Trp Asp Ser Ser Gln Thr Leu Val Val

1 5 10

<210> 42

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 42

Ser Ser Ser Asn Ile Gly Ala Gly Tyr Ser

1 5 10

<210> 43

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 43

Gly Gln Ser

1

<210> 44

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 44

Trp Asp Ser Ser Gln Thr Leu Val

15

<210> 45

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 45

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Ser Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Gln Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ser

85 90 95

Gln Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 46

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 46

cagtcagtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgcaccg gctctagctc taatatcggc gctggctata gcgtgcactg gtatcagcag 120

ctgcccggca ccgcccctaa gctgctgatc tacggtcagt cagagcggcc tagcggcgtg 180

cccgataggt ttagcggctc taagtcaggc actagcgcta gtctggctat caccggcctg 240

caggctgagg acgaggccga ctactactgt cagtcctggg actctagtca gaccctggtg 300

gtgttcggcg gaggcactaa gctgaccgtg ctg 333

<210> 47

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 47

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Ser Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Gln Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ser

85 90 95

Gln Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 48

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 48

cagtcagtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgcaccg gctctagctc taatatcggc gctggctata gcgtgcactg gtatcagcag 120

ctgcccggca ccgcccctaa gctgctgatc tacggtcagt cagagcggcc tagcggcgtg 180

cccgataggt ttagcggctc taagtcaggc actagcgcta gtctggctat caccggcctg 240

caggctgagg acgaggccga ctactactgt cagtcctggg actctagtca gaccctggtg 300

gtgttcggcg gaggcactaa gctgaccgtg ctgggtcagc ctaaggctgc ccccagcgtg 360

accctgttcc cccccagcag cgaggagctg caggccaaca aggccaccct ggtgtgcctg 420

atcagcgact tctacccagg cgccgtgacc gtggcctgga aggccgacag cagccccgtg 480

aaggccggcg tggagaccac cacccccagc aagcagagca acaacaagta cgccgccagc 540

agctacctga gcctgacccc cgagcagtgg aagagccaca ggtcctacag ctgccaggtg 600

acccacgagg gcagcaccgt ggaaaagacc gtggccccaa ccgagtgcag c 651

<210> 49

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 49

Ser Tyr Val Val His

15

<210> 50

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 50

Arg Thr Arg His Ser Asp Met Gly Tyr Ala Thr Ser Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 51

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 51

Val Glu Arg Ser Lys Ser Gly Phe Asp Asn

1 5 10

<210> 52

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 52

Gly Phe Thr Phe Ser Ser Tyr

15

<210> 53

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 53

Arg His Ser Asp Met Gly Tyr Ala

15

<210> 54

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 54

Val Glu Arg Ser Lys Ser Gly Phe Asp Asn

1 5 10

<210> 55

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 55

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Gly Arg Thr Arg His Ser Asp Met Gly Tyr Ala Thr Ser Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Glu Arg Ser Lys Ser Gly Phe Asp Asn Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 56

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 56

caggtgcagc tggtggaatc aggcggcgga ctggtcaagc ctggcggtag cctgagactg 60

agctgcgctg ctagtggctt caccttctct agctacgtgg tgcactgggt cagacaggcc 120

cctggtaaag gcctggagtg ggtcggacgg actagacact cagatatggg ctacgctact 180

agctacgccg ctcccgtgaa gggccggttc actatctcta gggacgactc taagaacacc 240

ctgtacctgc agatgaatag cctgaaaacc gaggacaccg ccgtctacta ctgcgctaga 300

gtggaacggt ctaagtcagg cttcgataac tggggtcagg gcaccctggt caccgtgtct 360

agc 363

<210> 57

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 57

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Gly Arg Thr Arg His Ser Asp Met Gly Tyr Ala Thr Ser Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Glu Arg Ser Lys Ser Gly Phe Asp Asn Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 58

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 58

caggtgcagc tggtggaatc aggcggcgga ctggtcaagc ctggcggtag cctgagactg 60

agctgcgctg ctagtggctt caccttctct agctacgtgg tgcactgggt cagacaggcc 120

cctggtaaag gcctggagtg ggtcggacgg actagacact cagatatggg ctacgctact 180

agctacgccg ctcccgtgaa gggccggttc actatctcta gggacgactc taagaacacc 240

ctgtacctgc agatgaatag cctgaaaacc gaggacaccg ccgtctacta ctgcgctaga 300

gtggaacggt ctaagtcagg cttcgataac tggggtcagg gcaccctggt caccgtgtct 360

agcgctagca ctaagggccc aagtgtgttt cccctggccc ccagcagcaa gtctacttcc 420

ggcggaactg ctgccctgggg ttgcctggtg aaggactact tccccgagcc cgtgacagtg 480

tcctggaact ctggggctct gacttccggc gtgcacacct tccccgccgt gctgcagagc 540

agcggcctgt acagcctgag cagcgtggtg acagtgccct ccagctctct gggaacccag 600

acctatatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gagagtggag 660

cccaagagct gcgacaagac ccacacctgc cccccctgcc cagctccaga actgctggga 720

gggccttccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc 780

cccgaggtga cctgcgtggt ggtggacgtg tcccacgagg acccagaggt gaagttcaac 840

tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccagaga ggagcagtac 900

aacagcacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960

aaagaataca agtgcaaagt ctccaacaag gccctgccag ccccaatcga aaagacaatc 1020

1080

gagatgacca agaaccaggt gtccctgacc tgtctggtga agggcttcta ccccagcgat 1140

atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccca 1200

1260

tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320

acccagaagt ccctgagcct gagccccggc aag 1353

<210> 59

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 59

Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Ser Val His

1 5 10

<210> 60

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 60

Gly Gln Ser Glu Arg Pro Ser

15

<210> 61

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 61

Gln Ser Trp Asp Ser Ser Gln Thr Leu Val Val

1 5 10

<210> 62

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 62

Ser Ser Ser Asn Ile Gly Ala Gly Tyr Ser

1 5 10

<210> 63

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 63

Gly Gln Ser

1

<210> 64

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 64

Trp Asp Ser Ser Gln Thr Leu Val

15

<210> 65

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 65

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Ser Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Gln Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ser

85 90 95

Gln Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 66

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 66

cagtcagtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgcaccg gctctagctc taatatcggc gctggctata gcgtgcactg gtatcagcag 120

ctgcccggca ccgcccctaa gctgctgatc tacggtcagt cagagcggcc tagcggcgtg 180

cccgataggt ttagcggctc taagtcaggc actagcgcta gtctggctat caccggcctg 240

caggctgagg acgaggccga ctactactgt cagtcctggg actctagtca gaccctggtg 300

gtgttcggcg gaggcactaa gctgaccgtg ctg 333

<210> 67

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 67

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Ser Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Gln Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ser

85 90 95

Gln Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 68

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 68

cagtcagtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgcaccg gctctagctc taatatcggc gctggctata gcgtgcactg gtatcagcag 120

ctgcccggca ccgcccctaa gctgctgatc tacggtcagt cagagcggcc tagcggcgtg 180

cccgataggt ttagcggctc taagtcaggc actagcgcta gtctggctat caccggcctg 240

caggctgagg acgaggccga ctactactgt cagtcctggg actctagtca gaccctggtg 300

gtgttcggcg gaggcactaa gctgaccgtg ctgggtcagc ctaaggctgc ccccagcgtg 360

accctgttcc cccccagcag cgaggagctg caggccaaca aggccaccct ggtgtgcctg 420

atcagcgact tctacccagg cgccgtgacc gtggcctgga aggccgacag cagccccgtg 480

aaggccggcg tggagaccac cacccccagc aagcagagca acaacaagta cgccgccagc 540

agctacctga gcctgacccc cgagcagtgg aagagccaca ggtcctacag ctgccaggtg 600

acccacgagg gcagcaccgt ggaaaagacc gtggccccaa ccgagtgcag c 651

<210> 69

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 69

Ser Tyr Val Val His

15

<210> 70

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 70

Arg Ile Arg Leu Glu Thr His Gly Tyr Ala Ala Glu Tyr Ala Ala Ser

1 5 10 15

Val Lys Gly

<210> 71

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 71

Val Glu Arg Ser Lys Ser Gly Phe Asp Asn

1 5 10

<210> 72

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 72

Gly Phe Thr Phe Ser Ser Tyr

15

<210> 73

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 73

Arg Leu Glu Thr His Gly Tyr Ala

15

<210> 74

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 74

Val Glu Arg Ser Lys Ser Gly Phe Asp Asn

1 5 10

<210> 75

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 75

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Gly Arg Ile Arg Leu Glu Thr His Gly Tyr Ala Ala Glu Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Glu Arg Ser Lys Ser Gly Phe Asp Asn Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 76

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 76

caggtgcagc tggtggaatc aggcggcgga ctggtcaagc ctggcggtag cctgagactg 60

agctgcgctg ctagtggctt caccttctct agctacgtgg tgcactgggt cagacaggcc 120

cctggtaaag gcctggagtg ggtcggacgg attagactgg aaactcacgg ctacgccgcc 180

gagtacgccg ctagtgtgaa gggccggttc actatctcta gggacgactc taagaacacc 240

ctgtacctgc agatgaatag cctgaaaacc gaggacaccg ccgtctacta ctgcgctaga 300

gtggaacggt ctaagtcagg cttcgataac tggggtcagg gcaccctggt caccgtgtct 360

agc 363

<210> 77

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 77

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Gly Arg Ile Arg Leu Glu Thr His Gly Tyr Ala Ala Glu Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Glu Arg Ser Lys Ser Gly Phe Asp Asn Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 78

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 78

caggtgcagc tggtggaatc aggcggcgga ctggtcaagc ctggcggtag cctgagactg 60

agctgcgctg ctagtggctt caccttctct agctacgtgg tgcactgggt cagacaggcc 120

cctggtaaag gcctggagtg ggtcggacgg attagactgg aaactcacgg ctacgccgcc 180

gagtacgccg ctagtgtgaa gggccggttc actatctcta gggacgactc taagaacacc 240

ctgtacctgc agatgaatag cctgaaaacc gaggacaccg ccgtctacta ctgcgctaga 300

gtggaacggt ctaagtcagg cttcgataac tggggtcagg gcaccctggt caccgtgtct 360

agcgctagca ctaagggccc aagtgtgttt cccctggccc ccagcagcaa gtctacttcc 420

ggcggaactg ctgccctgggg ttgcctggtg aaggactact tccccgagcc cgtgacagtg 480

tcctggaact ctggggctct gacttccggc gtgcacacct tccccgccgt gctgcagagc 540

agcggcctgt acagcctgag cagcgtggtg acagtgccct ccagctctct gggaacccag 600

acctatatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gagagtggag 660

cccaagagct gcgacaagac ccacacctgc cccccctgcc cagctccaga actgctggga 720

gggccttccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc 780

cccgaggtga cctgcgtggt ggtggacgtg tcccacgagg acccagaggt gaagttcaac 840

tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccagaga ggagcagtac 900

aacagcacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960

aaagaataca agtgcaaagt ctccaacaag gccctgccag ccccaatcga aaagacaatc 1020

1080

gagatgacca agaaccaggt gtccctgacc tgtctggtga agggcttcta ccccagcgat 1140

atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccca 1200

1260

tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320

acccagaagt ccctgagcct gagccccggc aag 1353

<210> 79

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 79

Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Ser Val His

1 5 10

<210> 80

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 80

Gly Gln Ser Glu Arg Pro Ser

15

<210> 81

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 81

Gln Ser Trp Asp Ser Ser Gln Thr Leu Val Val

1 5 10

<210> 82

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 82

Ser Ser Ser Asn Ile Gly Ala Gly Tyr Ser

1 5 10

<210> 83

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 83

Gly Gln Ser

1

<210> 84

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 84

Trp Asp Ser Ser Gln Thr Leu Val

15

<210> 85

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 85

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Ser Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Gln Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ser

85 90 95

Gln Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 86

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 86

cagtcagtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgcaccg gctctagctc taatatcggc gctggctata gcgtgcactg gtatcagcag 120

ctgcccggca ccgcccctaa gctgctgatc tacggtcagt cagagcggcc tagcggcgtg 180

cccgataggt ttagcggctc taagtcaggc actagcgcta gtctggctat caccggcctg 240

caggctgagg acgaggccga ctactactgt cagtcctggg actctagtca gaccctggtg 300

gtgttcggcg gaggcactaa gctgaccgtg ctg 333

<210> 87

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 87

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Ser Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Gln Ser Glu Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Ser Ser

85 90 95

Gln Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 88

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 88

cagtcagtcc tgactcagcc ccctagcgtc agcggcgctc ccggtcagag agtgactatt 60

agctgcaccg gctctagctc taatatcggc gctggctata gcgtgcactg gtatcagcag 120

ctgcccggca ccgcccctaa gctgctgatc tacggtcagt cagagcggcc tagcggcgtg 180

cccgataggt ttagcggctc taagtcaggc actagcgcta gtctggctat caccggcctg 240

caggctgagg acgaggccga ctactactgt cagtcctggg actctagtca gaccctggtg 300

gtgttcggcg gaggcactaa gctgaccgtg ctgggtcagc ctaaggctgc ccccagcgtg 360

accctgttcc cccccagcag cgaggagctg caggccaaca aggccaccct ggtgtgcctg 420

atcagcgact tctacccagg cgccgtgacc gtggcctgga aggccgacag cagccccgtg 480

aaggccggcg tggagaccac cacccccagc aagcagagca acaacaagta cgccgccagc 540

agctacctga gcctgacccc cgagcagtgg aagagccaca ggtcctacag ctgccaggtg 600

acccacgagg gcagcaccgt ggaaaagacc gtggccccaa ccgagtgcag c 651

<210> 89

<211> 139

<212> PROTEIN

<213> Homo sapiens

<400> 89

Ser Ala Ser Ser Arg Arg Arg Gln Gln Ser Arg Asn Arg Ser Thr Gln

1 5 10 15

Ser Gln Asp Val Ala Arg Val Ser Ser Ala Ser Asp Tyr Asn Ser Ser

20 25 30

Glu Leu Lys Thr Ala Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gln

35 40 45

Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala

50 55 60

Asn Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn

65 70 75 80

Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Leu Met Asn Pro

85 90 95

Glu Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile

100 105 110

Ser Val Leu Tyr Phe Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr

115 120 125

Arg Asn Met Val Val Arg Ala Cys Gly Cys His

130 135

<210> 90

<211> 139

<212> PROTEIN

<213> Homo sapiens

<400> 90

Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys

1 5 10 15

Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser

20 25 30

Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg

35 40 45

Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala

50 55 60

Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn

65 70 75 80

Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro

85 90 95

Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile

100 105 110

Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr

115 120 125

Arg Asn Met Val Val Arg Ala Cys Gly Cys His

130 135

<210> 91

<211> 138

<212> PROTEIN

<213> Homo sapiens

<400> 91

Ala Ala Asn Lys Arg Lys Asn Gln Asn Arg Asn Lys Ser Ser Ser His

1 5 10 15

Gln Asp Ser Ser Arg Met Ser Ser Val Gly Asp Tyr Asn Thr Ser Glu

20 25 30

Gln Lys Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp

35 40 45

Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Phe

50 55 60

Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala

65 70 75 80

Thr Asn His Ala Ile Val Gln Thr Leu Val His Leu Met Phe Pro Asp

85 90 95

His Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser

100 105 110

Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg

115 120 125

Asn Met Val Val Arg Ser Cys Gly Cys His

130 135

<210> 92

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 92

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Pro Phe Gly Asn Ala Met Asp Ile Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 93

<211> 444

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 93

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Pro Phe Gly Asn Ala Met Asp Ile Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 94

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 94

Tyr Ile Asn Pro Tyr Asn Arg Gly Thr Lys Tyr Asn Glu Asn Phe Lys

1 5 10 15

<210> 95

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 95

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Arg Gly Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Pro Phe Gly Asn Ala Met Asp Ile Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 96

<211> 444

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 96

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Arg Gly Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Pro Phe Gly Asn Ala Met Asp Ile Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 97

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: Synthetic

label 6xHis"

<400> 97

His His His His His His His

15

<210> 98

<211> 15

<212> PROTEIN

<213> Homo sapiens

<400> 98

Gln Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys Pro

1 5 10 15

<210> 99

<211> 13

<212> PROTEIN

<213> Homo sapiens

<400> 99

Val Ser Ser Ala Ser Asp Tyr Asn Ser Ser Glu Leu Lys

1 5 10

<---

Claims (199)

1. A method of treating anemia in a patient with a BMP6 antagonist, comprising selectively administering a therapeutically effective amount of the BMP6 antagonist to the patient based on a biological sample from the patient having a ferritin level greater than or equal to 500 ng/mL, wherein said BMP6 antagonist is an antibody to BMP6 or its antigen-binding fragment containing: (i) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively; (ii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively; (iii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively; (iv) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively; (v) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively; (vi) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively; (vii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or (viii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively. 2. A method for selectively treating anemia in a patient with a BMP6 antagonist, comprising: a) performing an analysis of a biological sample from the patient in relation to the level of ferritin and b) thereafter selectively administering to the patient a therapeutically effective amount of said BMP6 antagonist, wherein the ferritin level is greater than or equal to 500 ng/mL, and wherein said BMP6 antagonist is an anti-BMP6 antibody or antigen-binding fragment thereof, comprising: (i) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively; (ii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively; (iii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively; (iv) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively; (v) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively; (vi) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively; (vii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or (viii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively. 3. A method for selectively treating anemia in a patient using a BMP6 antagonist, comprising: a) performing an analysis of a biological sample from the patient in relation to the level of ferritin; b) thereafter selecting a patient for treatment with a BMP6 antagonist based on a biological sample from the patient having a ferritin level greater than or equal to 500 ng/mL; And c) thereafter administering to the patient a therapeutically effective amount of said BMP6 antagonist, wherein said BMP6 antagonist is an anti-BMP6 antibody or antigen-binding fragment thereof, comprising: (i) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively; (ii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively; (iii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively; (iv) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively; (v) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively; (vi) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively; (vii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or (viii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively. 4. A method for predicting the likelihood that a patient having anemia will respond to treatment with a BMP6 antagonist, comprising assaying a biological sample from the patient for ferritin, where a ferritin level greater than or equal to 500 ng/mL is indicative of increased likelihood that the patient will respond to treatment with a BMP6 antagonist, wherein said BMP6 antagonist is an anti-BMP6 antibody or antigen-binding fragment containing: (i) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively; (ii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively; (iii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively; (iv) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively; (v) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively; (vi) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively; (vii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or (viii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively. 5. The method according to any one of paragraphs. 1-4, further comprising the step of obtaining a biological sample from the patient, wherein the step of obtaining is performed before the step of performing the analysis. 6. The method according to any one of paragraphs. 1-5, where the ferritin level represents the ferritin protein level. 7. The method according to any one of paragraphs. 2-6, wherein the step of performing the assay comprises a technique selected from the group consisting of immunoassay, immunohistochemistry, ELISA, flow cytometry, Western blotting, HPLC, and mass spectrometry. 8. The method according to any one of paragraphs. 1-7, where the anemia is anemia associated with a chronic disease. 9. The method of claim 8, wherein the chronic disease is chronic kidney disease, cancer, or inflammation. 10. The method according to any one of paragraphs. 1-9 wherein the patient is receiving or has been treated with an erythropoiesis stimulating agent (ESA). 11. The method of claim 10, wherein the ESA is erythropoietin (EPO). 12. The method according to any one of paragraphs. 1-11, where the anemia is anemia with EPO hyporeactivity. 13. The method according to any one of paragraphs. 1-12, where the anemia is anemia due to lack of iron, such as anemia due to functional lack of iron. 14. The method according to any one of paragraphs. 1-13, where the patient is a patient on chronic hemodialysis. 15. The method according to any one of paragraphs. 1-14 further comprising reducing the patient's iron requirement, reducing the patient's EPO dose, or reducing both the patient's iron requirement and the patient's EPO dose compared to said EPO dose requirement and/or iron requirement. in the absence of treatment with a therapeutically effective amount of a BMP6 antagonist. 16. The method according to any one of paragraphs. 1-15, where the biological sample is synovial fluid, blood, blood serum, feces, blood plasma, urine, tear, saliva, cerebrospinal fluid, leukocyte sample, or tissue sample. 17. The method of claim 16 wherein the biological sample is blood serum or blood. 18. The method according to claim 17, where the biological sample is blood serum. 19. The method according to any one of paragraphs. 1-18, where the antibody to BMP6 or its antigen-binding fragment contains: (a) the VH sequence under SEQ ID NO: 75; (b) the VH sequence under SEQ ID NO: 35; (c) the VH sequence under SEQ ID NO: 55 or (d) VH sequence under SEQ ID NO: 15. 20. The method according to any one of paragraphs. 1-19, where the antibody to BMP6 or its antigen-binding fragment contains: (a) the VL sequence under SEQ ID NO: 85; (b) the VL sequence under SEQ ID NO: 45; (c) the VL sequence under SEQ ID NO: 65 or (d) VL sequence under SEQ ID NO: 25. 21. The method according to any one of paragraphs. 1-20, wherein the anti-BMP6 antibody or antigen-binding fragment thereof contains: (a) the VH sequence of SEQ ID NO: 75 and the VL sequence of SEQ ID NO: 85; (b) the VH sequence of SEQ ID NO: 35 and the VL sequence of SEQ ID NO: 45; (c) the VH sequence of SEQ ID NO: 55 and the VL sequence of SEQ ID NO: 65, or (d) the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 25. 22. The method according to any one of paragraphs. 1-21, where the antibody to BMP6 or its antigen-binding fragment contains: (a) the sequence of the heavy chain under SEQ ID NO: 77; (b) the sequence of the heavy chain under SEQ ID NO: 37; (c) the heavy chain sequence of SEQ ID NO: 57 or (d) the heavy chain sequence of SEQ ID NO: 17. 23. The method according to any one of paragraphs. 1-22, where the BMP6 antibody or antigen-binding fragment contains: (a) the light chain sequence of SEQ ID NO: 87; (b) the light chain sequence of SEQ ID NO: 47; (c) a light chain sequence of SEQ ID NO: 67 or (d) the light chain sequence of SEQ ID NO: 27. 24. The method according to any one of paragraphs. 1-23, where the anti-BMP6 antibody or antigen-binding fragment contains: (a) the heavy chain sequence of SEQ ID NO: 77 and the light chain sequence of SEQ ID NO: 87; (b) the heavy chain sequence of SEQ ID NO: 37 and the light chain sequence of SEQ ID NO: 47; (c) a heavy chain sequence of SEQ ID NO: 57 and a light chain sequence of SEQ ID NO: 67, or (d) the heavy chain sequence of SEQ ID NO: 17 and the light chain sequence of SEQ ID NO: 27. 25. The method according to any one of paragraphs. 1-24, wherein the anti-BMP6 antibody, or antigen-binding fragment thereof, comprises a backbone selected from IgM and IgG. 26. The method according to any one of paragraphs. 1-25, wherein the anti-BMP6 antibody or antigen-binding fragment thereof is an IgG selected from IgG1, IgG2 and IgG3 or IgG4. 27. The method according to any one of paragraphs. 1-26, wherein the anti-BMP6 antibody, or antigen-binding fragment thereof, is selected from the group consisting of monoclonal antibody, chimeric antibody, single chain antibody, Fab, and scFv. 28. The method according to any one of paragraphs. 1-27, where the antibody to BMP6 or its antigennegative fragment is a component of the immunoconjugate. 29. The method according to any one of paragraphs. 1-28, wherein the anti-BMP6 antibody or antigen-binding fragment thereof has an altered effector function due to a mutation in the Fc region. 30. The method according to any one of paragraphs. 1-29, wherein the antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.01 mg/kg to 15 mg/kg. 31. The method according to any one of paragraphs. 1-30, wherein the antibody or antigen-binding fragment thereof is administered to said patient more than once. 32. The method according to any one of paragraphs. 1-31, where the antibody or antigen-binding fragment is administered: a) intravenously or b) subcutaneously. 33. The method according to any one of paragraphs. 1-32, where the ferritin level is less than or equal to 1900 ng/ml, less than or equal to 1800 ng/ml, less than or equal to 1700 ng/ml, less than or equal to 1600 ng/ml, less than or equal to 1500 ng/ml, less than or is equal to 1400 ng/ml, less than or equal to 1300 ng/ml, less than or equal to 1200 ng/ml, less than or equal to 1100 ng/ml or less than or equal to 1000 ng/ml. 34. The method according to any one of paragraphs. 1-33, where the ferritin level is less than or equal to 1500 ng/ml. 35. The method according to any one of paragraphs. 1-34, where the ferritin level is greater than or equal to 600 ng/ml, greater than or equal to 700 ng/ml, greater than or equal to 800 ng/ml, greater than or equal to 900 ng/ml, greater than or equal to 1000 ng/ml, greater than or equal to 1100 ng/mL, greater than or equal to 1200 ng/mL, greater than or equal to 1300 ng/mL, greater than or equal to 1400 ng/mL, greater than or equal to 1500 ng/mL, greater than or equal to 1600 ng/mL, greater than or equal to 1700 ng /ml greater than or equal to 1800 ng/ml or greater than or equal to 1900 ng/ml. 36. The method according to any one of paragraphs. 1-35, where the ferritin level is greater than or equal to 600 ng/ml, greater than or equal to 700 ng/ml, greater than or equal to 800 ng/ml, greater than or equal to 900 ng/ml, greater than or equal to 1000 ng/ml, greater than or equal to 1100 ng/ml, greater than or equal to 1200 ng/ml, greater than or equal to 1300 ng/ml or greater than or equal to 1400 ng/ml. 37. The use of a BMP6 antagonist in the treatment of anemia in a patient, characterized in that the patient is administered a therapeutically effective amount of the BMP6 antagonist based on the fact that the biological sample from the patient has a ferritin level greater than or equal to 500 ng/ml, where said BMP6 antagonist is an anti-BMP6 antibody or antigen-binding fragment thereof, containing: (i) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively; (ii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively; (iii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively; (iv) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively; (v) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively; (vi) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively; (vii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or (viii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively. 38. The use of a BMP6 antagonist in the treatment of anemia in a patient, characterized in that: a) a patient is selected for treatment with a BMP6 antagonist based on a biological sample from the patient having a ferritin level greater than or equal to 500 ng/mL; And b) the patient is then administered a therapeutically effective amount of said BMP6 antagonist, wherein said BMP6 antagonist is an anti-BMP6 antibody or antigen-binding fragment thereof, comprising: (i) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively; (ii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively; (iii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively; (iv) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively; (v) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively; (vi) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively; (vii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or (viii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively. 39. The use of a BMP6 antagonist in the treatment of anemia in a patient, characterized in that: a) analyzing a biological sample from the patient for ferritin; b) the patient is selectively administered a therapeutically effective amount of a BMP6 antagonist based on the biological sample from the patient having a ferritin level greater than or equal to 500 ng/mL, wherein said BMP6 antagonist is an anti-BMP6 antibody or an antigen-binding fragment thereof, comprising: (i) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively; (ii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively; (iii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively; (iv) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively; (v) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively; (vi) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively; (vii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or (viii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively. 40. The use of a BMP6 antagonist in the treatment of anemia in a patient, characterized in that: a) analyzing a biological sample from the patient for ferritin; b) a patient is selected for treatment with a BMP6 antagonist based on a biological sample from the patient having a ferritin level greater than or equal to 500 ng/mL; And c) a therapeutically effective amount of a BMP6 antagonist is selectively administered to the patient, wherein said BMP6 antagonist is an anti-BMP6 antibody or antigen-binding fragment thereof, comprising: (i) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 79, 80 and 81, respectively; (ii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 82, 83 and 84, respectively; (iii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 39, 40 and 41, respectively; (iv) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 42, 43 and 44, respectively; (v) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 59, 60 and 61, respectively; (vi) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 62, 63 and 64, respectively; (vii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 19, 20 and 21, respectively, or (viii) the sequences HCDR1, HCDR2 and HCDR3 under SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 under SEQ ID NOs: 22, 23 and 24, respectively. 41. Application according to any one of paragraphs. 37-40, where the ferritin level is the ferritin protein level. 42. Application according to any one of paragraphs. 37-41, wherein said assay comprises a technique selected from the group consisting of immunoassay, immunohistochemistry, ELISA, flow cytometry, Western blotting, HPLC, and mass spectrometry. 43. Application according to any one of paragraphs. 37-42, where the anemia is anemia associated with a chronic disease. 44. The use of claim 43 wherein the chronic disease is chronic kidney disease, cancer, or inflammation. 45. Application according to any one of paragraphs. 37-44 wherein the patient is receiving or has received treatment with an erythropoiesis stimulating agent (ESA). 46. Use according to claim 45, wherein ESA is erythropoietin (EPO). 47. Application according to any one of paragraphs. 37-46, where the anemia is anemia with EPO hyporeactivity. 48. Application according to any one of paragraphs. 37-47, where the anemia is anemia due to iron deficiency, such as anemia due to functional iron deficiency. 49. Application according to any one of paragraphs. 37-48, where the patient is a patient on chronic hemodialysis. 50. Application according to any one of paragraphs. 37-49, further comprising reducing the patient's iron requirement, reducing the patient's EPO dose, or reducing both the patient's iron requirement and the patient's EPO dose compared to said EPO dose requirement and/or iron requirement. in the absence of treatment with a therapeutically effective amount of a BMP6 antagonist. 51. Application according to any one of paragraphs. 37-50, where the biological sample is synovial fluid, blood, blood serum, feces, blood plasma, urine, tear, saliva, cerebrospinal fluid, leukocyte sample, or tissue sample. 52. Use according to claim 51, where the biological sample is blood serum or blood. 53. Use according to claim 52, where the biological sample is blood serum. 54. Application according to any one of paragraphs. 37-53, where the BMP6 antibody or antigen-binding fragment contains: (a) the VH sequence under SEQ ID NO: 75; (b) the VH sequence under SEQ ID NO: 35; (c) the VH sequence under SEQ ID NO: 55 or (d) VH sequence under SEQ ID NO: 15. 55. Application according to any one of paragraphs. 37-54, wherein the anti-BMP6 antibody or antigen-binding fragment thereof contains: (a) the VL sequence under SEQ ID NO: 85; (b) the VL sequence under SEQ ID NO: 45; (c) the VL sequence under SEQ ID NO: 65 or (d) VL sequence under SEQ ID NO: 25. 56. Application according to any one of paragraphs. 37-55, where the BMP6 antibody or antigen-binding fragment contains: (a) the VH sequence of SEQ ID NO: 75 and the VL sequence of SEQ ID NO: 85; (b) the VH sequence of SEQ ID NO: 35 and the VL sequence of SEQ ID NO: 45; (c) the VH sequence of SEQ ID NO: 55 and the VL sequence of SEQ ID NO: 65, or (d) the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 25. 57. Application according to any one of paragraphs. 37-56, wherein the anti-BMP6 antibody or antigen-binding fragment thereof contains: (a) the sequence of the heavy chain under SEQ ID NO: 77; (b) the sequence of the heavy chain under SEQ ID NO: 37; (c) the heavy chain sequence of SEQ ID NO: 57 or (d) the heavy chain sequence of SEQ ID NO: 17. 58. Application according to any one of paragraphs. 37-57, wherein the anti-BMP6 antibody or antigen-binding fragment thereof contains: (a) the light chain sequence of SEQ ID NO: 87; (b) the light chain sequence of SEQ ID NO: 47; (c) a light chain sequence of SEQ ID NO: 67 or (d) the light chain sequence of SEQ ID NO: 27. 59. Application according to any one of paragraphs. 37-58, wherein the anti-BMP6 antibody or antigen-binding fragment thereof contains: (a) the heavy chain sequence of SEQ ID NO: 77 and the light chain sequence of SEQ ID NO: 87; (b) the heavy chain sequence of SEQ ID NO: 37 and the light chain sequence of SEQ ID NO: 47; (c) a heavy chain sequence of SEQ ID NO: 57 and a light chain sequence of SEQ ID NO: 67, or (d) the heavy chain sequence of SEQ ID NO: 17 and the light chain sequence of SEQ ID NO: 27. 60. Application according to any one of paragraphs. 37-59, wherein the anti-BMP6 antibody or antigen-binding fragment thereof contains a backbone selected from IgM and IgG. 61. Application according to any one of paragraphs. 37-60, wherein the anti-BMP6 antibody or antigen-binding fragment thereof is an IgG selected from IgG1, IgG2 and IgG3 or IgG4. 62. Application according to any one of paragraphs. 37-61, wherein the anti-BMP6 antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibody, chimeric antibody, single chain antibody, Fab, and scFv. 63. Application according to any one of paragraphs. 37-62, wherein the anti-BMP6 antibody or antigen-binding fragment thereof is a component of the immunoconjugate. 64. Application according to any one of paragraphs. 37-63, wherein the anti-BMP6 antibody or antigen-binding fragment thereof has an altered effector function due to a mutation in the Fc region. 65. Application according to any one of paragraphs. 37-64, wherein the antibody or antigen-binding fragment thereof is administered at a dose ranging from 0.01 mg/kg to 15 mg/kg. 66. Application according to any one of paragraphs. 37-65, wherein the antibody or antigen-binding fragment thereof is administered to said patient more than once. 67. Application according to any one of paragraphs. 37-66, where the antibody or antigen-binding fragment is administered: a) intravenously or b) subcutaneously. 68. Application according to any one of paragraphs. 37-67, where the ferritin level is less than or equal to 1900 ng/ml, less than or equal to 1800 ng/ml, less than or equal to 1700 ng/ml, less than or equal to 1600 ng/ml, less than or equal to 1500 ng/ml, less than or is equal to 1400 ng/ml, less than or equal to 1300 ng/ml, less than or equal to 1200 ng/ml, less than or equal to 1100 ng/ml or less than or equal to 1000 ng/ml. 69. Application according to any one of paragraphs. 37-68, where the ferritin level is less than or equal to 1500 ng/ml. 70. Application according to any one of paragraphs. 37-69, where the ferritin level is greater than or equal to 600 ng/ml, greater than or equal to 700 ng/ml, greater than or equal to 800 ng/ml, greater than or equal to 900 ng/ml, greater than or equal to 1000 ng/ml, greater than or equal to 1100 ng/mL, greater than or equal to 1200 ng/mL, greater than or equal to 1300 ng/mL, greater than or equal to 1400 ng/mL, greater than or equal to 1500 ng/mL, greater than or equal to 1600 ng/mL, greater than or equal to 1700 ng /ml greater than or equal to 1800 ng/ml or greater than or equal to 1900 ng/ml. 71. Application according to any one of paragraphs. 37-70, where the ferritin level is greater than or equal to 600 ng/ml, greater than or equal to 700 ng/ml, greater than or equal to 800 ng/ml, greater than or equal to 900 ng/ml, greater than or equal to 1000 ng/ml, greater than or equal to 1100 ng/ml, greater than or equal to 1200 ng/ml, greater than or equal to 1300 ng/ml or greater than or equal to 1400 ng/ml.

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