EA039579B1 - Methods and compositions for antibodies targeting bmp6 - Google Patents

Abstract

The invention relates to antibodies and antigen-binding fragments thereof to human BMP6 and compositions and methods of use thereof.

Description

Related Applications

This patent application claims priority to US 62/094716, filed December 19, 2014, and US 62/181803, filed June 19, 2015, the contents of which are hereby incorporated by reference in their entirety.

Sequence list

The invention contains a sequence listing which has been provided electronically in ASCII format and is hereby incorporated by reference in its entirety. The specified ASCII copy, created on December 16, 2015, is designated PAT056599-WO-PCT_SL.txt and has a size of 68301 bytes.

Introduction

The present invention relates to antibodies and antigen-binding fragments to human BMP6, as well as compositions containing them and methods of using them.

State of the art

Anemia is common in patients with chronic kidney disease (CKD) and is associated with a lower quality of life and a higher risk of adverse disease outcomes, including cardiovascular disease and death. Several treatments for anemia in CKD patients include the use of erythropoiesis-stimulating drugs (ESDs), supplemental oral and intravenous iron supplements, and blood transfusions. However, many patients do not respond well to such treatment or require higher doses of ESP and/or iron. Large doses of iron can also cause toxicity associated with the formation of oxygen radicals and allergic reactions. Such treatments may not be effective enough because they do not fully address the root cause of the anemia, i.e. violation of iron absorption and mobilization of iron from the depot.

Attempts to control erythropoietin resistance are currently being made by co-administration of 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 data suggest that elevated hepcidin levels correlate with poor response to ESP during hemodialysis. Hepcidin-lowering agents may thus be an effective strategy to reduce the severity of ESP-refractory anemia in this group of patients and in other forms of anemia of chronic disease (ACD) characterized by iron restriction.

Thus, methods that reduce blood levels of hepcidin should increase iron absorption, promote the release of sequestered iron, and stimulate erythropoiesis in a patient with ESP-refractory anemia seen in patients with chronic kidney disease.

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

The essence of the invention

The present invention relates to isolated BMP6-binding molecules (e.g., BMP6-binding antibodies or antigen-binding fragments thereof), pharmaceutical compositions comprising such molecules, methods for preparing such molecules and compositions, as well as methods for their use in lowering hepcidin levels and in the treatment of anemia.

In one aspect, the invention provides an isolated anti-BMP6 antibody or antigen-binding fragment thereof, comprising any 1, 2, 3, 4, 5, or 6 CDR regions of any of the antibodies in Table 1. 1.

In one aspect, the invention provides an isolated anti-BMP6 antibody or antigen-binding fragment thereof comprising 6 CDR regions of antibody 3 as described in Table 1. 1.

In one aspect, the invention provides an isolated anti-BMP6 antibody or antigen-binding fragment thereof comprising 6 CDR regions of antibody 5 as described in Table 1. 1.

In one aspect, the invention provides an isolated anti-BMP6 antibody or antigen-binding fragment thereof comprising 6 CDRs of antibody 6 as described in Table 1. 1.

In one aspect, the invention provides an isolated anti-BMP6 antibody or antigen-binding fragment thereof, comprising 6 CDR regions of antibody 7 as described in Table 1. 1.

In one aspect of the present invention, an isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 9, 10 and 11, respectively; and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 19, 20 and 21, respectively.

In one aspect of the present invention, an isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 12, 13 and 14, respectively; and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 22, 23 and 24, respectively.

In one aspect of the present invention, an isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

- 1 039579 the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 39, 40 and 41, respectively.

In one aspect of the present invention, an isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the sequences HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 32, 33, and 34, respectively; and the sequences LCDR1, LCDR2, and LCDR3, in SEQ ID NOs: 42, 43, and 44, respectively.

In one aspect of the present invention, an isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO:

49, 50, and 51, respectively, and the sequences LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 59, 60, and 61, respectively.

In one aspect of the present invention, an isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the sequences HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 52, 53, and 54, respectively; and the sequences LCDR1, LCDR2, and LCDR3, in SEQ ID NOs: 62, 63, and 64, respectively.

In one aspect of the present invention, an isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 69, 70 and 71, respectively; and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 79, 80 and 81, respectively.

In one aspect of the present invention, an isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the sequences HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 72, 73, and 74, respectively; and the sequences LCDR1, LCDR2, and LCDR3, in SEQ ID NOs: 82, 83, and 84, respectively.

In one embodiment of the present invention, an isolated antibody or antigen-binding fragment thereof that binds human BMP6 includes:

the VH (heavy chain variable domain) sequence in SEQ ID NO: 15;

the VH sequence in SEQ ID NO: 35;

the VH sequence of SEQ ID NO: 55 or the VH sequence of SEQ ID NO: 75.

In one embodiment of the present invention, an isolated antibody or antigen-binding fragment thereof that binds human BMP6 comprises a VH sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity with one from the VH sequences described above.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the sequence of VL (light chain variable domain) in SEQ ID NO: 25;

the VL sequence in SEQ ID NO: 45;

the VL sequence of SEQ ID NO: 65 or the VL sequence of SEQ ID NO: 85.

In one embodiment of the present invention, an isolated antibody or antigen-binding fragment thereof that binds human BMP6 comprises a VL sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity with one from the VL sequences described above.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the VH sequence in SEQ ID NO: 15 and the VL sequence in SEQ ID NO: 25;

the VH sequence in SEQ ID NO: 35 and the VL sequence in SEQ ID NO: 45;

the VH sequence of SEQ ID NO: 55 and the VL sequence of SEQ ID NO: 65, or the VH sequence of SEQ ID NO: 75 and the VL sequence of SEQ ID NO: 85.

In one embodiment of the present invention, an isolated antibody or antigen-binding fragment thereof that binds human BMP6 comprises a VH sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, by

- 2 039579 at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity with one of the VH sequences described above, and include a VL sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least at least 98% or at least 99% sequence identity, but less than 100% sequence identity with one of the VL sequences described above. In an embodiment, VH and VL are derived from the same antibody listed in Table 1. 1.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the heavy chain sequence in SEQ ID NO: 17;

the heavy chain sequence in SEQ ID NO: 37;

the heavy chain sequence of SEQ ID NO: 57 or the heavy chain sequence of SEQ ID NO: 77.

In one embodiment of the present invention, an isolated antibody or antigen-binding fragment thereof that binds human BMP6 comprises a heavy chain sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity with one of the heavy chain sequences described above.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes:

the light chain sequence in SEQ ID NO: 27;

the light chain sequence in SEQ ID NO: 47;

the light chain sequence of SEQ ID NO: 67 or the light chain sequence of SEQ ID NO: 87.

In one embodiment of the present invention, an isolated antibody or antigen-binding fragment thereof that binds human BMP6 comprises a light chain sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity with one of the light chain sequences described above.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, binds human BMP6 and includes a heavy chain and a light chain, where:

the heavy chain sequence in SEQ ID NO: 17 and the light chain sequence in SEQ ID NO: 27;

the heavy chain sequence in SEQ ID NO: 37 and the light chain sequence in SEQ ID NO: 47;

heavy chain sequence in SEQ ID NO: 57 and light chain sequence in SEQ ID

NO: 67 or heavy chain sequence in SEQ ID NO: 77 and light chain sequence in SEQ ID

NO: 87.

In one embodiment of the present invention, an isolated antibody or antigen-binding fragment thereof that binds human BMP6 comprises a heavy chain sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity with one of the heavy chain sequences described above, and include a light chain sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity with one of the light chain sequences, op written above. In an embodiment, the heavy chain and light chain are derived from the same antibody listed in Table 1. 1.

In one embodiment, the present invention provides an isolated antibody or antigennegative fragment thereof that specifically binds to BMP6, wherein said antibody or antigennegative fragment thereof comprises at least one complementarity determining (CDR) sequence having at least 90, 95, 97, 98 % or at least 99% sequence identity with any one or more of:

- 3 039579 sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 9, 10 and 11, respectively, and LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NOs: 19, 20 and 21, respectively;

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 22, 23 and 24, respectively;

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

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

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

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

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 79, 80 and 81, respectively, or the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 82, 83, and 84, respectively.

In one embodiment of the present invention, the isolated monoclonal antibody, or antigen-binding fragment thereof, specifically binds to BMP6 and includes at least one complementarity determining (CDR) sequence that is identical to any one or more of:

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 19, 20 and 21, respectively;

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 22, 23 and 24, respectively;

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

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

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

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

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 79, 80 and 81, respectively, or the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 82, 83, and 84, respectively.

In one embodiment of the present invention, isolated antibodies or binding fragments thereof specifically bind to BMP6, wherein said antibodies comprise at least one heavy chain CDR sequence selected from the group consisting of:

HCDR1, HCDR2, and HCDR3 sequences HCDR1, HCDR2, and HCDR3 sequences HCDR1, HCDR2, and HCDR3 sequences HCDR1, HCDR2, and HCDR3 sequences HCDR1, HCDR2, and HCDR3 sequences HCDR1, HCDR2, and HCDR3 sequences HCDR1, HCDR2, and HCDR3 sequences in in in in in in in

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

12, 13

29, 30

32, 33

49, 50

52, 53

69, 70

72, 73 and and and and and and and and

9, 10 and 11.

In one embodiment of the present invention, isolated antibodies or binding fragments thereof specifically bind to BMP6, wherein said antibodies comprise at least one light chain CDR sequence selected from the group consisting of:

antigen for less sequences LCDR1, LCDR2 and LCDR3 sequences LCDR1, LCDR2 and LCDR3 sequences LCDR1, LCDR2 and LCDR3 sequences LCDR1, LCDR2 and LCDR3 sequences LCDR1, LCDR2 and LCDR3 sequences LCDR1, LCDR2 and LCDR3 sequences LCDR1, LCDR2 and LCDR3 sequences LCDR1, LCDR2 and LCDR3 in in in in in in in in

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

19, 20

22, 23

39, 40

42, 43

59, 60

62, 63

79, 80

82, 83 and and and and and and and and respectively;

respectively;

respectively;

respectively;

respectively;

respectively;

respectively and respectively.

In embodiments, the isolated antibody, or antigen-binding fragment thereof, is monoclonal.

In one embodiment, the present invention provides isolated antibodies, or antigen-binding fragments thereof, that specifically bind to BMP6, wherein said antibody

- 4 039579 has an affinity constant (KA) of at least about 1x10 ⁷ M ^-1 , 10 ⁸ M ^-1 , 10 ⁹ M ^-1 , 10 ¹⁰ M ^-1 or 10 ¹¹ M ^-1 . In one embodiment, the present invention provides isolated antibodies, or antigen-binding fragments thereof, that specifically bind to BMP6, wherein said antibody has an affinity constant (KA) of at least 1x10 ⁷ M ^-1 , 10 ⁸ M ^-1 , 10 ⁹ M ^-1 , 10 ¹⁰ M ^-1 or 10 ¹¹ M ^-1 .

In one embodiment of the present invention, an isolated antibody or antigen-binding fragment thereof specifically binds to BMP6, wherein said antibody or antigen-binding fragment thereof binds to BMP6 with a Kd of not greater than about 1 nM or not greater than about 0.1 nM. In one embodiment of the present invention, the isolated antibody or antigen-binding fragment thereof specifically binds to BMP6, wherein said antibody or antigen-binding fragment thereof binds to BMP6 with a Kd of not greater than 1 nM or not greater than 0.1 nM. In one embodiment of the present invention, the isolated antibody or antigen-binding fragment thereof specifically binds to BMP6, wherein said antibody or antigen-binding fragment thereof binds to BMP6 with a Kd<1 nM or <0.1 nM. In one embodiment, Kd corresponds to a BiaCore measurement.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, specifically binds to BMP6 and inhibits BMP6 activity.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, specifically binds to BMP6 and cross-competes with (cross-blocks) the antibody described in Table 1. 1 below. In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, bind to the same BMP6 epitope, cross-compete with the antibody described in Table 1. 1 below.

In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, have at least about 100, 500, or 1000 times greater affinity for human BMP6 than for any of: human BMP2, human BMP5, or human BMP7. In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, have at least 100, 500, or 1000 times greater affinity for human BMP6 than for any of: human BMP2, human BMP5, or human BMP7. In embodiments, the BMP6 specificity is as measured by ELISA.

In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, have at least about 100, 500, or 1000 times greater affinity for human BMP6 than for human BMP2. In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, have at least 100, 500, or 1000 times greater affinity for human BMP6 than for human BMP2. In embodiments, the BMP6 specificity is as measured by ELISA.

In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, have at least about 100, 500, or 1000 times greater affinity for human BMP6 than for human BMP5. In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, have at least 100, 500, or 1000 times greater affinity for human BMP6 than for human BMP5. In embodiments, the BMP6 specificity is as measured by ELISA.

In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, have at least about 100, 500, or 1000 times greater affinity for human BMP6 than for human BMP7. In one embodiment of the present invention, the isolated antibodies, or antigen-binding fragments thereof, have at least 100, 500, or 1000 times greater affinity for human BMP6 than for human BMP7. In embodiments, the BMP6 specificity is as measured by ELISA.

In one embodiment of the present invention, the antibody or antigen-binding fragment does not have any detectable binding to human BMP2 or BMP5 (eg, in ELISA).

In one embodiment of the present invention, the antibody or antigen-binding fragment does not have any detectable activity against human BMP2 (eg, in ELISA).

In one embodiment of the present invention, the antibody or antigen-binding fragment does not have any detectable activity against human BMP5 (eg, in ELISA).

In one embodiment, the antibodies and antigen-binding fragments thereof according to the invention that specifically bind to BMP6 are isolated monoclonal antibodies. In one embodiment, the antibodies and antigen-binding fragments thereof according to the invention that specifically bind to BMP6 are isolated human monoclonal antibodies. In one embodiment, the antibodies and antigen-binding fragments thereof according to the invention that specifically bind to BMP6 are humanized monoclonal antibodies. In one embodiment, the antibodies and antigen-binding fragments thereof according to the invention that specifically bind to BMP6 are isolated chimeric antibodies. In one embodiment, the antibodies and antigen-binding fragments thereof of the invention comprise a human heavy chain constant region and a human light chain constant region.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, that specifically binds to BMP6 is a single chain antibody.

In one embodiment of the present invention, the isolated antibody or antigen-binding fragment thereof that specifically binds to BMP6 is a Fab fragment.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, that specifically binds to BMP6 is a scFv.

In one embodiment, the antibodies of the invention are IgM or IgG. In one embodiment of the present invention, the IgG is IgG1, IgG2, IgG3, or IgG4. In one embodiment, the IgG is IgG1.

In one embodiment of the present invention, the isolated antibodies or antigen-binding fragments thereof comprise a backbone in which the amino acids have been replaced by an antibody backbone from the corresponding human germline VH or VL sequences.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, is a component of an immunoconjugate. In one embodiment, the immunoconjugate may include an isolated antibody or antigen-binding fragment thereof, and any of the following as non-limiting examples: an enzyme, toxin, hormone, growth factor, or drug.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, has an altered effector function as a result of a mutation in the Fc region.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof cross-block the antibody or isolated antigen-binding fragment listed in Table 1. 1.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, inhibits BMP6-induced hepcidin expression in liver cells (eg, liver cell lines and/or primary human liver cells in vitro). In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, inhibits BMP6-induced hepcidin expression in liver cells (eg, liver cell lines and/or primary human liver cells in vitro) by at least about 50%. For example, an isolated antibody or antigen-binding fragment thereof inhibits BMP6-induced hepcidin expression in liver cells by at least about 50%, 60%, 70%, 80%, 90%, or 100%. Hepcidin expression can be measured, as non-limiting examples, by measuring the amount of hepcidin mRNA or protein levels. In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, inhibits BMP6-induced hepcidin expression in liver cells (eg, liver cell lines and/or primary human liver cells in vitro) by at least 50%. For example, an isolated antibody or antigen-binding fragment thereof inhibits BMP6-induced hepcidin expression in liver cells by at least 50%, 60%, 70%, 80%, 90%, or 100%. Hepcidin expression can be measured, as non-limiting examples, by measuring the amount of hepcidin mRNA or protein levels.

In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, reduces the activity of human BMP6 in vitro. In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, reduces in vitro human BMP6 activity as measured in the HEP3BBRE-Luc reporter gene assay.

In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds BMP6 and that binds a human BMP6 epitope comprising the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 92 or amino acids 88-102 of SEQ ID NO: 89). In one aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, that specifically binds BMP6 and that binds a human BMP6 epitope consisting of the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 92 or amino acids 88102 in SEQ ID NO: 89). In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, has at least about 100-fold greater

- 6 039579 affinity for the human BMP6 epitope consisting of the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 92 or amino acids 88-102 in SEQ ID NO: 89) than for: (a) the human BMP7 epitope consisting of the sequence QTLVHFINPETVPKP (SEQ ID NO : 93 or amino acids 88102 in SEQ ID NO: 90) or (b) a human BMP5 epitope consisting of the sequence QTLVHLMFPDHVPKP (SEQ ID NO: 94 or amino acids 87-101 in SEQ ID NO: 91). In one embodiment of the present invention, the isolated antibody or antigen-binding fragment thereof has at least 100-fold greater affinity for the human BMP6 epitope consisting of the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 92 or amino acids 88-102 of SEQ ID NO: 89) than to: (a) a human BMP7 epitope consisting of the sequence QTLVHFINPETVPKP (SEQ ID NO: 93 or amino acids 88-102 of SEQ ID NO: 90) or (b) a human BMP5 epitope consisting of the sequence QTLVHLMFPDHVPKP (SEQ ID NO: 94 or amino acids 87-101 in SEQ ID NO: 91). Such an isolated antibody or antigen-binding fragment thereof may include, as a non-limiting example: (a) the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 79, 80 and 81, respectively, or (b) the sequences HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 72, 73, and 74, respectively, and the sequences LCDR1, LCDR2, and LCDR3, in SEQ ID NOs: 82, 83, and 84, respectively. In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, comprises the VH sequence of SEQ ID NO: 75; and the VL sequence in SEQ ID NO: 85. In an embodiment, the affinity corresponds to the measurement using BiaCore.

In one embodiment of the present invention, the isolated antibody or antigen-binding fragment thereof has at least about 500-fold greater affinity for a human BMP6 epitope consisting of the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 92 or amino acids 88-102 of SEQ ID NO: 89), than to: (a) a human BMP7 epitope consisting of the sequence QTLVHFINPETVPKP (SEQ ID NO: 93 or amino acids 88-102 of SEQ ID NO: 90) or (b) a human BMP5 epitope consisting of the sequence QTLVHLMFPDHVPKP (SEQ ID NO: 94 or amino acids 87-101 in SEQ ID NO: 91). Such an isolated antibody or antigen-binding fragment thereof may include, as non-limiting examples: (a) the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 79, 80 and 81, respectively, or (b) the sequences HCDR1, HCDR2, and HCDR3 in SEQ ID NOs: 72, 73, and 74, respectively, and the sequences LCDR1, LCDR2, and LCDR3, in SEQ ID NOs: 82, 83, and 84, respectively. In one embodiment of the present invention, the isolated antibody, or antigen-binding fragment thereof, comprises the VH sequence of SEQ ID NO: 75; and the VL sequence in SEQ ID NO: 85. In an embodiment, the affinity corresponds to the measurement using BiaCore.

In another aspect, the invention relates to a composition, for example, a pharmaceutical composition, comprising an isolated antibody or antigen-binding fragment according to any of the previous aspects or embodiments.

In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

In one embodiment, the composition further comprises an additional therapeutic agent.

In one embodiment of the present invention, an additional therapeutic agent reduces the activity of BMP6.

In one embodiment of the present invention, the additional therapeutic agent is an siRNA, an antibody or antigen-binding fragment or small molecule thereof.

In one embodiment of the present invention, the additional therapeutic agent is selected from the group consisting of: an erythropoiesis-stimulating drug (ESD) and an iron supplement (eg, an iron supplement or an IV iron supplement).

In one embodiment of the present invention, the additional therapeutic agent is an erythropoiesis-stimulating drug (ESD), such as EPO.

In one embodiment, the selected antibody or antigennegative fragment described in table. 1 may be administered to a patient in combination with a therapeutic method or procedure, such as those described in the present invention or known in the art. Selected antibody or antigennegative fragment described in table. 1 may be administered before, after, or at the same time as the method or procedure.

In one embodiment, the therapeutic method or procedure is a blood transfusion. In one embodiment, the therapeutic method or procedure is dialysis. In one embodiment, the therapeutic method or procedure is the administration of an ESP, such as an EPO. In one embodiment, the therapeutic method or procedure is the administration of an iron preparation, such as an intravenous iron preparation. In one embodiment, the therapeutic method or procedure is the administration of an ESP, such as EPO, and the administration of an iron preparation, such as an intravenous iron preparation. In one embodiment, the therapeutic method or process

- 7 039579 a combination of any of the previous ones is foolish.

In another aspect, the present invention includes a nucleic acid (polynucleotide) encoding any of the antibodies or antigen-binding fragments thereof described in the present invention. In one embodiment, the present invention provides a nucleic acid that encodes the isolated antibody or antigen-binding fragment described in Table. 1, including any one or more of:

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 19, 20 and 21, respectively;

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 22, 23 and 24, respectively;

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

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

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

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

the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 79, 80 and 81, respectively, or the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 82, 83, and 84, respectively.

In one embodiment, the present invention relates to a nucleic acid (polynucleotide) that encodes an isolated antibody or antigen-binding fragment described in table. 1, comprising an amino acid sequence comprising a sequence selected from the group consisting of:

heavy chain sequences in SEQ ID NO: 17;

the VH sequences in SEQ ID NO: 15;

light chain sequences in SEQ ID NO: 27;

the VL sequences in SEQ ID NO: 25;

heavy chain sequences in SEQ ID NO: 37;

the VH sequences in SEQ ID NO: 35;

light chain sequences in SEQ ID NO: 47;

the VL sequences in SEQ ID NO: 45;

heavy chain sequences in SEQ ID NO: 57;

the VH sequences in SEQ ID NO: 55;

light chain sequences in SEQ ID NO: 67;

the VL sequences in SEQ ID NO: 65;

heavy chain sequences in SEQ ID NO: 77;

the VH sequences in SEQ ID NO: 75;

light chain sequences in SEQ ID NO: 87; and VL sequences in SEQ ID NO: 85.

In one embodiment, the present invention relates to a nucleic acid (polynucleotide) that encodes an isolated antibody or antigen-binding fragment described in table. 1, comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from the group consisting of:

heavy chain sequences in SEQ ID NO: 17;

the VH sequences in SEQ ID NO: 15;

light chain sequences in SEQ ID NO: 27;

the VL sequences in SEQ ID NO: 25;

heavy chain sequences in SEQ ID NO: 37;

the VH sequences in SEQ ID NO: 35;

light chain sequences in SEQ ID NO: 47;

the VL sequences in SEQ ID NO: 45;

heavy chain sequences in SEQ ID NO: 57;

the VH sequences in SEQ ID NO: 55;

light chain sequences in SEQ ID NO: 67;

the VL sequences in SEQ ID NO: 65;

heavy chain sequences in SEQ ID NO: 77;

the VH sequences in SEQ ID NO: 75;

light chain sequences in SEQ ID NO: 87 and

- 8 039579 of the VL sequence in SEQ ID NO: 85.

In one embodiment, the present invention relates to a nucleic acid (polynucleotide) that encodes an isolated antibody or antigen-binding fragment described in table. 1, where the nucleic acid includes a sequence selected from the group consisting of:

heavy chain sequences in SEQ ID NO: 18;

heavy chain sequences in SEQ ID NO: 38;

heavy chain sequences in SEQ ID NO: 58;

heavy chain sequences in SEQ ID NO: 78;

light chain sequences in SEQ ID NO: 28;

light chain sequences in SEQ ID NO: 48;

light chain sequences in SEQ ID NO: 68;

light chain sequences in SEQ ID NO: 88;

the VH sequences in SEQ ID NO: 16;

the VH sequences in SEQ ID NO: 36;

the VH sequences in SEQ ID NO: 56;

the VH sequences in SEQ ID NO: 76;

the VL sequences in SEQ ID NO: 26;

the VL sequences in SEQ ID NO: 46;

the VL sequences of SEQ ID NO: 66; and the VL sequences of SEQ ID NO: 86.

In another aspect, the present invention also relates to a vector comprising such nucleic acids or polynucleotides.

In another aspect, the present invention also relates to a host cell comprising such nucleic acids or polynucleotides. In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell. In one embodiment of the present invention, the isolated host cells comprise a vector comprising such nucleic acids or polynucleotides.

In one embodiment, the present invention relates to an isolated host cell comprising: (1) a recombinant nucleic acid segment encoding the heavy chain of the antibodies of the invention, and (2) a second recombinant nucleic acid segment encoding the light chain of the antibodies of the invention; wherein said DNA segments are suitably operably linked to the first and second promoters and are capable of being expressed in said host cell. In another embodiment of the present invention, the isolated host cells comprise a recombinant DNA segment encoding the heavy chain and light chain of the antibodies of the invention, respectively, wherein said DNA segment is operably linked to a promoter and is capable of being expressed in said host cells. In one embodiment, the host cells are a non-human mammalian cell line. In one embodiment, the antibody or antigennegative fragment thereof is a human monoclonal antibody or antigennegative fragment thereof.

The present invention relates to the use of an anti-BMP6 antibody or antigen-binding fragment thereof, polynucleotide, vector or host cell as described in the present invention in the preparation of a medicament. The present invention relates to an antibody or antigennegative fragment thereof, as described in the present invention, for use as a drug. The present invention relates to an antibody, or antigen-binding fragment thereof, as described in the present invention, for use in therapy. The present invention provides an antibody, or antigen-binding fragment thereof, as described herein, for use in the treatment of anemia, such as anemia of 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 embodiments, the anemic patient has been or is being treated with an erythropoiesis-stimulating drug (ESP), such as erythropoietin (EPO).

In another aspect, the present invention provides methods for using antibodies and antigen-binding fragments thereof, and compositions comprising such antibodies and antigen-binding fragments thereof, as described herein. In one aspect, the invention provides a method for reducing hepcidin activity or levels in a patient, comprising the step of administering to the patient an anti-BMP6 antibody or antigen-binding fragment thereof, as described herein. In one embodiment of this method, the activity or level of hepcidin is reduced by at least 50%. In an embodiment, the patient is anemic. In embodiments, the anemia is anemia of chronic disease (ACD), such as anemia of chronic kidney disease (CKD), anemia of cancer, or anemia of inflammation. In an embodiment, the anemia is erythropoiesis-stimulating drug (ESD) resistant anemia or iron deficiency anemia.

The present invention relates to a method for treating anemia in a patient, comprising the step of

- 9 039579 administration to a patient of an antibody or antigen-binding fragment described in the present invention. In embodiments, the anemia is anemia of chronic disease (ACD), such as anemia of chronic kidney disease (CKD), anemia of cancer, or anemia of inflammation. In an embodiment, the anemia is erythropoiesis-stimulating drug (ESD) resistant anemia or iron deficiency anemia. In embodiments, the patient has been or is being treated with an erythropoiesis-stimulating drug (ESP), such as erythropoietin (EPO). In embodiments, the anemia is EPO-hyporeactive anemia. In embodiments, the anemia is iron deficiency anemia. In embodiments, the patient is a chronic hemodialysis patient.

In another embodiment, the present invention provides a method for inhibiting BMP6 in a patient, wherein the method comprises the step of administering to the patient an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to the invention. In embodiments, the patient is anemic. In embodiments, the anemia is anemia of chronic disease (ACD), such as anemia of chronic kidney disease (CKD), anemia of cancer, or anemia of inflammation. In an embodiment, the anemia is erythropoiesis-stimulating drug (ESD) resistant anemia or iron deficiency anemia. In embodiments, the patient has been or is being treated with an erythropoiesis-stimulating drug (ESP), such as erythropoietin (EPO). In embodiments, the anemia is EPO-hyporeactive anemia. In embodiments, the anemia is iron deficiency anemia. In embodiments, the patient is a chronic hemodialysis patient.

The present invention also relates to a method for reducing BMP6 activity in a cell, comprising the step of contacting the cell with an antibody or antigen-binding fragment of the invention.

The present invention further relates to a method for increasing serum iron levels, transferrin saturation (TSAT), reticulocyte hemoglobin (CHr), reticulocyte count, red blood cell count, hemoglobin and/or hematocrit in a patient, comprising the step of administering to the patient an effective amount of an antibody or antigen-binding antibody thereof. fragment according to the invention.

The present invention further relates to a method for increasing or maintaining a hemoglobin level in a patient, comprising administering to the patient an antibody, or antigen-binding fragment thereof, described in the present invention. In embodiments, the patient is anemic. In embodiments, the anemia is anemia of chronic disease (ACD), such as anemia of chronic kidney disease (CKD), anemia of cancer, or anemia of inflammation. In an embodiment, the anemia is erythropoiesis-stimulating drug (ESD) resistant anemia or iron deficiency anemia. In embodiments, the patient has been or is being treated with an erythropoiesis-stimulating drug (ESP), such as erythropoietin (EPO). In embodiments, the anemia is EPO-hyporeactive anemia. In embodiments, the anemia is iron deficiency anemia. In embodiments, the patient is a chronic hemodialysis patient. In embodiments, the method further comprises reducing the patient's required dose of iron, decreasing the patient's required EPO dose, or reducing the patient's required iron dose and desired EPO dose, compared to said required EPO dose and/or required dose of iron in the absence of treatment. an antibody or antigen-binding fragment described in the present invention. In embodiments, the hemoglobin level increases or remains at at least about 10.0, at least about 11.0, or at least about 12.0 g/dl. In embodiments, the hemoglobin level increases or remains at a level of at least 10.0, at least 11.0, or at least 12.0 g/dL.

In any of the above methods, the step of administering to a patient an antibody, or an antigen-binding fragment thereof, as described herein, includes the step of administering to a patient a composition that comprises the antibody, or antigen-binding fragment thereof, as described in the present invention.

In any of the above methods, the antibody or its antigen-binding fragment can be administered at a dose of 0.001-0.1 mg/kg, for example, at a dose of 0.001, 0.0016, 0.0025, 0.0040, 0.0063, 0.01, 0.016 , 0.025, 0.040, 0.063 or 0.1 mg/kg. In any of the above methods, the antibody or antigen-binding fragment thereof may be administered at a dose of from about 0.001 to about 0.1 mg/kg, for example, at a dose of about 0.001 mg/kg, about 0.0016 mg/kg, about 0.0025 mg/kg. kg, approximately 0.0040 mg/kg, approximately 0.0063 mg/kg, approximately 0.01 mg/kg, approximately 0.016 mg/kg, approximately 0.025 mg/kg, approximately 0.040 mg/kg, approximately 0.063 mg/kg, or approximately 0.1 mg/kg.

In embodiments, the antibody or antigen-binding fragment thereof is administered intravenously. In embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously. In embodiments, the antibody or antigen-binding fragment thereof is administered by infusion over a period of

- 10 039579 about 30 to about 60 minutes.

In another aspect, the present invention relates to an antibody or antigennegative fragment to BMP6, including the CDRs listed in table. 1. The present invention relates to an antibody or antigennegative fragment to VMP6 listed in table. 1. The present invention relates to a selected polynucleotide encoding an antibody or antigennegative fragment to BMP6, including the CDR listed in table. 1. In one embodiment of the present invention, the polynucleotide or nucleic acid is isolated. In one embodiment of the present invention, the antibody, or antigen-binding fragment thereof, is isolated.

Definitions

Unless otherwise indicated, all technical and scientific terms used in the present description have the same meaning, by which they are usually understood by those of ordinary skill in the art to which the present invention pertains.

BMP6 as used herein means bone morphogenetic protein 6 (BMP6) 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 also known as: BMP-6; VGR; VGR1; External ID: 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; localization (UCSC): Xp 6: 7.73-7.88 Mb; species: mouse: Entrez: 12161; Ensemble: ENSMUSG00000039004; UniProt: P20722; RefSeq (mRNA): NM_007556; RefSeq (protein): NP_031582; localization (UCSC): Xp 13: 38.35-38.5 MB. As described in the present invention, an antibody and an antigen-binding fragment thereof that bind to BMP6 bind to the BMP6 protein.

BMP2 as used herein means bone morphogenetic protein 2 (BMP2) or a gene or nucleic acid encoding BMP2. BMP2 is also known as: BDA2; and BMP2A; External ID OMIM: 112261 MGI: 88177 HomoloGene: 926 GeneCards: BMP2 gene. species: human; Entrez: 650; Ensemble: ENSG00000125845; UniProt: P12643; RefSeq (mRNA): NM_001200; RefSeq (protein): NP_001191; localization (UCSC): Xp 20: 6.75-6.76 MB. Species: mouse; Entrez: 12156; Ensemble: ENSMUSG00000027358; UniProt: P21274; RefSeq (mRNA): NM_007553; RefSeq (protein): NP_031579; localization (UCSC): Xp 2: 133.55-133.56 MB. As described in the present invention, an antibody and an antigen-binding fragment thereof that bind to BMP2 bind to the BMP2 protein.

BMP5 as used herein means bone morphogenetic protein 5 (BMP5) or a gene or nucleic acid encoding BMP5. BMP5 also known as: MGC34244; External ID OMIM: 112265 MGI: 88181 HomoloGene: 22412 GeneCards: BMP5 gene. Species: human; Entrez: 653; Ensemble: ENSG00000112175; UniProt: P22003; RefSeq (mRNA): NM_021073; RefSeq (protein): NP_066551; localization (UCSC): Xp 6: 55.62-55.74 MB. Species: mouse; Entrez: 12160; Ensemble: ENSMUSG00000032179; UniProt: P49003; RefSeq (mRNA): NM_007555; RefSeq (protein): NP_031581; localization (UCSC): Xp 9: 75.78-75.9 MB. As described in the present invention, an antibody and an antigen-binding fragment thereof that bind to BMP5 bind to the BMP5 protein.

BMP7 as used herein means bone morphogenetic protein 7 (BMP7) or a gene or nucleic acid encoding BMP7. BMP7 is also known as: osteogenic protein 1; OR-1; External ID OMIM: 112267 MGI: 103302 HomoloGene: 20410 GeneCards: BMP7 gene. Species: human; Entrez: 655; Ensemble: ENSG00000101144; UniProt: P18075; RefSeq (mRNA): NM_001719; RefSeq (protein): NP_001710; localization (UCSC): Xp 20: 55.74-55.84 MB. Species: mouse; Entrez: 12162; Ensemble: ENSMUSG00000008999; UniProt: P23359; RefSeq (mRNA): NM_007557; RefSeq (protein): NP_031583; localization (UCSC): Xp 2: 172.87-172.94 MB. As described in the present invention, an antibody and an antigen-binding fragment thereof that bind to BMP7 bind to the BMP7 protein.

Hepcidin means the gene for hepcidin or the protein hepcidin, a peptide hormone. Hepcidin is also known as: HAMP (hepcidin antimicrobial protein or peptide); NERS; HFE2B; LEAP1 (LEAP-1); PLTR; OMIM: 606464; HomoloGene: 81623; GeneCards: HAMP gene; Entrez 57817; Ensembl ENSG00000105697; UniProt P81172; RefSeq (mRNA) NM_021175; RefSeq (protein) NP_066998; localization (UCSC) Xp 19: 35.77-35.78 MB. 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.

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 reduced ability of the blood to carry oxygen.

- 11 039579

Anemia can be diagnosed using any method known in the art, including, as a non-limiting example, in men with hemoglobin below about 130-140 g/l (13-14 g/dl) and in women below about 120- 130 g/l (12-13 g/dl). Janz et al. 2013

Emerg. 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, BMP6 antagonist antibody, and the like. (and their antigennegative fragments) include antibodies (and their antigennegative fragments) that bind to the BMP6 protein.

The terms antibody, antigen-binding fragment, antigen-binding portion, and the like. when used herein, include full antibodies and any antigen-binding fragment (ie, antigen-binding portion) or single chains thereof. A natural antibody is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked 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 regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are most conserved, termed framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from N-terminus to C-terminus 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 binding of immunoglobulin to self tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

The terms antigen-binding fragment, antigen-binding fragment thereof, antigen-binding portion of an antibody, and the like. as used herein, refers 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 may provide fragments of an intact antibody. Examples of binding fragments encompassed by the term antigen-binding portion of an antibody include the Fab fragment , monovalent fragment consisting of VL, VH, CL, and CH1 domains; F(ab) ₂ fragment, bivalent fragment, including two Fab fragments linked by a disulfide bridge in the hinge region; Fd fragment, consisting of VH and CH1 domains; Fv a fragment consisting of the VL and VH domains of one arm of an antibody, a single domain fragment (dAb) of an antibody (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain, and an isolated complementarity determining region (CDR).

In addition, although the two domains of the Fv fragment, VL and VH, encode separate genes, they can be connected using recombinant methods, using an artificial peptide linker that allows them to be synthesized as a single protein chain in which the VL and VH regions are paired. to form monovalent molecules (known as single chain Fv (scFv); 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 an antibody. Such antibody fragments are prepared using standard methods known to those skilled in the art, with the fragments tested 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 into scaffolds based on polypeptides 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-chain molecules comprising 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): 10571062; 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 single antigenic sites. At each antigenic site, the antibody variable arm interacts through weak non-covalent forces with the antigen 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. It depends on three main factors: the affinity of the antibody for the epitope; valency of antigen and antibody; and structural arrangement of interacting parts. Ultimately, these factors determine the specificity of an antibody, that is, the likelihood that a particular antibody will bind to a particular epitope on an antigen.

- 12 039579

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. Natural amino acids are amino acids encoded by the genetic code, as well as those amino acids that undergo subsequent modification, such as hydroxyproline, gamma carboxyglutamate and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as the naturally occurring amino acid, i.e. an alpha carbon atom bonded to hydrogen, a carboxyl group, an amino group, and an R group, for example, 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 functions similarly to a naturally occurring amino acid.

The term binding specificity as used herein refers to the ability of a single antibody paratope to react with only one antigenic determinant. The paratope 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 interaction between one antigenic determinant and one paratope on an antibody. This is the sum of the attractive and repulsive forces acting between the antigenic determinant and the antibody paratope.

Specific binding between two molecules means binding with an equilibrium constant (KA or K _A ) of at least 1x10 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, BMP6 binding antibody) refers to a binding reaction that detects the presence of a cognate antigen (eg, human BMP6 protein) in a heterogeneous population of proteins and other biologicals. In addition to the equilibrium constant (KA) noted above, a BMP6-binding antibody of the invention typically also has a dissociation rate constant (Kd or KD or KD) of approximately 1x10' ² s ^-1 , 1x10 ^-3 s ^-1 or less , and binds to BMP6 with an affinity that is at least twice that of its binding affinity to a non-specific antigen (eg, 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 5x10 ² M ^-1 , at least 10 ³ M- ¹ , at least 5x10 ³ M- ¹ , at least 10 ⁴ M- ¹ , at least 5x10 ⁴ M ^-1 , at least 105 M ^-1 , at least 5x105 M ^-1 , at least 106 M ^-1 , at least 5x106 M ^-1 , at least 10 ⁷ M ^-1 , at least 5x10 ⁷ M ^-1 , at least 10 ⁸ M ^-1 , at least 5x10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 5x10 ⁹ M ^-1 , at least 10 ¹⁰ M ^-1 , at least 5x10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 5x10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , at least 5x10 ¹² M ^-1 , at least 10 ¹³ M ^-1 , at least 5x10 ¹³ M ^-1 , at least 10 ¹⁴ M ^-1 , at least 5x10 ¹⁴ M ^-1 , at least at least 10 ¹⁵ M ^-1 or at least 5x1015 M ^-1 .

The term chimeric antibody (or antigen-binding fragment thereof) is an antibody molecule (or antigen-binding fragment thereof) in which: or an altered class, effector function, and/or species, or a completely different molecule that confers new properties on the chimeric antibody, eg, an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced, or replaced by 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 replacing with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen, while reducing antigenicity in humans compared to the original mouse antibody.

The term conservatively modified variant refers 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, substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode one protein or another. For example, all codons GCA, GCC, GCG, and GCU code for the amino acid alanine. Thus, at each position at which an alanine is defined 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 of the varieties of conservative modality.

- 13 039579 fixed variations. Each nucleic acid sequence in the present description that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art will appreciate that every codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be changed to produce a functionally identical molecule. Thus, every silent variation of a nucleic acid that encodes a polypeptide is implicitly implied in every sequence described.

In the case of polypeptide sequences, conservatively modified variants include single substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Tables of conservative substitutions that present functionally similar amino acids are known in the art. Such conservatively modified variants are provided additionally and do not exclude polymorphic variants, interspecies homologues and alleles according to the 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 cause a significant change in the binding characteristics of an antibody containing an amino acid sequence.

The term block, as used herein, refers to stopping or preventing an interaction or process, such as stopping ligand-dependent or ligand-independent signaling.

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

The terms cross-block, cross-block, cross-block, compete, cross-compete, and related terms are used interchangeably herein to refer to the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to BMP6 in a standard competitive binding assay.

The ability or extent 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 be said to cross-block according to the invention can be determined using standard competitive binding assays. One suitable assay involves the use of BiaCore technology (eg, using the BIAcore 3000 instrument (BiaCore, Uppsala, Sweden)), which allows the degree of interactions to be measured using surface plasmon resonance technology. Another assay uses an ELISA-based method to measure cross-blocking.

The term neutralizes means that the antibody, when bound to its target, reduces the activity, level, or stability of the target; for example, an anti-BMP6 antibody, when bound to BMP6, neutralizes BMP6 to reduce, at least in part, the activity, level, or stability of BMP6, eg, signaling, or its role in hepcidin levels and anemia.

The term epitope means a protein determinant capable of specific 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 structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

The term epitope includes any protein determinant capable of specifically binding to an immunoglobulin or otherwise interacting with a molecule. Epitopic determinants usually consist of reactive surface groups of molecules such as BMP6 amino acids or carbohydrate or sugar side chains and may have specific three-dimensional structural 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 points of interaction between a protein and an interacting molecule (such as an antibody) are located linearly along the primary amino acid sequence of the (contiguous) protein.

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

The term human antibody (or antigen-binding fragment thereof) as used herein is intended to include antibodies (and antigen-binding fragments thereof) having variable regions in which 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 mutant variants of human germline sequences. Human antibodies and antigen-binding fragments thereof according to the invention may include amino acid residues not encoded by human sequences (eg, mutations introduced by random or site-specific in vitro mutagenesis or somatic mutation in vivo).

The phrases monoclonal antibody or composition of a monoclonal antibody (or antigen-binding fragment thereof) as used herein refers to polypeptides, including antibodies, antibody fragments, bispecific antibodies, etc., that have substantially identical amino acid sequences or are derived from the same genetic source. This term also includes preparations of antibody molecules of the same molecular composition. A monoclonal antibody composition exhibits one 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 the framework and CDR regions are derived from human sequences. In one embodiment, human monoclonal antibodies are synthesized by a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

The phrase recombinant human antibody (or antigen-binding fragment thereof), as used herein, includes all human antibodies (and antigen-binding fragments thereof) that are produced, expressed, generated, or isolated by genetic engineering, such as antibodies isolated from an animal (e.g., , mouse) that is transgenic or transchromosomal for human immunoglobulin genes, or a hybridoma derived from it, antibodies isolated from a host cell transformed to express a human antibody, e.g., from a transfectome, antibodies isolated from a recombinant, combinatorial library of human antibodies , and antibodies produced, expressed, constructed or isolated by any other means which involve splicing all or part of the human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from germline human immunoglobulin sequences. In one embodiment, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, in the case of an animal transgenic for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences which, while derived from and related to human germline VH and VL sequences, may not naturally exist within the in vivo human germline antibody repertoire.

A humanized antibody (or antigen-binding fragment thereof) as used herein is an antibody (or antigen-binding fragment thereof) that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remaining portions of the antibody with their human counterparts (ie, the constant region as well as the framework portions 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 engineering technology include, without limitation, the Hoth technology disclosed in US Pat. No. 5,766,886.

The terms identical or percent identity, in relation to two or more nucleic acid sequences or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Two sequences are substantially identical if two sequences contain a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65, 70, 75, 80, 85, 90, 95, or 99% identity over the specified area, or, if not specified, throughout the entire sequence), when compared and aligned with the best match in the comparison window or specified area, when measured using one of the following sequence comparison algorithms or manual alignment and visual review. Not necessarily the identity is present 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). Optionally, the identity is present 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, typically one sequence acts as a reference sequence against which the analyzed sequences are compared. When using the sequence comparison algorithm, the analyzed and reference sequences are entered into the computer, if necessary, the coordinates of the subsequences are determined and the software parameters of the sequence analysis algorithm are set. Default program parameters may be used, or alternative parameters may be set. The sequence comparison algorithm then calculates percent sequence identity for the analyzed sequences relative to the reference sequence, based on the program parameters.

A comparison window as used herein includes reference to a segment of any number of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, in which the sequence can be compared to reference sequence with the same number of contiguous positions, after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm of Smith and Waterman (Smith and Waterman (1970) Adv. Appl. Math. 2:482c), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), the Pearson and Lipman similarity search method (Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual review (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 in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990 respectively. The program for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) while identifying short words of length W in the requested sequence that meets or satisfies some threshold score T with a positive value when aligned with a word of the same length in the sequence from the database. T is called the closest word evaluation threshold (Altschul et al., supra). Such initial closest word matches act as similar points to initiate searches to find longer HSP sequences containing them. The matched words are extended in both directions along each sequence for as long as the total alignment score can increase. The cumulative score is 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, a substitution matrix is used to calculate the cumulative score. The continuation of matching words in each direction is stopped if: the cumulative alignment score decreases by X from its maximum achieved value; the cumulative score drops to or below zero due to the accumulation of one or more negatively scoring residual alignments; or the end of any sequence has been reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses wordlength (N) 11, expectation (E) 10, M=5, N=-4, and comparison of both strands as default parameters. For amino acid sequences in the BLASTP program, the default parameters are wordlength 3 and expectation (E) 10, and the BLOSUM62 substitution matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignments ( B) 50, expectation (E) 10, M=5, N=-4 and comparison of both chains.

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

- 16 039579 significantly less than about 0.001.

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

Other than the percent sequence identity noted 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 raised against the polypeptide encoded by the second nucleic acid. as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example, when 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 complements, 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.

The term isolated antibody (or antigen-binding fragment thereof), as used herein, refers to an antibody (or antigen-binding fragment thereof) that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds BMP6 is essentially free of contains antibodies that specifically bind antigens other than BMP6). In addition, the isolated antibody may be substantially free of other cellular material and/or chemical compounds.

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

The term Kassoc or Ka or KA or K _A as used herein is intended to refer to the rate of association in a particular antibody-antigen interaction, while the term Kdis or Kd as used herein is assumed to refer to the rate of dissociation. specific 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 (ie, Kd/Ka) and expressed in 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.

The terms monoclonal antibody (or antigen-binding fragment thereof) or monoclonal antibody composition (or antigen-binding fragment thereof) as used herein refers to the production of an antibody molecule (or antigen-binding fragment thereof) of a single molecular composition. A monoclonal antibody composition exhibits monospecific binding and affinity for a particular epitope.

The term nucleic acid is used interchangeably with the term polynucleotide and refers to deoxyribonucleotides or ribonucleotides and their polymers, either 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, and non-naturally occurring, that have similar binding properties as the reference nucleic acid, and that are metabolized similarly to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses appropriate conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as well as the sequence as expressly specified. In particular, as detailed below, degenerate codon substitutions can be generated by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixed base 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.

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Probes 8:91-98, 1994).

The term operably linked refers to a functional relationship between two or more polynucleotide segments (eg, DNA). Typically, this refers to the functional relationship between the transcription control sequence and the 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. Typically, transcription control promoter sequences that are operably linked to the transcribed sequence are in physical contact with the transcribed sequence, ie. they are cis-acting. However, some transcriptional control sequences, such as enhancers, should not physically contact or be in close proximity to the coding sequences whose transcription they enhance.

As used herein, the term "optimized" means that the nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the producing cell or organism, typically a eukaryotic cell, such as a Pichia cell, a Chinese Hamster Ovary (CHO) cell, or a human cell. The optimized nucleotide sequence is designed to fully or as much as possible preserve the amino acid sequence originally encoded by the original nucleotide sequence, which is also known as the parent sequence. The optimized sequences in the present invention were designed with codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic cells or prokaryotic cells is also contemplated by the present invention. 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. These terms refer to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding natural amino acid, as well as to natural amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses corresponding conservatively modified variants.

The term recombinant human antibody (or antigen-binding fragment thereof) as used herein includes all human antibodies (and antigen-binding fragments thereof) that are produced, expressed, generated, or isolated by recombinant techniques, such as antibodies isolated from an animal (e.g., mice). ) that is transgenic or transchromosomal for human immunoglobulin genes, or a hybridoma derived therefrom, antibodies isolated from a host cell transformed to express a human antibody, such as from a transfectoma, antibodies isolated from a recombinant, combinatorial library of human antibodies, and antibodies produced, expressed, generated or isolated by any other means which involve splicing all or part of the human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from germline human immunoglobulin sequences. In one embodiment, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, in the case of an animal transgenic for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions recombinant antibodies are sequences which, while derived from and related to human germline VH and VL sequences, may not exist naturally within the in vivo human germline antibody repertoire.

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 the particular cell in question, but also to the progeny of such a cell. Since some modifications may occur in subsequent generations, either through mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term host cell as used herein.

The term patient includes human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals such as non-human primates, sheep, dog, cow, chicken, amphibians and reptiles. Except where noted separately, the terms patient or patient are used interchangeably herein.

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The term treatment includes the administration of compositions or antibodies for the purpose of preventing or delaying the onset of symptoms, complications, or biochemical manifestations of a disease (eg, anemia), alleviating symptoms, or arresting or inhibiting the further development of a disease, condition, or disorder. Treatment may be prophylactic (with the aim of preventing or delaying the onset of a disease or preventing 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 used 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 double-stranded DNA circular loop 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. Some 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 be inserted into the genome of a host cell when introduced into the host cell, and thus replicate along with the genome of the host organism. In addition, some 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). Typically, expression vectors that can be used in recombinant DNA techniques are often in the form of plasmids. In the present description, the plasmid and the vector can be used interchangeably, since the plasmid is the most commonly used form of the vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), that have equivalent functions.

The term hematocrit, as used herein, is also known as sedimented cell volume (CCV) or erythrocyte volume fraction (EVF) and is the volume (%) of erythrocytes in the blood. It is usually about 45% for men and about 50% for women. It is considered an integral part of the results of a complete blood count of a person, along with the concentration of hemoglobin, the number of leukocytes and the number of platelets. In one embodiment, anemia refers to an abnormally low hematocrit, as opposed to polycythemia, which is an abnormally high hematocrit.

Brief description of the figures

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 an ELISA binding assay in which the binding of antibody 7 to human BMP6, human BMP7, human BMP5, mouse BMP6, hBaffR, BSA and Neu was examined. In this figure and various other figures, and elsewhere in the present description: Ab 5=Antibody 5; Ab 6=Antibody 6 and Ab 7=Antibody 7.

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

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

In FIG. 4 shows the therapeutic use of an anti-BMP6 antibody in ESP-resistant anemia in a mouse model of inflammation. Top: Experimental scheme of VA-induced ESP-resistant anemia in an inflammation model. 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 HDxMS epitope mapping (hydrogen/deuterium exchange coupled to mass spectrometry). The BMP6 epitope bound by antibody 7 is shown (human BMP6 residues 88-102 (QTLVHLMNPEYVPKP (SEQ ID NO: 92))). Using HDxMS, antibody 676, a humanized version of a commercially available anti-BMP6 antibody, was found to bind to an epitope consisting of residues 23-35 of human BMP6 (VSSASDYNSSELK (SEQ ID NO: 95)).

In FIG. 6 shows the protocol of the 1st part of the clinical program to study the safety and efficacy of antibodies to BMP6.

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

In FIG. 8 shows the protocol of the 2nd part of the clinical program to study the safety and efficacy of antibodies to BMP6.

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

In FIG. 10 shows the dose-dependent effect of antibody 7 on serum biomarkers of iron metabolism in rats. Shown is a quantitative analysis of the concentration of hepcidin in serum after a single injection of antibody 7 or control (solvent) at the indicated dose. The panel on the left shows an enlarged image of the action during the first 24 hours after administration.

In FIG. 11 shows the dose-dependent effect of antibody 7 on serum iron in rats. Quantitative analysis of serum iron concentration after a single injection of antibody 7 or control (solvent) at the indicated dose is shown. The panel on the left shows an enlarged image of the action during the first 24 hours after administration.

In FIG. 12 shows the concentration-time profile of a single iv dose of antibody 7 (3 mg/kg) in cynomolgus monkeys. The graph shows the total concentration of antibody 7 (free and BMP6-bound).

In FIG. 13 shows the serum concentration of hepcidin and Fe in male cynomolgus monkeys after a single intravenous injection of antibody 7 at a dose of 3 mg/kg. Data from three different monkeys are shown, as well as the average.

Detailed description of the invention

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to the BMP6 protein, as well as pharmaceutical compositions, methods for preparing and methods of using such antibodies and compositions.

Antibodies to BMP6 and their antigen-binding fragments

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to human BMP6.

BMP6, a secreted growth factor ligand of the BMP protein family, has been identified as an important endogenous regulator of hepatic expression of hepcidin, a hormone that regulates iron metabolism. Without wishing to be bound by any particular theory, the present description proposes an anti-BMP6 antagonist antibody as a hepcidin-lowering therapy that is expected to be effective in patients with iron deficiency anemia by overcoming erythropoiesis-stimulating drug (ESP) resistance, which is a significant contributor to the severity of underlying disease and is often a predictor of poor outcome.

Examples of such antibodies against human BMP6 are antibodies 3, 5, 6 and 7, the sequences of which are listed in table. 1.

All 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).

To provide new evidence that intervention in this pathway may lead to improved functional outcomes, we investigated the ability of a BMP6-specific antibody, Antibodies 5-7, to modulate serum biomarkers of iron metabolism in normal mice and rats and help reverse ESP-resistant anemia in murine models of anemia in inflammation. It was found that a single injection of anti-BMP6 antibody into animals resulted in a sustained increase in serum iron levels accompanied by a potent suppression of circulating hepcidin. In addition, therapeutic treatment of mice subjected to inflammation-induced anemia significantly improved erythropoietic parameters in response to concurrent treatment with erythropoietin.

As used herein, inhibition of BMP6 signaling in a mouse inflammatory anemia model significantly improved iron-dependent erythrocyte parameters.

The BMP6 antagonist antibodies disclosed in the present invention represent a novel therapeutic approach for the safe improvement of erythropoietin desensitized anemia. Without wishing to be bound by any particular theory, it is assumed herein that this may result from the mobilization and availability of depot iron required in the erythroid compartment.

In one embodiment, the present invention provides isolated antibodies, or antigen-binding fragments thereof, that bind with 100, 500, or 1000 times higher affinity to human BMP6 protein than either human BMP5 protein or human BMP7 protein. BMP6 specificity without binding to BMP7 is important because BMP6 knockout is not lethal in mice. However, BMP7 knockout mice die after birth with kidney, eye, and bone damage. Single knockouts of either gene do not cause changes in cardiogenesis, however, double knockout of BMP6 and BMP7 has shown several defects and delays in heart development; embryos died from heart failure. BMP7 is important in preventing the development of chronic heart disease associated with fibrosis. Thus, cross-reactivity of an anti-BMP6 antibody with BMP7 is not desirable. The antibodies of the present invention are specific for BMP6 as compared to BMP7; see, for example, Table. 4A. In FIG. 1B also shows evidence of binding specificity for human BMP6 versus human BMP2, BMP5 and BMP7 proteins.

In contrast, a commercially available anti-BMP6 antibody (R&D Systems), for example, has been shown to have strong cross-reactivity to BMP7 in a reporter gene assay and simultaneously inhibit BMP6 and BMP7.

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

Examples of such antibodies against human BMP6 are antibodies 3, 5, 6 and 7, the sequences of which are listed in table. 1.

Mature antibody 7 was obtained by removing PTM at the germline level from NOV0442_VL (YGQ), which was obtained from the original IgG hit NOV0442 (VH3_3-15, VΠ_1e). Antibody 7 binds with high affinity to human BMP6 in an ELISA binding assay, with a 500-fold selectivity over human BMP7 (ie affinity for human BMP6 is 500 times greater than for human BMP7). This antibody also has no detectable activity against human BMP2 or BMP5. The BMP6 peptide recognized by the parent IgG NOV0442 and antibody 7 is shown in FIG. 5. The peptide comprises the amino acids QTLVHLMNPEYVPKP (SEQ ID NO: 92) of human BMP6. Unlike IgG NOV0442 and antibody 7, humanized mAb 507 (R&D Systems) binds to the sequence VSSASDYNSSELK (SEQ ID NO: 95) 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. BMP6 binding to BMPR1A is maximally inhibited by 59%; binding to BMPR1B maximally inhibited by 85%; and binding to RGM-c is maximally inhibited by 72%. A single treatment of rats at a dose of 10 mg/kg resulted in long-term suppression of circulating hepcidin. The estimated minimum effective dose in mice is less than or equal to 0.1 mg/kg. Serum iron also showed an increase and hepcidin showed a decrease after a single dose of 3 mg/kg antibody in monkeys. In mice, when using the Brucella abortus antigen to model anemia, the result of treatment with antibody 7 (2 mg/kg) is consistent with a clinically significant erythropoietic response to long-term EPO therapy with a gradual increase in hemoglobin >2.0 g/dl from baseline.

In antibody 7, a potential post-translational modification site was removed by mutation of N51Q in LCDR2 to increase the homogeneity of the subsequent product. An antibody derived from the VH3/lambda1 scaffold was designed to match the nearest human germline sequence: in VH with the V40A mutation, in VL with the D1Q, I2S mutations and the introduction of amino acids Y49 and G50 to restore the scaffold, which originally lacked these 2 residues.

This work led to the creation of antibody 7 (=NOV0958=NOV0806_VH[V40A]_VL [D1Q, I2S, Y49, G50, N51Q]).

All antibodies 3, 5, 6 and 7 show high specificity for human BMP6 protein compared to human BMP2, BMP5 or BMP7 proteins. The epitope of all these antibodies is predicted to be the same, since they are all derived from the same parent Fab before affinity maturation. Antibody 3, for example, shares a Fab clone with both antibodies 5 and 7 prior to HCDR2 affinity maturation. Antibody 5 derived from NOV0442 (VH3_3-15, VI1_1e)^NOV0442_VL (YGQ)^(HCDR2 affinity maturation)^Antibody 5. Antibody 3 derived from NOV0442 (VH3_3-15, VI1_1e)^NOV0442_VL (YGS)^(HCDR2 affinity maturation) ^Antibody 3. Additional details regarding the preparation of the antibodies described in the present invention are provided in the examples.

The present invention relates to antibodies that specifically bind BMP6 (for example, human BMP6 protein), while these antibodies include the VH domain listed in table. 1. The present invention also relates to antibodies that specifically bind to the BMP6 protein, while these antibodies include a VH CDR having the amino acid sequence of any of the VH CDRs listed in table. 1. In particular, the invention relates to antibodies that specifically bind to the BMP6 protein, while these antibodies include (or, alternatively, consist of) one, two, three, four, five or more VH CDRs having the amino acid sequence of any of VH CDR listed in Table. 1.

The invention also relates to antibodies and their antigennegative fragments that specifically bind to BMP6, while these antibodies or their antigennegative fragments include (or, alternatively, consist of) the VH amino acid sequence listed in table. 1, where 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, an addition, a substitution, or a deletion). The invention also relates to antibodies and their antigennegative fragments that specifically bind to BMP6, while these antibodies or their antigennegative fragments include (or, alternatively, consist of) the VH amino acid sequence listed in table. 1, where there are no more than 10 amino acids in the framework sequence (for example, a sequence that is not

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CDR) have been mutated (where the mutation is, as various non-limiting examples, an addition, a substitution, or a deletion).

The invention also relates to antibodies and their antigennegative fragments that specifically bind to BMP6, while these antibodies or their antigennegative fragments include (or, alternatively, consist of) the VH amino acid sequence listed in table. 1, where no more than about 20 amino acids in a 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 invention also relates to antibodies and their antigennegative fragments that specifically bind to BMP6, while these antibodies or their antigennegative fragments include (or, alternatively, consist of) the VH amino acid sequence listed in table. 1, where 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 invention also relates to antibodies and their antigennegative fragments that specifically bind to BMP6, while these antibodies or antigennegative fragments include (or, alternatively, consist of) the VL amino acid sequence listed in table. 1, where 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, an addition, a substitution, or a deletion). The invention also relates to antibodies and their antigennegative fragments that specifically bind to BMP6, while these antibodies or their antigennegative fragments include (or, alternatively, consist of) the VL amino acid sequence listed in table. 1, where 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, an addition, a substitution, or a deletion).

The invention also relates to antibodies and their antigennegative fragments that specifically bind to BMP6, while these antibodies or antigennegative fragments include (or, alternatively, consist of) the VL amino acid sequence listed in table. 1, where no more than about 20 amino acids in a 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 invention also relates to antibodies and their antigennegative fragments that specifically bind to BMP6, while these antibodies or their antigennegative fragments include (or, alternatively, consist of) the VL amino acid sequence listed in table. 1, where 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 relates to antibodies and their antigennegative fragments that specifically bind to the BMP6 protein, while these antibodies or their antigennegative fragments include the VL domain listed in table. 1. The present invention also relates to antibodies and their antigennegative fragments that specifically bind to the BMP6 protein, while these antibodies or their antigennegative fragments include a VL CDR having the amino acid sequence of any of the VL CDRs listed in table. 1. In particular, the invention relates to antibodies and their antigen-binding fragments that specifically bind to the BMP6 protein, while these antibodies or their antigen-binding fragments include (or, alternatively, consist of) one, two, three or more VL CDRs having the amino acid sequence of any of the VL CDRs listed in Table. 1.

Other antibodies and their antigen-binding fragments according to the invention include amino acids that have been mutated, and at the same time have at least 60, 70, 80, 90 or 95% identity in the CDR regions with the CDR regions presented in the sequences described in table . 1. 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.

The present invention also relates to 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 exemplary heavy chain and light chain nucleic acid sequences of antibodies 3, 5, 6, and 7).

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Table 1

Examples of anti-BMP6 antibodies of the present invention

ANTIBODY 3 SEQID NO: Kabat HCDR1 SYWH 9 Kabat HCDR2 RIKDHKQGYTTAYAASVKG 10 Kabat HDDR3 VERSKSGFDN eleven Chothia HCDR1 GFTFSSY 12 Chothia HCDR2 KDHKQGYT 13 Chothia HDDR3 VERSKSGFDN 14 vh QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAPGKGLEWVGRIKDHKQGYTTAYAASVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSS 15 DNA VH caggtgcaattggtggaaagcggcggtggcctggtgaa accaggcggcagcctgcgcctgagctgcgccgcctccg gattcaccttttcttcttacgttgttcattgggtgcgc caggccccgggcaaaggtctcgagtgggtgggccgtat caaagaccacaaacagggctacactactgcttatgccg cctctgtgaaaggccgctttaccattagccgcgatgat tcgaaaaacaccctgtatctgcaaatgaacagcctgaa aaccgaagatacggccgtgtattattgcgcgcgtgttg aacgttctaaatctggtttcgataactggggccaaggc accctggtgactgttagctca 16 heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAPGKGLEWVGRIKDHKQGYTTAYAASVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WD VS Η E D P E VK FNW YVD GVE VHNAKT KPREEQYNSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQ P RE PQVYT L P P S REEMT KNQVS LT CLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 17 heavy chain DNA caggtgcaattggtggaaagcggcggtggcctggtgaa accaggcggcagcctgcgcctgagctgcgccgcctccg gattcaccttttcttcttacgttgttcattgggtgcgc caggccccgggcaaaggtctcgagtgggtgggccgtat caaagaccacaaacagggctacactactgcttatgccg cctctgtgaaaggccgctttaccattagccgcgatgat tcgaaaaacaccctgtatctgcaaatgaacagcctgaa aaccgaagatacggccgtgtattattgcgcgcgtgttg aacgttctaaatctggtttcgataactggggccaaggc accctggtgactgttagctcagcctccaccaagggtcc atcggtcttccccctggcaccctcctccaagagcacct ctgggggcacagcggccctgggctgcctggtcaaggac tacttccccgaaccggtgacggtgtcgtggaactcagg cgccctgaccagcggcgtgcacaccttcccggctgtcc tacagtcctcaggactctactccctcagcagcgtggtg accgtgccctccagcagcttgggcacccagacctacat ctgcaacgtgaatcacaagcccagcaacaccaaggtgg acaagagagttgagcccaaatcttgtgacaaaactcac acatgcccaccgtgcccagcacctgaactcctgggggg accgtcagtcttcctcttccccccaaaacccaaggaca ccctcatgatctcccggacccctgaggtcacatgcgtg 18

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gtggtggacgtgagccacgaagaccctgaggtcaagtt caactggtacgtggacggcgtggaggtgcataatgcca agacaaagccgcgggaggagcagtacaacagcacgtac cgggtggtcagcgtcctcaccgtcctgcaccaggactg gctgaatggcaaggagtacaagtgcaaggtctccaaca aagccctcccagcccccatcgagaaaaccatctccaaa gccaaagggcagccccgagaaccacaggtgtacaccct gcccccatcccgggaggagatgaccaagaaccaggtca gcctgacctgcctggtcaaaggcttctatcccagcgac atcgccgtggagtgggagagcaatgggcagccggagaa caactacaagaccacgcctcccgtgctggactccgacg gctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgt gatgcatgaggctctgcacaaccactacacgcagaaga gcctctccctgtctccgggtaaa Kabat LCDR1 TGSSSNIGAGYSVH 19 Kabat LCDR2 GSSERPs 20 Kabat LCDR3 QSWDSSQTLW 21 Chothia LCDR1 SSSNIGAGYS 22 Chothia LCDR2 GSS 23 Chothia LCDR3 WDSSQTLV 24 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGSSERPSGVPDRFSGSSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVL 25 DNA VL CAGAGCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGC ACCGGGCCAGCGCGTGACCATTAGCTGTACCGGCAGCA GCAGCAACATTGGTGCTGGTTACTCTGTGCATTGGTAC CAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTA TGGTAGCTCTGAACGCCCGAGCGGCGTGCCGGATCGCT TTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCG ATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTA CTGCCAGTCTTGGGACTCTTCTCAGACTCTGGTTGTGT TTGGCGGCGGCACGAAGTTAACCGTCCTA 26 light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGSSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNTVKYAASSYCSLTPEQHEGSHEAPTE 27 light chain DNA CAGAGCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGC ACCGGGCCAGCGCGTGACCATTAGCTGTACCGGCAGCA GCAGCAACATTGGTGCTGGTTACTCTGTGCATTGGTAC CAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTA TGGTAGCTCTGAACGCCCGAGCGGCGTGCCGGATCGCT TTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCG ATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTA CTGCCAGTCTTGGGACTCTTCTCAGACTCTGGTTGTGT TTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCAGCCC AAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTC TGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTC TCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCC TGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGA GAG СAC СACAC С С T С СAAACAAAG СAACAACAAGTAC G CGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGG AAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGA AGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAT GTTCA 28 ANTIBODY 5 Kabat HCDR1 SYWH 29 Kabat HCDR2 RIKRESSSYTTMYAAPVKG thirty Kabat HDDR3 VERSKSGFDN 31

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Chothia HCDR1 GFTFSSY 32 Chothia HCDR2 KRESSSYT 33 Chothia HDDR3 VERSKSGFDN 34 vh QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAPGKGLEWVGRIKRES S SYTTMYAAPVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSS 35 DNA VH CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAA GCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTG GCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGA CAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGAT TAAGAGAGAGTCCTCTAGCTACACTACTATGTACGCCG CTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGAC TCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAA AACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG AACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGC 36 heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAPGKGLEWVGRIKRES S SYTTMYAAPVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WD VS Η E D P E VK FNW YVD GVE VHNAKT KPREEQYNSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 37 heavy chain DNA CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAA GCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTG GCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGA CAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGAT TAAGAGAGAGTCCTCTAGCTACACTACTATGTACGCCG CTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGAC TCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAA AACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG AACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCC AAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTT CCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGC TGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATAT CTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGG ACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCAC ACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGG GCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACA CCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCA AGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTG GCTGAACGGCAAAGAATACAAGTGC AAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGT CCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGAT ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACG GCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT 38

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GATGCACGAGGCCCTGCACAACCACTACACCCCAGAAGT CCCTGAGCCTGAGCCCCGGCAAG Kabat LCDR1 TGSSSNIGAGYSVH 39 Kabat LCDR2 GQSERPS 40 Kabat LCDR3 QSWDSSQTLW 41 Chothia LCDR1 SSSNIGAGYS 42 Chothia LCDR2 GQS 43 Chothia LCDR3 WDSSQTLV 44 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVL 45 DNA VL CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTG 46 light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSQVTPEQW KSHREKSCHESCHE 47 light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCT AAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAG CGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCC TGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGA GAG САС САС С С С СAG СAAG СAGAG СAACAACAAGTAC G CCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGG AAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGA GGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGT GCAGC 48 ANTIBODY 6 Kabat HCDR1 SYWH 49 Kabat HCDR2 RT RH S DMGYAT S YAAPVKG 50 Kabat HDDR3 VERSKSGFDN 51 Chothia HCDR1 GFTFSSY 52 Chothia HCDR2 RHSDMYA 53 Chothia HDDR3 VERSKSGFDN 54 vh QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAP GKGLEWVGRT RH S DMGYAT S YAAPVKGRFTIS RDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSS 55 DNA VH CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAA GCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTG GCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGA CAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGAC TAGACACTCAGATATGGGCTACGCTACTAGCTACGCCG 56

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CTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGAC TCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAA AACGAGGACACCGCCGTCTACTACTGCCGCTAGAGTGG AACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGC heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAP GKGLEWVGRTRH S DMGYAT S YAAPVKGRFTIS RDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WD VS Н Е D Р Е VK FNW YVD GVE VHNAKT KPREEQYNSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 57 heavy chain DNA CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAA GCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTG GCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGA CAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGAC TAGACACTCAGATATGGGCTACGCTACTAGCTACGCCG CTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGAC TCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAA AACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG AACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCC AAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTT CCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGC TGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATAT CTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGG ACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCAC ACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGG GCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACA CCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCA AGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTG GOTGAACGGCAAAGAATACAAGTGC AAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGT CCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGAT ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACG GCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT GATGCACGAGGCCCTGCACAACCACTACACCCAGAAGT CCCTGAGCCTGAGCCCCGGCAAG 58 Kabat LCDR1 TGSSSNIGAGYSVH 59 Kabat LCDR2 GQSERPS 60 Kabat LCDR3 QSWDSSQTLW 61 Chothia LCDR1 SSSNIGAGYS 62 Chothia LCDR2 GQS 63 Chothia LCDR3 WDSSQTLV 64 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVL 65

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DNA VL CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTG 66 light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSQVTPEQW KSHREKSCHESCHE 67 light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCT AAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAG CGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCC TGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGA GAG САС САС С С С СAG СAAG СAGAG СAACAACAAGTAC G CCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGG AAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGA GGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGT GCAGC 68 ANTIBODY 7 Kabat HCDR1 SYWH 69 Kabat HCDR2 RIRLETHGYAAEYAASVKG 70 Kabat HDDR3 VERSKSGFDN 71 Chothia HCDR1 GFTFSSY 72 Chothia HCDR2 RLETHGYA 73 Chothia HDDR3 VERSKSGFDN 74 vh QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAPGKGLEWVGRIRLETHGYAAEYAASVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSS 75 DNA VH CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAA GCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTG GCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGA CAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGAT TAGACTGGAAACTCACGGCTACGCCGCCGAGTACGCCG CTAGTGTGAAGGGCCGGTTCACTATCTCTAGGGACGAC ТCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAA AACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG AACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGC 76 heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAPGKGLEWVGRIRLETHGYAAEYAASVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV 77

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WD VS Η E D Ρ E VK FNWYVD GVE VHNAKT KPREEQYNSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQ R RE R QVYT L R R S RE EMT KNQVS LT S LVKG FY PSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVLSTVCSQHEALK heavy chain DNA CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAA GCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTG GCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGA CAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGAT TAGACTGGAAACTCACGGCTACGCCGCCGAGTACGCCG CTAGTGTGAAGGGCCGGTTCACTATCTCTAGGGACGAC ТCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAA AACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG AACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCC AAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTT CCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGC TGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATAT CTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGG ACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCAC ACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGG GCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACA CCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCA AGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTG GCTGAACGGCAAAGAATACAAGTGC AAAGTСТССААСА AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGT CCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGAT ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACG GCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT GATGCACGAGGCCCTGCACAACCACTACACCCAGAAGT CCCTGAGCCTGAGCCCCGGCAAG 78 Kabat LCDR1 TGSSSNIGAGYSVH 79 Kabat LCDR2 GQSERPS 80 Kabat LCDR3 QSWDSSQTLW 81 Chothia LCDR1 SSSNIGAGYS 82 Chothia LCDR2 GQS 83 Chothia LCDR3 WDSSQTLV 84 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVL 85 DNA VL CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTG 86 light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVLGQP 87 KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECS light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCT AAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAG CGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCC TGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGA GAC САС САС С С С СAG СAAG СAGAG СAACAACAAGTAC G CCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGG AAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGA GGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGT GCAGC 88

Other antibodies and antigen-binding fragments thereof according to the invention include those in which

- 29 039579 second amino acids or nucleic acids encoding amino acids have been mutated, and at the same time have at least 60, 70, 80, 90 or 95% identity with the sequences described in table. 1. 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 with substantially the same therapeutic activity.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds human BMP6 and comprises the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 9, 10 and 11, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NO : 19, 20 and 21, respectively.

In another specific embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds human BMP6 and comprises the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 12, 13 and 14, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NO : 22, 23 and 24, respectively.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds human BMP6 and comprises the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 29, 30 and 31, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in 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 human BMP6 and comprises the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 32, 33 and 34, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NO : 42, 43 and 44, respectively.

In another specific embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds human BMP6 and comprises the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in 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 human BMP6 and comprises the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in 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 human BMP6 and comprises the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 69, 70 and 71, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NO : 79, 80 and 81, respectively.

In another specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds human BMP6 and comprises the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 72, 73 and 74, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NO : 82, 83 and 84, respectively.

Since each of these antibodies can bind to BMP6, the sequences of VH, VL, full length light chain and full length heavy chain (amino acid sequences and nucleotide sequences encoding amino acid sequences) can be mixed and combined to obtain other BMP6-binding antibodies and their antigen-binding fragments according to invention. Such mixed and combined BMP6-binding antibodies can be tested using binding assays known in the art (eg, ELISA and other assays described in the examples section). When mixing and combining these strands, the VH sequence from a particular VH/VL pair must 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, a 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 relates to BMP6-binding antibodies, which include CDR1, CDR2 and CDR3 regions of the heavy chain and light chain, as described in table. 1, or combinations thereof. CDR regions were determined using the Kabat system (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. 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 the CDR1, 2 and 3 regions provide the antigen-binding specificity primarily, the VH CDR1, 2 and 3 sequences and the VL sequences of CDR1, 2 and 3 can be mixed and combined (i.e., .e. CDR-regions from times

- 30 039579 personal antibodies can be mixed and combined, although each antibody must contain VH CDR1, 2 and 3 and VL CDR1, 2 and 3) to obtain other BMP6-binding molecules according to the invention. Such mixed and combined BMP6-binding antibodies can be tested using binding assays known in the art and described in the examples (eg, ELISA assays). When VH CDR sequences are mixed and combined, the CDR1, CDR2 and/or CDR3 sequence from the particular VH sequence must be replaced with structurally similar CDR sequence(s). Similarly, when VL CDR sequences are mixed and combined, the CDR1, CDR2 and/or CDR3 sequence from a particular VL sequence must be replaced with structurally similar CDR sequence(s). It will be apparent to one of ordinary skill in the art that new VH and VL sequences can be created by mutating one or more sequences of VH and/or VL CDR regions with structurally similar sequences from the CDR sequences shown herein for the monoclonal antibodies of the present invention.

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

In one embodiment, the antibody that specifically binds to BMP6 is the antibody described in Table 1. 1.

As used herein, a human antibody includes 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 that uses human germline immunoglobulin genes. . Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest, or screening a human immunoglobulin gene library presented on a bacteriophage with an antigen of interest. A human antibody that is the product of or derived from a human germline immunoglobulin sequence can be determined 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. e. having the highest % identity) with the 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, for example, to natural somatic mutations or the intentional introduction of site-directed mutations. However, in the VH or VL framework regions, the selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that define the human antibody as human when compared to the amino acid sequences germline immunoglobulin from other species (eg, murine germline sequences). In some 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 in amino acid sequence to the amino acid sequence encoded by germline immunoglobulin genome. Typically, a recombinant human antibody will show no more than 10 amino acid differences with the amino acid sequence encoded by the germline human immunoglobulin gene in the VH or VL framework regions. In some cases, a human antibody may show no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequences encoded by the germline immunoglobulin gene.

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Members of the BMP family and hepcidin

In one embodiment, the invention relates to an antibody, or a binding fragment thereof, that specifically binds to BMP6, is an antibody. In one embodiment, the antibody or its binding fragment is described in table. 1.

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

BMP6, a secreted growth factor ligand of the BMP family, is a 30 kDa disulfide-linked homodimer in its mature active form. This 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) are a family of secreted signaling molecules that can cause ectopic bone growth. BMP proteins are part of the transforming growth factor beta (TGF-beta) superfamily. BMP proteins were originally identified by the ability of a demineralized bone extract to induce intracartilaginous osteogenesis in vivo at an extraskeletal site. Due to its expression at an early stage of embryogenesis, it is suggested that the BMP encoded by this gene plays a role in early development. In addition, the fact that this BMP is closely related to BMP5 and BMP7 has led to the suggestion of a possible bone growth inducing activity. An additional function of BMP6 has been determined 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 the main regulator of hepcidin, a small peptide secreted in the liver, which is the main regulator of iron metabolism in mammals. Hepcidin regulates both the amount of iron absorbed in the duodenum from food and the iron released by reticuloendothelial cells. Hepcidin levels are elevated by a variety of stimulatory factors, including inflammation and iron overload, and are lowered by anemia, hypoxia, and iron deficiency.

Without wishing to be bound by any particular theory, it is proposed herein that an anti-BMP6 antagonist antibody as a hepcidin-lowering therapy is expected to be effective in patients with iron deficiency anemia by overcoming erythropoiesis-stimulating drug (ESD) resistance, which is a significant contributor to into the severity of the underlying disease and is often a predictor of poor outcome. When interacting with the BMPR1 and BMPR2 receptors, it causes 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 an anti-BMP6 antibody to mice reduced hepcidin levels and increased iron levels.

In the prior art, BMP6 is also described, for example, in: 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 the development of bone and cartilage tissue. It is involved in the hedgehog signaling pathway, the TGF-beta signaling pathway, and in the interaction of cytokines with cytokine receptors. It is also involved in cardiac myocyte differentiation and epithelial-mesenchymal transition. BMP2 plays many important roles, as noted 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 also described, inter alia, in: 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 BMP proteins, it is known for its ability to induce bone and cartilage development. BMP5 is expressed in the trabecular meshwork and optic disc and may play a role in development and normal function. It is also expressed in the lungs 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.

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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 phosphorylation of SMAD1 and SMAD5, which in turn cause the transcription of numerous osteogenic genes.

As noted above, BMP6 knockout mice are viable and fertile, and show normal bone and cartilage development. However, BMP7 knockout mice die after birth with kidney, eye, and bone damage. Single knockouts of any of the genes do not cause changes in cardiogenesis, however, double knockout of BMP6 and BMP7 has demonstrated several defects and delays in cardiac development; embryos died from heart failure. BMP7 is important in preventing the development 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 (antimicrobial protein or hepcidin peptide).

A recent gene duplication event in mouse evolution resulted in two similar hepcidin genes, hepcidin 1 and hepcidin 2, in mice, Ilyin et al. 2003 FEBS Lett. 542:22-26. Mouse hepcidin 2 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, while it is necessary for the regulation of iron accumulation in macrophages and for iron absorption in the intestine. 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 25-ac peptide is secreted mainly in the liver and is considered the main regulator of iron metabolism. 20- and 22-aa metabolites 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 are rich in cysteines, which form intramolecular bonds that stabilize their beta sheet structures.

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

The 25-ac peptide hepcidin is secreted primarily in 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, thus functionally reducing iron absorption. Inhibition of ferroportin also interferes with the release of iron from macrophages; therefore, hepcidin maintains iron homeostasis. Hepcidin activity is also partly responsible for the iron sequestration seen in anemia associated with 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 leads to cardiomyopathy, cirrhosis, and endocrine insufficiency.

Most cases of juvenile hemochromatosis are due to mutations in hemojewelin, a regulator of hepcidin production.

Genetically modified mice engineered to overexpress hepcidin die shortly after birth with severe iron deficiency, indicating a central and indispensable role in the regulation of iron metabolism. The first evidence linking hepcidin to inflammatory anemia came from investigators' analysis of tissues from two patients with liver tumors and severe microcytic anemia that 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 a violation of the normal absorption of iron contributes to the development of iron deficiency and iron deficiency anemia. Treatment will depend on hepcidin levels, as oral treatment is likely to be ineffective if hepcidin blocks absorption in the small intestine.

In one embodiment, administration of an anti-BMP6 antibody or binding fragment thereof reduces the activity and/or level of hepcidin and thus may be useful in the treatment of anemia. In one embodiment, the invention relates to a method for reducing hepcidin activity or levels in a patient, comprising the step of administering to the patient an antibody or antigen-binding antibody thereof.

- 33 039579 fragments to BMP6. 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. This includes any disease associated with hepcidin and/or mutation and/or overexpression of wild-type and/or mutant hepcidin and/or diseases where disease progression is enhanced or prognosis is impaired in the presence of hepcidin and/or as a result of mutation and/or overexpression of wild-type and/or mutant hepcidin, and/or reduced renal elimination of hepcidin in the urine.

Non-limiting examples of hepcidin-associated diseases include: anemia, iron deficiency erythropoiesis, hypoferremia, impaired dietary iron absorption, iron sequestration, anemia in inflammation (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, poor growth and/or hair loss. In one embodiment, the patient is a dialysis patient. In one embodiment, the hepcidin-associated disease is anemia and the patient is a dialysis patient. The prevalence of iron deficiency and ESP-refractory anemia is high in the group of people requiring chronic hemodialysis.

Anemia includes, but is not limited to, anemia of chronic disease (ACD), anemia of chronic kidney disease (CKD), anemia of cancer, erythropoiesis-stimulating drug (ESP) resistant anemia, and/or iron deficiency anemia.

Anemia in CKD is a common and early complication of chronic kidney disease. Cancer anemia is caused by hematologic malignancies and some 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 cause the progression of diabetic neuropathy, cardiovascular disease, retinopathy, and other disorders. Cancer-associated 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, decreased erythropoiesis, and shortened erythrocyte life cycle. Some treatments for anemia include ESP, erythropoietin, iron supplementation, or blood transfusions.

Hepcidin is a key hormone involved in iron homeostasis. A high level of hepcidin was associated with iron-deficient erythropoiesis in ACD. BMP6 is known to upregulate hepcidin expression.

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

Homologous antibodies

In yet another embodiment, the present invention relates to an antibody or antigennegative fragment, including amino acid sequences that are homologous to the sequences described in table. 1, while the specified antibody binds to BMP6 and retains the required functional properties of those antibodies that are described in table. 1.

For example, the invention relates to 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 comprises 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 comprises 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 the lysis of red blood cells in a hemolytic test, where the hemolytic test is known in the art. In a specific example, such antibodies have an IC ₅₀ value in the hemolytic test of 20-200 pM 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 shown in Table 1. 1. In one embodiment, the amino acid sequences of VH and/or VL may be identical except for an amino acid substitution at no more than 1, 2, 3, 4, or 5 amino acid positions. An antibody having VH and VL regions that have a high (ie, 80% or more) identity with the VH and VL regions described in table. 1 can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoded in

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SEQ ID NO: 16; 36; 56 or 76; and 26; 46; 66 or 86, respectively, followed by testing the encoded altered antibody for retention of function using the functional assays described in the present invention.

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% identical to the sequences shown in Table 1. 1. An antibody having a full-length heavy chain and a full-length light chain with high (ie, 80% or more) identity with full-length heavy chains of any of SEQ ID NO: 18; 38; 58 or 78 and full-length light chains of 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 retention of function using the functional assays described in the present invention.

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 shown in Table 1. 1.

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 shown in Table 1. 1.

As used herein, the percentage of identity between two sequences depends on the number of identical positions that the sequences have in common (i.e., % identity is equal to the number of identical positions / total number of positions x100), taking into account the number of gaps and the length of each gap that is required to be entered for optimal alignment of two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the present invention may also be used as a query sequence to search public databases, for example, to determine 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 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 such CDR sequences have specific antibody-based amino acid sequences. described in the present invention, or conservative modifications thereof, and where the antibodies retain the desired functional properties of BMP6-binding antibodies and their antigen-binding fragments according to the invention. Thus, the invention relates to an isolated monoclonal antibody or a functional antigen-binding fragment thereof, consisting of a heavy chain variable region, including the sequences CDR1, CDR2, and CDR3, and a light chain variable region, including the sequences CDR1, CDR2, and CDR3, where: the CDR1 chains include 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 CDR2 heavy chain variable region 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 CDR3 heavy chain variable region 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 CDR1 light chain variable region 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 CDR2 light chain variable region 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 CDR3 light chain variable region 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 hemolytic test.

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, wherein one or more of these sequences have specific amino acid sequences based on the antibodies described herein, or conservative modifications thereof, and where the antibodies retain the desired functional properties of BMP6-binding antibodies and their antigen-binding fragments according to the invention. Thus, the invention relates to 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 conservative modifications thereof; and the full-length light chain has amino acid sequences selected from the group of SEQ ID NO: 28; 48; 68 or 88, and conservative modifications thereof; the antibody specifically binds to BMP6; and the antibody inhibits erythrocyte lysis in a hemolytic test as described in the present invention. In a certain embodiment, such antibodies have an IC ₅₀ value in the hemolytic test of 20-200 pM using human BMP6-depleted serum, which is reconstituted with 100 pM human BMP6.

Antibodies that bind to the same epitope

The present invention relates to antibodies that bind to the same epitope as BMP6-binding antibodies listed in table. 1. The epitope bound by antibody 7 is shown in FIG. 5. Additional antibodies can thus be defined 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 invention in BMP6 binding assays. The ability of a test antibody to inhibit the binding of antibodies and antigen-binding fragments thereof according to the present invention to the BMP6 protein demonstrates that the test antibody can compete with such an antibody for binding to BMP6; such an antibody, according to non-limiting theory, can bind to the same or related (eg, structurally similar or spatially closest) epitope on BMP6 as the antibody with which it competes. In a particular embodiment, the 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 obtained and isolated as described in the present invention.

Once a desired epitope has been determined on an antigen, antibodies to that epitope can be generated, for example, using the methods described herein. Alternatively, during the process of discovery, production and analysis of antibodies, information about the desired epitopes can be obtained. Antibodies can then be competitively screened for binding to the same epitope based on this information. An approach to achieve this is to conduct cross-competition assays to look for antibodies that compete with each other, eg antibodies compete for binding to an antigen. A high throughput method for sorting antibodies based on their cross-competition is described in international patent application WO 2003/48731. As will be appreciated by one of skill in the art, virtually anything that an antibody can specifically bind to can be an epitope. An epitope may include those residues to which the antibody binds.

Typically, antibodies specific for a particular target antigen 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 determined using any number of epitope mapping techniques 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, the 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 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 can be easily identified by determining the spatial structure of the BMP6 amino acids, for example, using hydrogen/deuterium exchange, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydrophobicity curves, such as those calculated using, for example, the Omiga program version 1.0 available from the Oxford Molecular Group. This computer program uses the Hopp/Woods method, Knorr 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 curves.

Engineered and Modified Antibodies An antibody of the invention can also be produced by using an antibody having one or more of the VH and/or VL sequences shown in the present invention as a starting material for constructing a modified antibody, where the modified antibody may have altered properties compared to original antibody. An antibody can be constructed by modifying one or more residues in one or both of the variable regions (ie, VH and VL), for example, in one or more CDR regions and/or in one or more framework regions. Additionally or alternatively, the antibody can be engineered by changing residues in the constant region(s), eg, to change the effector function(s) of the antibody.

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One type of variable region construction that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues that are located in the six complementarity determining regions (CDRs) of the heavy and light chains. Therefore, the amino acid sequences within the CDR regions are more diverse in individual antibodies than the sequences outside the CDR regions. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of certain native antibodies by constructing expression vectors that include CDR sequences from a specific native antibody grafted onto framework sequences from another antibody with different properties (see ., e.g., 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 5,225,539 (Winter) and U.S. Patents 5,530,101; 5,585,089; 5,693,762 and 6,180,370 (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 of 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, U.S. 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 into this application by reference.

Examples of framework sequences for use in antibodies and their antigen-binding fragments according to the invention are framework sequences that are structurally similar to the framework sequences used in selected antibodies and antigen-binding fragments according to the present invention, for example, consensus sequences and/or framework sequences used in monoclonal antibodies according to invention. The CDR1, 2 and 3 VH sequences and the CDR1, 2 and 3 VL sequences can be grafted onto framework regions that have the same sequence as the sequence 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 to be advantageous in some cases to mutate residues in the framework regions while maintaining or increasing the antigen-binding capacity of the antibody (see, for example, US Pat.

Another type of variable region modification is to mutate amino acid residues in the CDR1, CDR2, and/or CDR3 regions of the VH and/or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest, and is known as affinity maturation. . Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), wherein the effect on antibody binding or other functional property of interest can be assessed in in vitro or in vivo assays as described in the present invention and provided in the examples . Conservative modifications may be introduced (as discussed above). Mutations can be amino acid substitutions, additions, or deletions. In addition, as a rule, no more than one, two, three, four or five residues in the CDR region are changed.

Grafting of antigen-binding domains into alternative scaffolds or scaffolds

A variety of antibody/immunoglobulin scaffolds or scaffolds can be used, provided that the resulting polypeptide will include 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 animal immunoglobulins from other species, preferably having humanized aspects. Single heavy chain antibodies, such as those found in camelids, are of particular interest in this regard. Specialists continue to discover and create new frameworks, scaffolds and fragments.

In one aspect, the invention relates to a method for generating non-immunoglobulin antibodies using non-immunoglobulin scaffolds onto which the CDRs of the invention can be grafted. Known or future non-immunoglobulin scaffolds and scaffolds can be used provided they include a binding region specific for the target BMP6 protein. Known non-immunoglobulin scaffolds or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.), maxitel (Avidia, Inc., Mountain View, Calif.) , protein A (Affibody AG, Sweden) and affilin (gamma crystallin or ubiquitin) (SciI Proteins GmbH, Halle, Germany).

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Fibronectin scaffolds are based on a type III fibronectin domain (eg, type III fibronectin module ten (Fn3 domain 10)). The type III fibronectin domain has 7 or 8 beta strands that are distributed between two beta layers that pack opposite each other to form the protein core, and also contains loops (similar to CDR regions) that connect the beta strands to each other and are hydrophilic. There are at least three such loops at each edge of the beta-pleated sandwich, where the edge is the protein boundary perpendicular to the direction of the beta strands (see US Pat. No. 6,818,418). Such fibronectin-based scaffolds are not immunoglobulin, although the overall fold is very close to the smallest functional antibody fragment, the heavy chain variable region, which includes the entire antigen recognition unit in camel and llama IgG. Due to this structure, a non-immunoglobulin antibody mimics antigen-binding properties that are similar in nature and affinity to those of antibodies. Such scaffolds 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 where the loop regions of the molecule can be replaced by the CDR regions of the invention using standard cloning techniques.

Ankyrin technology is based on the use of ankyrin-based repeating proteins as scaffolds carrying variable regions that can be used to bind to various targets. The ankyrin repeat module is a 33 amino acid long polypeptide consisting of two antiparallel alpha helices and a beta fold. Variable region binding is usually optimized by ribosome display.

Avimers are derived from a naturally occurring A domain containing protein such as LRP-1. These domains are exploited due to the nature of protein-protein interactions, with over 250 proteins in humans being structurally based on A-domains. Avimers are composed of a number of different monomeric A domains (2-10) linked by an amino acid linker. Avimers that can bind to a target antigen can be prepared using the technique described, for example, in US Patent Application Publications 20040175756; 20050053973; 20050048512 and 20060008844.

Affiteles, affinity ligands, are small simple proteins consisting of a three-stranded scaffold of one of the IgG-binding domains of Protein A. Protein A, the surface protein of Staphylococcus aureus aureus. This framework domain consists of 58 amino acids, 13 of which are randomized to produce libraries of affitels with a large number of ligand variants (see, for example, US Pat. No. 5,831,012). Affitel molecules are similar to antibodies, in that they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. Despite its small size, the binding site of an affitel molecule is similar to that of an antibody.

Anticalins are products developed by Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and rigid proteins that are commonly involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several naturally occurring lipocalins are present in the tissues or fluids of the human body. The protein architecture resembles that of immunoglobulins, with hypervariable loops on top of a rigid scaffold. However, unlike antibodies or their recombinant fragments, lipocalins consist of a single polypeptide chain of 160-180 amino acid residues, being 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 multiple side chains. The shape of the binding site can thus be modified in a proprietary manner to recognize the desired target molecules of various shapes with high affinity and specificity. One lipocalin family protein, bilin-binding protein (BBP) from Pieris Brassicae, was used to generate anticalins by four-loop set mutagenesis. One example of a patent application that describes anticalins is PCT Publication WO199916873.

Affinins are small non-immunoglobulin proteins that are designed with specific affinity for proteins and small molecules. New affilin molecules can be selected very quickly from two libraries, each based on a different human-derived scaffold protein. Affilin molecules do not show structural homology with immunoglobulin proteins. Two affinity scaffolds are currently in use, one being gamma-crystallin, the human structural protein of the lens of the eye, and the other being a protein from the ubiquitin superfamily. Both human scaffolds are very small, exhibit thermal stability, and are virtually resistant to pH changes and denaturing agents. This high stability is mainly due to the extended beta sheet structure of these proteins. Examples of gamma crystallin-based proteins are described in WO 200104144 and examples of ubiquitin-like proteins are described in WO 2004106368.

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

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Antibodies that bind human BMP6 can be obtained using methods known in the art. For example, the humanization technology used to convert non-human antibodies into engineered human antibodies. US Patent Publication 20050008625 describes an in vivo method for replacing the variable region of a non-human antibody with a human variable region in the antibody while maintaining or improving binding properties compared to the non-human antibody. The method is based on epitope-driven replacement of the variable regions of a non-human reference antibody with a fully human antibody. The resulting human antibody usually differs in structure from the reference non-human antibody, but binds to the same epitope on the same antigen as the reference antibody. Briefly, a serial epitope-driven complementarity-switching method is performed by allowing cellular competition between a competitor and a library of diverse reference antibody (test antibodies) hybrids for binding to limited amounts of antigen in the presence of a reporter system that is responsive to test antibody binding to antigen. The competitor may be a reference antibody or a derivative thereof, such as a single chain Fv fragment. The competitor may also be a natural or artificial ligand for an antigen that binds to the same epitope as the reference antibody. The only requirement for a competitor is that it must bind to the same epitope as the reference antibody and compete with the reference antibody for antigen binding.

Test antibodies share one antigen-binding V region from a non-human reference antibody, with the other V region randomly selected from a broad source such as a library of human antibody repertoire. The common V-region from the reference antibody serves as a guide directing test antibodies to the same epitope on the antigen, and in the same orientation, so that selection proceeds in the direction of the highest antigen-binding match for the reference antibody.

Many types of reporter system can be used to detect the desired interactions between test antibodies and antigen. For example, reporter-complementing fragments can be associated with an antigen and a test antibody, respectively, with reporter activation by fragment complementation occurring only when the test antibody binds to the antigen. When fusions of a test antibody and an 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 Autoinhibited Reporter Reactivation System (RAIR) reactivator disclosed in US Patent Application 10/208730 (Publication 20030198971) or the Competitive Activation System disclosed in US Patent Application 10/076845 (Publication 20030157579) .

In the case of a serial epitope-directed complementarity replacement system, selection is made to find cells that express one test antibody along with competitor, antigen, and reporter components. In these cells, each antibody tested competes one-on-one 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 for 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 consisting 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 as the reference antibody. One or more hybrid antibodies selected in the first round will have an affinity for the antigen that is comparable to or greater than the affinity 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 replacements for the remaining V region of the non-human reference antibody using an extensive 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 differ in structure from the reference antibody, but which compete with the reference antibody for binding to the same antigen. Some selected human antibodies bind to the same epitope on the same antigen as the reference antibody. Of 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 murine or chimeric BMP6-binding antibodies described above as a reference antibody, this method can easily be 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 human BMP6-binding antibodies can also be purchased from companies that typically manufacture human antibodies, such as KaloBios, Inc. (Mountain View, Calif.).

Camelid antibodies

Antibody proteins derived from members of the camel and dromedary family (Camelus bactrianus and Calelus dromaderius), including New World members such as llama species (Lama paccos, Lama glama, and Lama vicugna), were investigated for size, structural complexity, and antigenicity in humans. Some IgG antibodies from this family of mammals have been found in nature to have no light chains, and thus are structurally different from the typical four-chain quaternary structure containing two heavy and two light chains, as 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 referred to as VHH, can be genetically engineered into a small protein with high affinity for the target, resulting in a low molecular weight antibody-based protein known as camelid nanobody. See US Pat. No. 5,759,808 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 camel antibodies and antibody fragments are commercially available from, for example, Ablynx, Ghent, Belgium. As with other non-human antibodies and their antigen-binding fragments, the amino acid sequence of a camel antibody can be altered recombinantly to produce a sequence that is more similar to the human sequence, ie. the nanobody can be humanized. Thus, the natural low antigenicity of camelid antibodies for humans can be further reduced.

The camel nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few millimicrons. One consequence of their small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally undetectable to larger antibody proteins, i.e. Camelid nanobodies can be used as reagents for the detection of antigens that are otherwise hidden using classical immunological methods, as well as possible therapeutic agents. Thus, another consequence of the small size is that the camel nanobody can inhibit 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 resembles the function of a classical small molecule drug than a natural one. antibodies.

The low molecular weight and compact size also result in camel nanobodies being extremely heat resistant, resistant to extreme pH and proteolytic degradation, and low antigenicity. Another implication is that camel nanobodies move easily from the cardiovascular system to tissues, and even cross the blood-brain barrier, and can treat disorders that affect neural tissue. Nanobodies can also facilitate drug transport across the blood-brain barrier. See U.S. Patent Application 20040161738 published August 19, 2004. Such properties, combined with low antigenicity in humans, indicate great therapeutic potential. In addition, these molecules can be fully expressed in prokaryotic cells such as E. coli and expressed as fusion proteins with bacteriophage and still be functional.

Thus, a feature of the present invention is a camel antibody or nanobody having high affinity for BMP6. In one embodiment, in the present invention, the camelid antibody or nanobody is naturally produced in a camelid animal, i. produced by camelids after immunization with BMP6 or a peptide fragment thereof using the methods described in the present invention for other antibodies. In an alternative, the BMP6-binding camelid nanobody is engineered, i. obtained by selecting, for example, from a library of phage presenting properly mutated camel nanobody proteins using BMP6-targeted sorting techniques as described in the examples below. Designed nanobodies can also be individually modified using genetic engineering methods to change the half-life in the recipient's body from 45 minutes to 2 weeks. In a particular embodiment, a camel antibody or nanobody is obtained by grafting heavy or light chain CDR sequences of human antibodies of the invention into a single domain antibody nanobody or framework sequences, as described, for example, in PCT/EP93/02214.

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Bispecific molecules and multivalent antibodies

In another aspect of the present invention, bispecific or multispecific molecules are provided comprising a BMP6 binding antibody or fragment thereof according to the invention. An antibody of the 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 molecules - targets. An antibody of the invention may actually be derivatized or linked to more than one functional molecule, resulting in 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 invention, an antibody of the invention may be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent linkage, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, in resulting in a bispecific molecule.

Thus, the present invention includes bispecific molecules comprising at least one first binding specificity for BMP6 and a second 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 an invention in which the bispecific molecule is multispecific, the molecule may further include a third binding specificity, in addition to the first and second target epitopes.

In one embodiment, the bispecific molecules of the invention include, as binding specificity, at least one antibody or antibody fragment, including, for example, Fab, Fab', F(ab')2, Fv, or single chain Fv. The antibody can also be a light chain or heavy chain dimer or any minimal fragment thereof, such as Fv or a single chain construct as described in US Pat. No. 4,946,778 (Ladner et al.).

Diabodies are bivalent, bispecific molecules in which the VH and VL domains are expressed on the same polypeptide chain linked by a linker that is too short to allow pairing between the two domains on the same chain. The VH and VL domains pair with the complementary domains of the other chain, resulting in two antigen-binding sites (see, e.g., Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poijak et al., 1994 Structure 2:11211123). Diabodies can be generated by expressing two polypeptide chains with the structure of either 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-stranded diabodies (scDb) are produced by joining two diabody-forming polypeptide chains with a linker approximately 15 amino acid residues long (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 to Fc to form a didiabody (see Lu et al., 2004 J. Biol. Chem., 279(4):2856-65).

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

The bispecific molecules of the present invention can be prepared by conjugating multiple binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be created separately and then conjugated to each other. In the case where the binding specificities are proteins or peptides, various cross-linking or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, cyanamide, N-acetyl-succinimidyl-5-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), ophenylenedimaleimide (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 229:8183, and Glennie et al., 1987 J. Immunol. 139:2367-2375. The conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, III.).

Where the binding specificities are antibodies, they can be conjugated by sulfhydryl linkage of the C-terminal hinge regions of the two heavy chains. In a specific embodiment, the modified hinge region contains an odd number of sulfhydryl residues, such as one, prior to conjugation.

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Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is mAbxmAb, mAbxFab, FabxF(ab')2 or a χFab ligand fusion protein. The bispecific molecule of the invention may be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may include at least two single chain molecules. Methods for obtaining bispecific molecules are described, for example, in US patent 5260203; US Pat. No. 5,455,030; US Pat. No. 4,881,175; US Pat. No. 5,132,405; US Pat. No. 5,091,513; US Pat. No. 5,476,786; US Pat. No. 5,013,653; US Pat. No. 5,258,498; and US Patent 5,482,858.

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

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

The trimerizing domain is described, for example, in Borean Pharma EP1012280 B1. Pentamerizing modules are described, for example, in PCT/EP97/05897.

Antibodies with extended half-life

The present invention provides antibodies that specifically bind to BMP6 that have an extended in vivo half-life.

Many factors can affect the in vivo half-life of a protein. Examples are renal filtration, liver metabolism, degradation by proteolytic enzymes (proteases), and immunogenic reactions (eg, protein neutralization by antibodies and uptake by macrophages and dendritic cells). A variety of strategies can be used to increase the half-life of the antibodies and antigen-binding fragments thereof of the present invention. For example, chemical binding to polyethylene glycol (PEG), reCODE PEG, antibody backbone, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate shields; genetic fusion with proteins that bind to serum proteins such as albumin, IgG, FcRn and transferrin; cross-linking (genetic or chemical) with other binding molecules that bind to serum proteins, such as nanobodies, Fab fragments, darpins, avimers, affitels and anticalins; genetic fusion with rPEG, albumin, albumin domain, albumin-binding proteins and Fc; or incorporation into nanocarriers, sustained release dosage forms, or medical devices.

To increase the duration of serum circulation of antibodies in vivo, inert polymeric molecules, such as high molecular weight PEG, can be attached to antibodies or their fragment through a multifunctional linker or without it, or by site-specific conjugation of PEG with N- or C- the end of antibodies, or through epsilon-amino groups present on 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 aldehyde derivative of PEG, under conditions in which one or more PEG moieties are bound to the antibody or antibody fragment. PEGylation can be performed by an acylation reaction or an alkylation reaction using a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol encompasses any of the forms of PEG that have been used to derivatize other proteins, such as mono(C ₁ -C ₁₀ ) alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. In one embodiment, the pegylated antibody is an aglycosylated antibody. Linear or branched polymer derivatization will be used which results in minimal loss of biological activity. The degree of conjugation can be accurately controlled by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to antibodies. Unreacted PEG can be separated from antibody-PEG conjugates using size exclusion or ion exchange chromatography. PEGylated antibodies can be assayed for binding activity as well as in vivo potency using methods known to those skilled in the art, such as the immunoassays described herein. Methods for pegylation of proteins are known in the art and can be applied to antibodies and antigen-binding fragments thereof according to the invention. See, for example, EP 0154316 (Nishimura et al.) and EP 0401384 (Ishikawa et al.).

Other modified PEGylation technologies include Reconstructive Chemical Orthogonal Directed Engineering (ReCODE PEG) technology, which allows the introduction of chemically specific side chains into biosynthetic proteins using a remodeling system that includes tRNA synthetase and tRNA. This technology allows more than 30 new amino acids to be incorporated into biosynthetic proteins in E. coli, yeast and mammalian cells. The tRNA inserts the regulatory amino acid at any position where an amber codon is present, converting the amber from a termination codon to a codon that signals the inclusion of the chemically specified amino acid.

Recombinant PEGylation (rPEG) technology can also be used to extend the serum half-life. This technology involves the genetic fusion of a terminal non-structural protein segment of 300-600 amino acids with an existing pharmaceutical protein. Because the apparent molecular weight of such a non-structural protein chain is approximately 15 times greater than 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 increase the duration of action and increase the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (sugar). When used to deliver protein and therapeutic peptide drugs, polysialic acid provides a protective microenvironment during conjugation. It increases the duration of action of the therapeutic protein in the bloodstream and prevents it from being recognized by the immune system. The PSA polymer is present naturally in the human body. It is used by some bacteria that appeared several million years ago to coat their cell walls with it. Such naturally polysialylated bacteria were able, through molecular mimicry, to evade the effects of the body's immune system. PSC, nature's most advanced camouflage technology, can be easily obtained from such bacteria in large quantities and with desired physical properties. Bacterial PSC is completely non-immunogenic, even when bound to proteins, as it is chemically identical to PSC in the human body.

Another technology involves the use of hydroxyethyl starch (HES) derivatives that bind to antibodies. HES is a modified natural polymer derived from waxy corn starch and can be broken down by body enzymes. HES solutions are usually used to replace the missing blood volume and improve the rheological properties of blood. GEkylation of an antibody provides an increase in the duration of the circulation half-life, increases the stability of the molecule, and also reduces renal clearance, which leads to an increase in biological activity. By changing various parameters, such as the molecular weight of the HES, a wide range of different HES-antibody conjugates can be obtained.

Antibodies having an extended in vivo half-life can also be made by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) to an IgG constant domain or FcRn binding fragment (preferably an Fc fragment or Fc hinge region). -domain). See, for example, International Application Publication WO 98/23289; publication of international application WO 97/34631; and US Patent 6,277,375.

In addition, antibodies can be conjugated to albumin so that the antibody or antibody fragment is more stable in vivo or has a longer in vivo half-life. Such methods are known in the art, see, for example, international applications WO 93/15199, WO 93/15200 and WO 01/77137; and European patent EP 413622.

Strategies for increasing half-life are particularly useful in the case of nanobodies, fibronectin-based binders, and other antibodies or proteins that require an extended in vivo half-life.

Antibody conjugates

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to BMP6, recombinantly fused or chemically conjugated (including covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or antigen-binding fragment thereof, preferably a polypeptide of at least 10, at least 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 form fusion proteins. In particular, the invention relates to fusion proteins comprising an antigen-binding fragment of an antibody described in the present invention (for example, Fab fragment, Fd-43 039579 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; European patents EP 307434 and EP 367166; publications of international applications 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 using gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively referred to as DNA shuffling) techniques. DNA shuffling can be used to alter the activity of antibodies and antigen-binding fragments of the invention (eg, higher affinity, lower dissociation rate antibodies and antigen-binding fragments thereof). 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 is hereby incorporated by reference in its entirety). Antibodies and their antigen-binding fragments or encoded antibodies and their antigen-binding fragments can be altered by random mutagenesis in low precision PCR, random insertion of nucleotides, or other methods prior to recombination. A polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to BMP6 can be recombined with one or more components, motifs, sections, moieties, domains, fragments, and so on. one or more heterologous molecules.

In addition, 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: 96), such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. sci. USA 86:821-824, for example, hexahistidine (SEQ ID NO: 96) provides a convenient purification of the fusion protein. Other peptide tags used for purification include, but are not limited to, a hemagglutinin (HA) tag that matches 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 thereof according to the present invention are conjugated to a diagnostic or detectable agent. Such antibodies can be used to monitor or predict the occurrence, development, progression and/or severity of a disease or disorder as part of a clinical research procedure, such as determining the effectiveness of a particular therapy. Such diagnosis and detection can be performed by combining the antibody with detectable substances, including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; 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 fluorescein, 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 ( ¹³¹ I, ¹²⁵ I, ¹²³ I and ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ In, ¹¹³ In , ¹¹² In and ⁿ¹ In), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Tl), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co , ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn and ¹¹⁷ Sn; and positron active metals used in various types of positron emission tomography, and non-radioactive paramagnetic metal ions.

The present invention also encompasses the use of antibodies and antigen-binding fragments thereof conjugated to a therapeutic molecule. The antibody, or antigen-binding fragment thereof, may be conjugated to a therapeutic molecule such as a cytotoxin, eg, a cytostatic or cell destroying agent, a therapeutic agent, or a radioactive metal ion, eg, sources of alpha radiation. A cytotoxin or cytotoxic agent includes any substance that is destructive to cells.

In addition, the antibody, its antigen-binding fragment can be conjugated to a therapeutic or drug molecule that modifies the biological response. Therapeutic or drug molecules should not be construed as being limited to classical chemical therapeutic agents. For example, the drug molecule 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 exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor,

- 44 039579 interferon alpha, interferon beta, nerve growth factor, platelet growth factor, tissue plasminogen activator, apoptotic agent, anti-angiogenic agent; or a biological response modifier such as, for example, a lymphokine.

In addition, the antibody may be conjugated to therapeutic molecules such as a radioactive metal ion, such as alpha radiation sources such as ²¹³ Bi, or macrocyclic chelating agents used to conjugate radiometal ions, including, but not limited to, ¹³¹ In, ¹³¹ Lu , ¹³¹ Y, ¹³¹ Ho, ¹³¹ Sm, with polypeptides. In one embodiment, the macrocyclic chelating agent is 1,4,7,10-tetraα3αcyclododecαn-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, incorporated by reference in their entirety.

Methods for conjugating therapeutic molecules to antibodies are known, see, for example, Amon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, 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, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Methods for producing antibodies according to the invention

Nucleic Acids Encoding Antibodies The invention relates to substantially purified nucleic acid molecules that encode polypeptides comprising segments or chain domains of the BMP6-binding antibodies described above. Some nucleic acids according to the invention include 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 NO: 26; 46; 66 or 86. In a certain embodiment, the nucleic acid molecules are identified in table. 1. Some other nucleic acid molecules of the invention include nucleotide sequences that are essentially identical (for example, at least 65%, 80%, 95%, or 99%) of the nucleotide sequences identified in table. 1. When expressed from suitable expression vectors, the polypeptides encoded by such polynucleotides are capable of exhibiting BMP6 antigen-binding ability.

The invention also relates to polynucleotides that encode at least one CDR region and usually all three CDR regions from the heavy or light chain of the BMP6-binding antibody shown in Table 1. 1. Some other polynucleotides encode all or substantially the entire sequence of the variable region of the heavy chain and/or light chain of the BMP6-binding antibody shown in Table. 1. Due to the degeneracy of the genetic code, multiple nucleic acid sequences encode each of the amino acid immunoglobulin sequences.

The nucleic acid molecules of the invention may encode both the variable region and the constant region of an antibody. Certain nucleic acid sequences of the invention include 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 shown in any of SEQ IDs. NO: 16; 36; 56 or 76. Some other nucleic acid sequences include 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 shown 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, the sequence described in the examples below) encoding a BMP6 binding antibody or binding fragment thereof. Direct chemical synthesis of nucleic acids can be performed using methods known in the art, such as the phosphotriester method (Narang et al., 1979, Meth. Enzymol. 68:90); phosphodiester method (Brown et al., Meth. Enzymol. 68:109, 1979); diethylphosphoramidite method (Beaucage et al., Tetra. Lett., 22:1859, 1981); and the solid support synthesis method of US Pat.

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.

The invention also relates to expression vectors and host cells for the production of BMP6 binding antibodies described above. Various expression vectors can be used to express polynucleotides encoding chains or binding fragments of BMP6 binding antibodies. 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 that can be used to express BMP6-binding polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1/His, pEBVHis A, B, and C, (Invitrogen, San Diego, Calif.), MPSV vectors, and many other vectors known in the art for expressing other proteins. Suitable viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40 vectors, 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 antigen-binding fragment of the BMP6-binding antibody chain. In one embodiment, an inducible promoter is used to prevent expression of the inserted sequences except under inducing conditions. Inducible promoters include, for example, the arabinose, lacZ, metallothionein or heat shock promoter. Cultures of transformed organisms can be propagated under non-stimulatory conditions without changing the composition of the population towards coding sequences whose expression products are better tolerated by host cells. In addition to promoters, other regulatory elements may also be or may be required for efficient expression of the antigen-binding fragment of the BMP6-binding antibody chain. Such 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 may also provide the position of a secretion signal sequence to produce a fusion protein with polypeptides encoded by the inserted BMP6 binding antibody sequences. Typically, the inserted BMP6 binding antibody sequences are linked to signal sequences prior to inclusion in the vector. Vectors that will be used to insert sequences encoding the light and heavy chain variable domains of a BMP6 binding antibody sometimes also encode constant regions or portions thereof. Such vectors allow the expression of variable regions as fusion proteins with constant regions, resulting in the production of intact antibodies and their antigen-binding fragments. Typically, such constant regions are human.

Host cells for the introduction and expression of BMP6-binding antibody chains can be prokaryotic or eukaryotic. E. coli is one of the prokaryotic hosts suitable for cloning and expression of the polynucleotides of the present invention. Other microorganism hosts suitable for use include bacilli such as Bacillus subtilis and other enterobacteria such as Salmonella, Serratia and various Pseudomonas species. For such 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 different known promoters will be present, such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. Promoters typically control expression, optionally with an operator sequence, and contain ribosome binding site sequences and the like. for the initiation and termination of transcription and translation. Other microorganisms, such as yeast, can also be used to express the BMP6 binding polypeptides of the invention. Insect cells can also be used 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 any hybridoma cell line expressing endogenous immunoglobulin genes (eg, myeloma hybridoma clone 1D6.C9 as described in the examples), or a mammalian cell line carrying an exogenous expression vector (eg, SP2 myeloma cells). 0 below). They include any normal mortal or normal or abnormal immortal animal or human cell. For example, a variety 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 discussed generally 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 as an origin of replication, a promoter and an enhancer (see, for example, Queen, et al., Immunol. Rev. 89:49-68, 1986), as well as necessary information processing sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription termination sequences. These expression vectors typically contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific and/or modulated or regulated. Suitable 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 promoter-enhancer combinations known in the art.

Methods for introducing expression vectors containing polynucleotide sequences of interest differ depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts (see generally 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, naked DNA, artificial virions, fusion with the structural protein VP22 of the herpes virus (Elliot and O 'Hare, Cell 88:223, 1997), drug-enhanced DNA uptake and ex vivo transduction. Long-term production of recombinant proteins in high yield often requires stable expression. For example, cell lines that stably express BMP6 binding antibody chains or binding fragments can be generated using expression vectors of the invention that contain viral origins or endogenous expression elements and a selectable marker gene. Following vector introduction, cells can be grown for 1-2 days in enriched media before being transferred to selective media. The purpose of the selectable marker is to confer resistance to selection, while its presence ensures the growth of cells that successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be propagated using tissue culture techniques appropriate for the particular cell type.

Obtaining monoclonal antibodies according to the invention

Monoclonal antibodies (mAbs) can be prepared in a variety of ways, including standard techniques for preparing monoclonal antibodies, for example, the standard somatic cell hybridization method of Kohler and Milstein (Kohler and Milstein, 1975 Nature 256:495). To obtain a monoclonal antibody, you can use a variety of methods, such as viral or oncogenic transformation of B-lymphocytes.

The system in which animals are used to produce hybridomas is the murine system. Obtaining hybridomas in mice is a well-established technique. Immunization protocols and methods for isolating immunized splenocytes for fusion are known in the art. Fusion participants (eg murine myeloma cells) and fusion techniques are also known.

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

- 47 039579

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

The HuMAb® mouse (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human immunoglobulin heavy (mu and gamma) and kappa light chain sequences and has targeted mutations that inactivate the endogenous mu and kappa chain loci (see e.g. , Lonberg, et al., 1994 Nature 368 (6474): 856-859). Thus, mice show 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 kappa IgG (Lonberg, N. et al., 1994, above, reviewed by 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 also 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. 152:29122920; Taylor, L. et al., 1994 International Immunology 579-591; and Fishwild, D. et al., 1996 Nature Biotechnology 14: 845-851, the contents of which are hereby expressly incorporated by reference. See also US Pat. Nos. 5,545,806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299; and 5770429 (Lonberg and Kay); US patent 5545807 (Surani et al.); PCT Publications WO 92/103918, WO 93/12227, WO 94/25585, WO 97/113852, WO 98/24884 and WO 99/45962 (Lonberg and Kay); and PCT Publication WO 01/14424 (Korman et al.).

In another embodiment, human antibodies of the invention can be induced 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 (Ishida et al.).

In addition, alternative systems based on transgenic animals expressing human immunoglobulin genes are available in the art and can be used to induce BMP6-binding antibodies and antigen-binding fragments thereof according to the invention. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in US patents 5939598; 6075181; 6114598; 6150584 and 6162963 (Kucherlapati et al.).

In addition, alternative systems based on transchromosomal animals expressing human immunoglobulin genes are available in the art and can be used to induce BMP6-binding antibodies of the invention. For example, mice carrying 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) that can be used to induce BMP6-binding antibodies of the invention.

Human monoclonal antibodies of the invention can also be generated using phage display techniques to screen human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are known in the art or described in the examples below. See, for example: US Pat. Nos. 5,223,409; 5403484; and 5571698 (Ladner et al.); US patents 5427908 and 5580717 (Dower et al.); US patents 5969108 and 6172197 (McCafferty et al.); and US Patents 5,885,793; 6521404; 6544731; 6555313; 6582915 and 6593081 (Griffiths et al.).

The human monoclonal antibodies of the invention can also be made using SCID mice in which human immunocytes have been reconstituted so that a human humoral immune response can be obtained when immunized. Such mice are described, for example, in US patents 5476996 and 5698767 (Wilson et al.).

Wireframe or Fc-region engineering

Engineered antibodies and antigen-binding fragments thereof according to the invention include antibodies and antigen-binding fragments in which modifications have been made to the framework residues in 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 method is to back-mutate one or more framework residues to the appropriate germline sequence. More specifically, an antibody that has been somatically mutated may contain framework residues that differ from the germline sequence from which

- 48 039579 an antibody occurs. Such residues can be determined by comparing the framework sequences of the antibody with the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can reverse mutate to produce a germline sequence, for example, by site-directed mutagenesis. It is contemplated that such back mutated antibodies are also included in the invention.

Another type of framework modification involves mutating one or more residues in the framework, or even 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 Publication 20030153043 (Carr et al.).

In addition to, or as an alternative to, modifications made to the framework or CDR regions, the antibodies of the invention may be genetically engineered to include modifications to the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life. , complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, an antibody of the invention may be chemically modified (eg, one or more chemical molecules may be attached to the antibody) or modified to change its glycosylation, again to change one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The numbering of residues in the Fc region corresponds to the numbering according to the Kabat EU index.

In one embodiment, the CH1 hinge region is altered such that the number of cysteine residues in the hinge region is altered, such as increased or decreased. This approach is also described in US patent 5677425 (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 hinge of the Fc region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domains of the Fc-hinge fragment such that the antibody has impaired binding to staphylococcal protein A (SpA) relative to SpA binding of the native Fc-hinge domain. This approach is described in more detail in US patent 6165745 (Ward et al.).

In another embodiment, the antibody is modified to increase its half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in US Pat. No. 6,277,375 (Ward et al.). Alternatively, to increase the biological half-life of an antibody, the antibody can be altered in the CH1 or CL region to include a rescue receptor binding epitope taken from two loops of the CH2 domain of the IgG Fc region as described in US Pat. Nos. 5,869,046 and 6,121,022 (Presta et al.).

In one embodiment, the Fc region is changed by replacing at least one amino acid residue with another 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 antigen-binding ability of the original antibody. 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 patents 5624821 and 5648260 (Winter et al.).

In another embodiment, one or more amino acids selected from amino acid residues may be replaced with another 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 6194551 (Idusogie et al.).

In another embodiment, one or more amino acid residues are changed to thereby alter the ability of the antibody to bind complement. This approach is also described in PCT publication WO 94/29351 (Bodmer et al.).

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

In another embodiment, the glycosylation of the antibody is altered. For example, an aglycosylated antibody (ie, the antibody has no glycosylation) can be made. Glycosylation can be modified, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be obtained, for example, by changing one or more glycosylation sites in the antibody sequence. For example, one or more amino acids can be made

- 49 039579 substitutions, which lead to the elimination of one or more glycosylation sites of the framework of the variable region with the elimination of glycosylation at this site. Such aglycosylation can increase the affinity of the antibody for the antigen. A similar approach is described in more detail in US patents.

5714350 and 6350861 (Co et al.).

Additionally or alternatively, an antibody can be designed that has an altered glycosylation pattern, such as a hypofucosylated antibody with reduced fucosyl residues or an antibody with increased GlcNAc branching structures. It has been demonstrated that such altered glycosylation profiles increase the ADCC capacity of antibodies. Such carbohydrate modifications can be obtained, for example, by expressing an antibody in a host cell with an altered glycosylation machinery. Cells with an altered glycosylation apparatus have been described in the art and can be used as host cells in which the recombinant antibodies of the invention can be expressed, resulting in an antibody with altered glycosylation. For example, EP 1176195 by Hang el al. describes a cell line with a functionally interrupted FUT8 gene that encodes a fucosyltransferase, and antibodies expressed in such a cell line show hypofucosylation. PCT publication WO 03/035835 by Presta describes another CHO cell line, LecI3 cells, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also leads to hypofucosylation of antibodies expressed in such a host cell (see (see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT publication WO 99/54342 (Umana el al.) describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g. cell lines exhibit increased GlcNac branching structures resulting in increased ADCC antibody activity (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Methods for constructing modified antibodies

As discussed above, BMP6 binding antibodies having VH and VL sequences or full length heavy and light chain sequences shown herein can be used to generate new BMP6 binding antibodies by modifying full length heavy chain and/or light chain sequences, VH sequences. and/or VL or constant region(s) attached to them. Thus, in another aspect of the invention, the structural features of a BMP6-binding antibody of the 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 invention, such as binding to human BMP6, as well as inhibition of one or more functional properties of BMP6 (eg, inhibition of erythrocyte lysis in a hemolytic test).

For example, one or more CDR regions of antibodies and their antigen-binding fragments of the present invention, or mutations thereof, can be combined recombinantly with known framework regions and/or other CDR regions to obtain additional recombinantly engineered BMP6-binding antibodies and their antigen-binding fragments of the 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, to generate a engineered antibody. Rather, the information contained in the sequence(s) is used as starting material to create a second generation sequence(s) derived from the original sequence(s), and then the second generation sequence(s) are generated and expressed as a protein.

An altered antibody sequence can also be obtained by screening antibody libraries having fixed CDR3 sequences or minimal essential binding determinants as described in US20050255552 and distinct CDR1 and CDR2 sequences. Screening can be performed according to any screening technology suitable for screening antibodies in 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 the altered antibody sequence(s) is an antibody that retains one, some, or all of the functional properties of the BMP6 binding antibodies described herein, wherein the functional properties include, but are not limited to, specific binding to human BMP6 protein; while the antibody inhibits the lysis of erythrocytes in the hemolytic test.

The functional properties of the modified antibodies can be examined using standard assays available in the art and/or described in the present invention, such as the assays presented in the examples (eg, ELISA assays).

- 50 039579

In one embodiment of the methods for constructing antibodies and their antigen-binding fragments according to the invention, mutations can be introduced randomly or selectively at all or part of the coding sequence of BMP6-binding antibodies, while the resulting modified BMP6-binding antibodies can be screened for binding activity and / or other functional properties as described in the present invention. Mutation methods have been described in the prior art. For example, PCT Publication WO 02/092780 by Short describes methods for generating and screening for antibody mutations using saturation mutagenesis, assembly of ligatable synthetic fragments, or a combination thereof. Alternatively, PCT publication WO 03/074679 (Lazar et al.) describes methods for using computer screening methods to optimize the physiochemical properties of antibodies.

Antibody assay according to the invention

Antibodies and their antigen-binding fragments according to the invention can be investigated using various functional assays. For example, they can be tested for the 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, with binding detected indirectly using various sandwich assay formats known in the art.

In one embodiment, BMP6-binding antibodies and antigen-binding fragments thereof according to the invention block the binding of a reference BMP6-binding antibody to or compete with a BMP6 polypeptide. They may be the fully human BMP6 binding antibodies described above. They may 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 binding of the reference antibody indicates that the BMP6-binding antibody in the assay binds to the same or similar epitope as the specific reference antibody, or to an epitope that is sufficiently close to the epitope bound by the reference BMP6-binding antibody. Such antibodies are particularly likely to have the general advantageous properties identified in the reference antibody. The ability to block or compete with a reference antibody can be determined, for example, using a competitive binding assay. Using a competition binding assay, a test antibody is tested for its ability to inhibit the 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 significantly inhibits binding of the reference antibody. 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 well-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 (ELISA), competitive sandwich assay (see Stahli et al., Methods in Enzymology 9:242-253, 1983); solid phase direct biotin-avidin ELISA (see Kirkland et al., J. Immunol. 137:3614-3619, 1986); solid-phase direct analysis using a label, solid-phase direct sandwich analysis using a label (see Harlow & Lane, supra); solid phase direct RIA using ¹²⁵ I as a label (see Morel et al., Molec. Immunol. 25:715, 1988); solid phase direct avidin-biotin ELISA (Cheung et al., Virology 176:546-552, 1990); and direct RIA using a label (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 BMP6 binding antibody, 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 antibody binding to the same epitope that the reference antibody binds and antibody binding to an adjacent epitope close enough to the epitope bound by the reference antibody that steric hindrance occurs.

To determine if selected BMP6-binding monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (eg, those of Pierce, Rockford, III.). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using ELISA plates coated with BMP6 polypeptide. Binding of the biotinylated mAb can be detected using a streptococcal avidin alkaline phosphatase conjugate test. To determine the isotype of purified BMP6

- 51 039579 binding antibodies can be performed by isotype-specific ELISA. For example, microtiter plate wells can be coated with 1 μg/ml anti-human IgG overnight at 4°C. After blocking with 1% BSA, the plates are reacted with 1 μg/ml or less of monoclonal BMP6 binding antibody or purified isotype controls at ambient temperature for one to two hours. The wells can then be reacted with alkaline phosphatase conjugated probes specific for either human IgG1 or human IgM. 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 the binding of monoclonal BMP6-binding 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 hour . After washing, the cells are reacted with fluorescein-labeled anti-human IgG antibody under the same conditions as when staining the primary antibody. Samples can be analyzed on a FACScan cytometer using forward and side scatter parameters for single cell sorting. An alternative analysis using fluorescence microscopy (in addition to or instead of flow cytometric analysis) can be used. Cells can be stained exactly as described above and examined using fluorescence microscopy. This method allows imaging of single cells, but may have reduced sensitivity depending on the density of the antigen.

BMP6-binding antibodies and antigen-binding fragments thereof according to the invention can also be tested for reactivity with a BMP6 polypeptide or antigenic fragment using Western blotting. Briefly, purified BMP6 polypeptides or fusion proteins or cell extracts from BMP6 expressing cells 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 treated with the monoclonal antibodies to be tested. Human IgG binding can be detected using an anti-human IgG antibody-alkaline phosphatase conjugate and displayed 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 relates to methods for treating a disease or disorder associated with increased BMP6 activity by administering to a patient an effective amount of the antibodies and antigen-binding fragments thereof according to the invention. In a particular embodiment, the present invention relates to a method for treating anemia by administering to a patient an effective amount of antibodies and antigen-binding fragments thereof according to the invention.

Antibodies and their antigen-binding fragments according to the invention can be used, among other things, to prevent the progression of anemia. They may also be used in combination with other treatments to treat anemic patients.

In one embodiment, the present invention provides methods for treating a BMP6-associated disease or disorder by administering to a patient an effective amount of the antibodies and antigen-binding fragments thereof of the invention. Examples of known BMP6-associated diseases or disorders include: anemia, including but not limited to: anemia of chronic disease (ACD), anemia of (e.g., associated with) chronic kidney disease (CKD), anemia of cancer, anemia of inflammation, erythropoiesis-stimulating drug (ESD) resistant anemia (e.g., erythropoietin (EPO) resistant anemia, ESP hyporeactive anemia (e.g., EPO hyporeactive anemia), functional iron deficiency anemia, and/or iron deficiency anemia.

In a particular embodiment, the present invention provides methods for treating a BMP6-associated disease or disorder by administering to a patient an effective amount of the antibodies and antigen-binding fragments thereof of the invention, wherein said disease or disorder is anemia. In an embodiment, the anemia is anemia of 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 of chronic disease) is ESP (eg, EPO) resistant anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is ESP (eg, EPO) hyporeactive anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a chronic hemodialysis (HD) patient. In embodiments, including any of the above embodiments, the patient has a kidney disease, such as end stage renal disease.

- 52 039579

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

In a particular embodiment, the present invention relates to methods for treating anemia by administering to a patient an effective amount of a composition comprising an antibody of the present invention. In a particular embodiment, the present invention relates to methods for treating anemia by administering to a patient an effective amount of a composition comprising an antibody of the present invention. In an embodiment, the anemia is chronic kidney disease anemia. In an embodiment, the anemia is ESP (eg, EPO) resistant anemia. In an embodiment, the anemia is ESP (eg, EPO) hyporeactive 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 patient is a chronic hemodialysis (HD) patient. In embodiments, including any of the above embodiments, the patient has a kidney disease, such as end stage renal disease.

In a particular embodiment, the present invention provides methods for treating a BMP6-associated disease or disorder by administering to a patient 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 patient is a treated ESP (eg, EPO) patient on chronic hemodialysis. In an embodiment, the patient is an ESP-treated (eg, EPO) chronic hemodialysis patient with chronic kidney disease.

In a particular embodiment, the present invention relates to methods for treating anemia.

In a particular embodiment, the present invention relates to a method for reducing the need for administering doses of ESP (eg, EPO) in a patient by administering to the patient an effective amount of an antibody or antigen-binding fragment thereof according to the invention. In an embodiment, the anemia is anemia of 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 of chronic disease) is ESP (eg, EPO) resistant anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is ESP (eg, EPO) hyporeactive anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a chronic hemodialysis (HD) patient. In embodiments, including any of the above embodiments, the patient has a kidney disease, such as end stage renal disease.

In a particular embodiment, the present invention relates to methods of reducing the need for administering doses of ESPs (eg, EPO) in a patient by administering to the patient an effective amount of an antibody or antigen-binding fragment of the invention, wherein said patient has functional iron deficiency anemia. In an embodiment, the patient is a treated ESP (eg, EPO) patient on chronic hemodialysis. In an embodiment, the patient is an ESP-treated (eg, EPO) chronic hemodialysis patient with chronic kidney disease.

In a particular embodiment, the present invention relates to a method of reducing the need for administration of doses of ESP (eg, EPO) in a patient by introducing to the patient an effective amount of a composition comprising an antibody according to the present invention. In an embodiment, the patient is anemic. In an embodiment, the anemia is anemia of 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 of chronic disease) is ESP (eg, EPO) resistant anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is ESP (eg, EPO) hyporeactive anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a chronic hemodialysis (HD) patient. In embodiments, including any of the above embodiments, the patient has

- 53 039579 kidney disease, such as end-stage renal disease.

In a particular embodiment, the present invention relates to methods for reducing the need for administering doses of ESPs (eg, EPO) in a patient by administering to the patient an effective amount of a composition comprising an antibody of the present invention, wherein said patient has functional iron deficiency anemia. In an embodiment, the patient is a treated ESP (eg, EPO) patient on chronic hemodialysis. In an embodiment, the patient is an ESP-treated (eg, EPO) chronic hemodialysis patient with chronic kidney disease.

In a particular embodiment, the present invention relates to a method for reducing the need for administering doses of an iron preparation (eg, an intravenous iron preparation) in a patient by administering to the patient an effective amount of antibodies and antigen-binding fragments thereof according to the invention. In an embodiment, the patient is anemic. In an embodiment, the anemia is anemia of 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 of chronic disease) is ESP (eg, EPO) resistant anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is ESP (eg, EPO) hyporeactive anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a chronic hemodialysis (HD) patient. In embodiments, including any of the above embodiments, the patient has a kidney disease, such as end stage renal disease.

In a particular embodiment, the present invention relates to methods for reducing the need for administering doses of an iron preparation (e.g., an intravenous iron preparation) in a patient by administering to the patient an effective amount of antibodies and antigen-binding fragments thereof according to the invention, where the specified patient has functional iron deficiency anemia. In an embodiment, the patient is a treated ESP (eg, EPO) patient on chronic hemodialysis. In an embodiment, the patient is an ESP-treated (eg, EPO) chronic hemodialysis patient with chronic kidney disease.

In a particular embodiment, the present invention relates to a method of reducing the need for administering doses of an iron preparation (eg, an intravenous iron preparation) in a patient by administering to the patient an effective amount of a composition comprising an antibody of the present invention. In an embodiment, the patient is anemic. In an embodiment, the anemia is anemia of 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 of chronic disease) is ESP (eg, EPO) resistant anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is ESP (eg, EPO) hyporeactive anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a chronic hemodialysis (HD) patient. In embodiments, including any of the above embodiments, the patient has a kidney disease, such as end stage renal disease.

In a particular embodiment, the present invention relates to methods of reducing the need for administering doses of an iron preparation (e.g., an intravenous iron preparation) in a patient by administering to the patient an effective amount of a composition comprising an antibody of the present invention, wherein said patient has functional iron deficiency anemia. In an embodiment, the patient is a treated ESP (eg, EPO) patient on chronic hemodialysis. In an embodiment, the patient is an ESP-treated (eg, EPO) chronic hemodialysis patient with chronic kidney disease.

In a particular embodiment, the invention relates to a method for reducing the need for administering doses of an iron preparation (for example, an intravenous iron preparation) in a patient and reducing the need for administering doses of an ESP (for example, EPO) in a patient, comprising administering an antibody or antigen-binding fragment of the invention or composition , including the specified antibody or antigennegative fragment. In an embodiment, the anemia is anemia of chronic disease. In an embodiment, the chronic disease is chronic kidney disease. In an embodiment, the chronic disease is cancer. In an embodiment, the patient is anemic. In an embodiment, the chronic disease is inflammation. In an embodiment, the anemia (eg, anemia of chronic disease) is ESP (eg, EPO) resistant anemia. In an embodiment, anemia (e.g., anemia of chronic disease

- 54 039579 vaniya) is ESP (for example, EPO) hyporeactive anemia. In an embodiment, the anemia (eg, anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a chronic hemodialysis (HD) patient. In embodiments, including any of the above embodiments, the patient has a kidney disease, such as end stage renal disease.

In an embodiment, the present invention relates to methods for mobilizing sequestered iron by administering to a patient an effective amount of the antibodies and antigen-binding fragments thereof according to the invention.

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

In an embodiment, the present invention relates to a method of improving (e.g., increasing) the hemoglobin level in an anemic patient while reducing the need for doses of erythropoietin and/or an iron preparation (e.g., an i.v. iron preparation), comprising administering an antibody or iron preparation to the patient. antigen-binding fragment according to the invention. In an embodiment, the anemia is anemia associated with a chronic disease. In an embodiment, improving the hemoglobin level includes improving to the level set in 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 hereby incorporated 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, eg, to about 11.0 to about 12.5 g/dl. In an embodiment, improving the hemoglobin level includes improving the hemoglobin level to at least 11.0 g/dL, for example, to a level of 11.0 to 12.5 g/dL.

In an embodiment, the present invention relates to a method of improving (e.g., increasing) hemoglobin levels in an anemic patient while reducing the need for doses of erythropoietin and/or iron (e.g., i.v. iron), comprising administering to the patient a composition comprising an antibody according to the invention. In an embodiment, the anemia is anemia associated with a chronic disease. In an embodiment, improving the hemoglobin level includes improving to the level set in 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; 2:279335, the contents of which are hereby incorporated 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, eg, to about 11.0 to about 12.5 g/dl. In an embodiment, improving the hemoglobin level includes improving the hemoglobin level to at least 11.0 g/dL, for example, to a level of 11.0 to 12.5 g/dL.

In an embodiment, the present invention relates to a method of maintaining hemoglobin levels in an anemic patient by reducing the need for erythropoietin and/or iron (e.g., intravenous iron) doses, comprising administering to the patient an antibody or antigen-binding fragment thereof according to the invention. In an embodiment, the anemia is anemia associated with a chronic disease. In an embodiment, improving the hemoglobin level includes improving to the level set in 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 hereby incorporated 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, eg, to about 11.0 to about 12.5 g/dl. In an embodiment, improving the hemoglobin level includes improving the hemoglobin level to at least 11.0 g/dL, for example, to a level of 11.0 to 12.5 g/dL.

In an embodiment, the present invention relates to a method of maintaining hemoglobin levels in an anemic patient by reducing the need for erythropoietin and/or iron (e.g., IV iron) doses, comprising administering to the patient a composition comprising an antibody of the invention. In an embodiment, the anemia is anemia associated with a chronic disease. In an embodiment, improving the hemoglobin level includes improving to the level set in 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 hereby incorporated 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, eg, to about 11.0 to about 12.5 g/dl. In an embodiment, the improvement

- 55 039579 hemoglobin level includes improving the hemoglobin level to at least 11.0 g/dl, for example to a level of from 11.0 to 12.5 g/dl.

In one embodiment, the selected antibody or antigennegative fragment described in table. 1 may be administered to a patient in combination with a therapeutic method or procedure, such as those described in the present invention or known in the art. Such a method or procedure includes, as non-limiting examples: administration of a therapeutically effective amount of an ESP (eg, EPO), erythropoietin, or an iron preparation, and blood transfusion. Treatment is usually continued intermittently for a week, a month, three months, six months or a year. In some patients, treatment is prescribed for the rest of the patient's life.

When the therapeutic agents of the present invention are administered together with another agent, the two agents may be administered sequentially in any order or simultaneously. In some aspects, an antibody of the present invention is administered to a patient who is also receiving therapy with a second agent or method (eg, ESP, erythropoietin, iron, blood transfusion). In other aspects, the binding molecule is administered in conjunction with surgical treatment.

Suitable agents for combined 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, siRNAs and small molecules to BMP6.

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

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US Patent 8,795,665; And

WO2010/056981;

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

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

Not et al. 2003 Cell. signal. 25:1372-1378;

Ikeda et al. 2012 PLoS 0040465;

Kautz et al. 2008 Blood 112: 1503;

Mi et al. 2011 J. Cancer Res. Clin. oncol. 137:245;

Xia et al. 2007 J. Biol. Chern. 282: 18129-18140;

Xia et al. 2008 Blood 111: 5195; And

Yang et al. 2009 Int. J Bioch. Cell biol. 41:853-861.

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Additional BMP6 inhibitors are known. Any of these may be used in combination with any antibody or antigen-binding fragment thereof disclosed in the present invention.

The combination therapy regimen may be additive, or it may lead to synergistic results (eg, a greater reduction in BMP6 activity than would be expected from 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 as described above using a BMP6 binding antibody of the invention and an antianemic agent or method such as ESP, erythropoietin, iron or blood transfusion.

Diagnostic applications

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

Diagnostic assays, such as competitive assays, rely on the ability of a labeled analog (tracer) 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, after which the radiotracer and analyte bound to the binding partner are separated from the unbound radiotracer and analyte. This separation is performed by draining (where the binding partner has previously been converted to an insoluble form) or centrifugation (where the binding partner has been precipitated after a competitive reaction). The amount of analyte in the test sample is inversely proportional to the amount of bound radiotracer, as measured by the amount of marker substance. Dose-response curves with known amounts of analyte are plotted and compared to test results to quantify the amount of analyte present in the test sample. These assays are referred to as ELISA systems when enzymes are used as detectable markers. In this format assay, competitive binding between antibodies and BMP6-binding antibodies results in binding of BMP6, preferably BMP6 epitopes of the invention, corresponding to the antibody count in the serum sample, more specifically neutralizing antibodies in the serum sample.

A significant advantage of this assay is that it directly measures neutralizing antibodies (ie, antibodies that interfere with the binding of BMP6, in particular its epitopes). Such an assay, especially in the form of an ELISA test, has significant applications in the clinical setting and in routine blood testing.

In clinical diagnosis or monitoring of patients with disorders associated with anemia, the detection of BMP6 proteins in comparison with levels in a corresponding biological sample of a healthy patient indicates a patient with disorders associated with anemia.

In vivo diagnosis or imaging is described in US2006/0067935. Briefly, these methods typically involve administering or administering to a patient a diagnostically effective amount of a BMP6 binding molecule that is operably linked to a non-invasively detectable marker or label. The marker antibody conjugate is allowed sufficient time to localize and bind to BMP6. The patient is then examined with a detection device to determine the detectable marker, resulting in an image of the localization 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 antibody marker binds to a tissue component. Detection of an elevated level of BMP6 proteins or a protein combination compared to a healthy person without anemia indicates a predisposition to and/or occurrence of disorders associated with anemia. These aspects of the invention are also intended for use in tissue imaging and combined diagnostic and therapeutic methods.

The invention also relates to the field of predictive medicine, in which diagnostic tests, prognostic tests, pharmacogenomic studies and monitoring clinical studies are used for prognostic purposes (for prognosis), in order to thus carry out prophylactic treatment of an individual.

The invention also relates to prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with dysregulation of BMP6 pathway activity. For example, mutations in the BMP6 gene can be analyzed in a biological sample. Such assays can be used for prognostic purposes to result in prophylactic treatment of an individual prior to the onset of a disorder characterized by or associated with BMP6, nucleic acid expression or activity.

In another aspect, the invention relates to methods for determining BMP6 nucleic acid expression or BMP6 activity in an individual, thereby selecting appropriate therapeutic agents.

- 57 039579 or prophylactic agents for the individual (referred to in the present description as pharmacogenomic studies). Pharmacogenomics allows selection of agents (eg, drugs) for therapeutic or prophylactic treatment of an individual based on the individual's genotype (eg, the genotype of an individual being tested to determine an individual's ability to respond to a particular agent).

In yet another aspect, the invention relates to a method for monitoring the effect of agents (eg, drugs) on BMP6 expression or activity in clinical studies.

Pharmaceutical compositions

The invention relates to pharmaceutical compositions comprising a BMP6-binding antibody or binding fragment thereof together 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 BMP6 associated disease (eg anemia). Pharmaceutical carriers improve or stabilize the composition, or facilitate the manufacture of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like which are physiologically compatible.

The pharmaceutical composition of the present invention may be administered in a variety of ways known in the art. The route of administration and/or route of administration will vary depending on the desired results. The administration may be intravenous, intramuscular, intraperitoneal or subcutaneous, or administration is carried out in the vicinity of the target site.

The pharmaceutically acceptable carrier must be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active substance, i.e. an antibody, bispecific and multispecific molecule, may be coated with a material to protect the compound from attack by acids and other external factors that may inactivate the compound.

The composition must be sterile and fluid. The desired fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases it is preferable to include isotonic substances, for example sugars, polyhydric alcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition a substance that delays absorption, such as aluminum monostearate or gelatin.

Pharmaceutical compositions according to the invention can be manufactured in accordance with known and routinely used 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 under GMP conditions. Typically, the pharmaceutical compositions of the invention will employ a therapeutically effective dose or effective dose of the BMP6 binding antibody. BMP6 binding antibodies are formulated into pharmaceutically acceptable dosage forms by standard methods known to those skilled in the art. Dosing regimens are adjusted to provide the optimum desired response (eg, therapeutic response). For example, a single dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased in accordance with the exigency of the therapeutic situation. It is most preferred to formulate parenteral compositions in unit dose form for ease of administration and dosing uniformity. Dosage form with a single dose, as used in the present description, refers to physically discrete units that are suitable for use as single doses for patients undergoing treatment; each unit contains a predetermined amount of active ingredient, calculated to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier.

Actual dosage levels of active ingredients in the pharmaceutical compositions of the present invention may be varied to provide an amount of active ingredient that is effective to achieve the desired therapeutic response in a particular patient, formulation and route of administration that is not toxic to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the potency of the particular compositions of the present invention employed, or of the respective ester, salt or amide, route of administration, time of administration, excretion rate of the particular compound employed, duration of treatment, other drugs, compounds and/or materials. used in combination with the specific compositions used, age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors.

A physician or veterinarian may begin to administer doses of the antibodies and antigen-binding fragments of the invention used in a pharmaceutical composition at lower levels than

- 58 039579 is necessary 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 the routes of administration, the target site, the psychological state of the patient, whether the patient is human or animal, other drugs used, and whether the treatment is prophylactic or therapeutic. Doses used in treatment should be titrated to optimize safety and efficacy. When the antibody is administered systemically, the dose varies from about 0.0001 to 100 mg/kg, and usually 0.01-15 mg/kg, based on the body weight of the recipient. An exemplary treatment regimen includes systemic administration once every two weeks or once a month, or once every 3-6 months. With intravitreal administration of the antibody, the dose varies from about 0.0001 to about 10 mg. An exemplary treatment regimen includes systemic administration once every two weeks, or once a month, or once every 3-6 months.

The antibody is usually administered multiple times. Dosing intervals may be weekly, monthly or yearly. The intervals may also be irregular, depending on the measurement of BMP6-binding antibody levels in the patient's blood. In some methods of systemic administration, the dose is adjusted to obtain a plasma antibody concentration of 1-1000 μg/ml, and in some methods 25-500 μg/ml. Alternatively, the antibody can be administered as a sustained release dosage form, in which case less frequent administration is required. The dose and frequency of administration vary depending on the half-life of the antibody in the patient's body. Typically, humanized antibodies exhibit a longer half-life than chimeric antibodies 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 long intervals over an extended period of time. Some patients continue treatment throughout their lives. In therapeutic applications, a relatively high dosage is sometimes required at relatively short intervals until the progression of the disease is slowed or halted, and preferably until the patient demonstrates a partial or complete reduction in the severity of the symptoms of the disease. After that, the patient can be assigned a prophylactic regimen.

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

Examples

The following examples are presented to further illustrate the invention without limiting its scope. Other variations of the invention will be apparent to one of ordinary skill in the art and are included within the scope of the appended claims.

Example 1

The examples describe, among other things, antibodies 3, 5, 6 and 7.

This describes the relationship between them and the original antibodies: _______________

original clone NOV0442 (VH3_3-15, Vll_le) removal RTM+Recovery FW-LCDR2 NOV0442_VL[Y49,G50,N51Q] NOV0442_VL [Y49,G50,N51S] Mature clone with new HCDR2 NOV0787 NOV0798 NOV0806 NOV0800 Engineered clone (with germline sequence) NOVO951 NOVO954 NOVO958 NOVO961 NVP number Antibody 5 Antibody 6 Antibody 7 Antibody 3

All clones are human IgG1 antibodies.

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List of abbreviations.

Reduction Description mk micro ak Amino acid AHZ Anemia in chronic disease Amp Ampicillin AR Alkaline phosphatase BMP Bone morphogenetic protein BMPR Bone morphogenetic protein receptor mon base pair BRE BMP sensing element BSA Bovine serum albumin Cam Chloramphenicol CDR Complementarity-determining region CKD chronic kidney disease su Javanese macaque Yes dalton DMEM Dulbecco's Modified Medium Needle DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleoside triphosphate DTT DL-Dithiotreitol eu ₅₀ Median effective concentration ECL Enhanced chemiluminescence E. coli Escherichia coli ED TA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay EPO Erythropoietin ESP Erythropoiesis-stimulating drug fab Antigen-binding antibody fragment EU Crystallizable Fragment F-DAS Final assessment of the development feasibility G1U Glucose HEK cells Human embryonic kidney cells HGB Hemoglobin HP Helper phage, VCSM13 HRP horseradish peroxidase Hu Human ic ₅₀ Median inhibitory concentration IgG Immunoglobulin G IPTG Isopropyl-β-D-thiogalactoside _KD Equilibrium dissociation constant ^of f Dissociation rate constant k _on Association rate constant LB Luria-Bertani LP Liquid phase Luc Luciferase M Molar min minutes MG Mass spectrometry MSD MESOScale discovery NaCl Sodium chloride Ni-NTA Nickel nitrilotriacetic acid O/n overnight OD Optical density PBS Phosphate buffered saline PBST Phosphate buffered saline with 0.1% Tween 20 PCR polymerase chain reaction

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PEG Polyethylene glycol pay Polyethyleneimine REM Protein Expression Medium Pen/Strep Penicillin/streptomycin PK/PD Pharmacokinetics/pharmacodynamics POD Peroxidase RTM Post-translational modification site RE Restrictase RGA Reporter analysis rpm Rpm CT Room temperature EN resonant units With seconds S-DAS Evaluation of the feasibility of development during selection SDS-PAGE Electrophoresis in polyacrylamide gel with sodium dodecyl sulfate SET Equilibrium titration of a solution SP solid phase SPR Surface plasmon resonance Tp Melting temperature UV Ultraviolet vh Heavy chain variable domain fragments VL Light chain variable domain fragments

Short review.

In this work, specific antibodies against human BMP6 were successfully identified using phage display using a human phage display library.

Introduction.

Anemia is common in patients with chronic kidney disease (CKD) and is associated with a lower quality of life and a higher risk of adverse outcomes, including cardiovascular disease and death.

The goal of anti-BMP6 antibodies as hepcidin-lowering therapy is to improve the condition of patients with iron deficiency anemia while increasing hemoglobin, reducing the need for erythropoiesis-stimulating drugs such as erythropoietin and intravenous iron preparations. Hepcidin-lowering agents may be an effective strategy to reduce the severity of ESP of hyporeactive anemia in this group of patients and in other forms of anemia in chronic diseases (ACD) characterized by iron deficiency.

The overall goal of the project is to identify and develop antibodies against BMP6 that can lower hepcidin levels and thus improve the condition of patients with iron deficiency anemia by reducing the need for erythropoiesis-stimulating drugs (ESPs). In this work, phage display was used to identify the BMP6-specific binding agent.

Preferred anti-BMP6 antibodies meet most or all of the following criteria.

Dissociation constants (KD values) of Fab fragments with respect to human BMP6 are below 1 nM.

The KD values for the BMP6 antigen of the cynomolgus monkey are no more than 5 times weaker than for the human BMP6.

The KD values for the mouse BMP6 antigen are no more than 5 times weaker than for the human BMP6.

Selectivity for human BMP6 compared to human BMP5 and human BMP7 by more than 100-fold difference.

The ability to bind and neutralize BMP6 signaling activity in the HEP3B-BRE-Luc reporter gene assay. HEP3B cells stably transfected with the BRE2-luc2 reporter gene were induced with hBMP proteins (R&D) and treated with anti-BMP6 antibodies. BrightGlo analysis was performed 24 hours after treatment.

The ability to inhibit BMP6-induced hepcidin expression in liver cell lines and primary human liver cells.

Low or moderate development risks. One final antibody format is human IgG1.

Antibodies were generated using a commercially available phage display library as previously described (Knappik et al. 2000 J. Mol. Biol. 296:57-86, Prassler et al. 2011 J. Mol. Biol. 413:261-278), in which uses Fab display technology on the phage surface (Rothe et al. 2008 J. Mol. Biol. 376:1182-1200). Sorting and initial ELISA screens were performed on hBMP6 (R&D Systems). ELISA screening for protein-binding hits led to the identification of hBMP6-specific binders. Antibody Fab fragments were then examined for cross-species cross-reactivity with the mouse BMP6 homolog and their binding affinity for human BMP6 was determined. The specificity of Fab fragments was also tested using hBMP2, hBMP5, and hBMP7 proteins in

ELISA. The BMP6-specific activity of the Fab fragments was also evaluated in a reporter gene assay.

Based on this initial characterization, several clones were converted to human IgG1 format and tested using the same binding, specificity and activity assays. The result of this functional analysis, combined with the profiles obtained from the development feasibility study, led to the selection of 3 clones for affinity maturation (NOV0429, NOV0441 and NOV0442).

Clones were randomized to either their LCDR3 or their HCDR2, generating 2 new Fab libraries per original clone. Solid phase sorts were performed using hBMP6 (Peprotech), as well as liquid phase sorting using randomly biotinylated hBMP6 (Peprotech). MSD-SET screening of E. coli Fab lysates, combined with a specific ELISA for hBMP5 and hBMP7 proteins, resulted in the identification of hBMP6-specific binders with improved affinity compared to the original clones. These improved derivatives were then converted to human IgG1 format and further tested in ELISA and RGA to evaluate their hBMP6-specific activity.

Mature antibody clones with the desired properties and good development feasibility profiles were selected for construction (deletion of potential PTM regions and resequencing according to germline type). The 28 resulting variants were generated as hIgG1 microscale expression and tested as previously described.

The result of this functional study, combined with the profiles obtained from the development feasibility study, led to the selection of the main candidates.

ELISA screening on plates directly coated with antigen.

Using ELISA screening, individual Fab clones were identified from the sort results when bound to the target antigen. Fab fragments were tested using Fab-containing E. coli whole lysates (see section 2.3.3).

Primary screening was performed using Maxisorp 384-well plates that were coated o/n at 4°C with hBMP6_RD at a concentration of 1.5 μg/ml in 50 mM citrate buffer, pH 4.7.

Secondary screening of major hits was performed using Maxisorp 96-well plates coated with hBMP6 RD (1.5 µg/mL in 50 mM citrate buffer, pH 4.7) and Maxisorb 96-well plates coated with hBMP7 (3 µg/mL in 50 mM citrate buffer, pH 4.7) to check specificity.

After washing, the plates were blocked for 2 h with 5% skim milk in PBS. Fab-containing E. coli lysates were added and binding was performed for 2 h at RT. To detect bound Fab fragments, the plates were washed 5 times with TBST and AP conjugated anti-human F(ab') ₂ antibody was added at a 1/5000 dilution. After incubation for 2 h at RT, the plates were washed 5 times with TBST and AttoPhos substrate was added according to the manufacturer's instructions. The plates were scanned in an ELISA spectrophotometer 10 min after the addition of the substrate.

Screening for SET after affinity maturation.

Affinity comparison was in principle performed as described below. To assess mature binders using Equilibrium Solution Titration based on the principles described by Haenel et al., 2005, a constant amount of diluted BEL extract was equilibrated overnight with various antigen concentrations.

The mixture was then transferred to MSD plates that had been pre-coated with antigen, and after incubation and washing, the appropriate MSD-Sulfo-labeled detection antibody was added.

Thereafter, the concentration of unbound Fab was determined by ECL detection using a Sector Imager 6000 (Meso Scale Discovery, Gaithersburg, MD, USA).

The results were processed using the XLfit program (IDBS) using an appropriate approximation model (Section 2.6.2.2) to evaluate affinities and thus identify the most successful clones improved by affinity maturation.

In vitro assays.

Evaluation of selectivity and cross-reactivity in ELISA.

To determine cross-species cross-reactivity of anti-BMP6 antibodies, recombinant human and mouse BMP6 proteins were bound on a plate and the ability of the antibodies to bind the recombinant proteins was determined by ELISA. Binding to the nearest human homologues, BMP5 and BMP7, was also evaluated to evaluate selectivity.

Antigenic reagents were applied to ELISA plates by direct immobilization at a concentration of 3-5 μg/ml o/n at 4°C. For loading, hBMP6 (Peprotech) was diluted in 50 mM Tris, pH 8.0. hBMP6, mBMP6 and hBMP5 (R&D Systems) were diluted in 50 mM citrate buffer, pH 4.7. hBMP7 (Peprotech) was diluted in 50 mM citrate buffer, pH 4.7. As an irrelevant antigen, hDKK1-His was applied at a concentration of 5 μg/ml in PBS.

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The next day, the antigen solutions were removed and the plates were washed three times with 100 μl TBST.

Each well was then blocked with 100 μl of 5% milk in TBST for 2 hours at RT. In subsequent experiments, blocking was performed with 100 μl of Superblock blocking buffer.

After washing the plates three times with 100 μl TBST, each antigen was incubated with 40 μl of purified Fab or IgG samples at 1 μM and 0.2 mM, respectively (in PBST buffer).

After incubation for 2 hours at RT, the plates were washed again three times and bound Fab/IgG were detected by adding 40 μl of secondary AP-conjugated anti-human IgG F(ab2) at a dilution of 1:5000. After 1 hour at RT, the signal was developed by adding 40 µl of AttoPhos substrate according to the manufacturer's protocol and the plates were analyzed immediately using an excitation wavelength of 430 nm and an emission wavelength of 535 nm on an ELISA plate spectrophotometer.

Affinity score.

ELISA binding curves.

The hBMP6 antigen (Peprotech) was loaded onto ELISA plates by direct immobilization at a concentration of 1.5 μg/ml in 50 mM Tris buffer, pH 8.0, and incubated o/n at 4°C. The next day, the antigen solution was removed and the plates were washed three times with 100 μl TBST. Each well was then blocked with 100 μl of 5% milk in TBST for 2 hours at RT. In subsequent experiments, blocking was performed with 100 μl of Superblock blocking buffer.

After washing the plates three times with 100 μg TBST, the antigen was incubated with 30 μl of purified Fab or IgG samples in a series of dilutions from 1 μM to 0.03 nM in PBST for Fab fragments, from 0.5 μM to 0.01 nM in PBST for IgG.

After incubation for 2 hours at RT, the plates were washed again three times and bound Fab/IgG were detected by adding 30 µl of secondary AP-conjugated anti-human IgG F(ab2) at a dilution of 1:5000. After 1 hour at RT, the signal was developed by adding 30 µl of AttoPhos substrate according to the manufacturer's protocol and the plates were analyzed immediately using an excitation wavelength of 430 nm and an emission wavelength of 535 nm on an ELISA plate spectrophotometer.

Equilibrium solution titration (SET) method for determination of K _D using Sector Imager 6000 (MSD).

Solution affinity determinations were generally performed as described in the literature (Friquet et al., 1985). To improve the sensitivity and accuracy of the SET method, it has been adapted from classical ELISA to ECL-based technology (Haenel et al., 2005).

mg/mL goat anti-human (Fab) ₂ fragment IgG specific antibody was labeled with MSD Sulfo-TAG™ NHS-Ester (Meso Scale Discovery, Gaithersburg, MD, USA) according to the manufacturer's instructions.

Experiments were performed in polypropylene microtiter plates and in PBS containing 0.5% (v/v) BSA and 0.02% (v/v) Tween20 as assay buffer. The unlabeled antigen was diluted in series 2n starting at a concentration at least 10 times higher than the expected KD. Wells without antigen were used to determine Bmax values; wells containing assay buffer only were used for background determination. After adding an appropriate amount of binder (antibody concentration at or below the expected KD, in a final volume of 60 μl), the mixture was incubated overnight at RT.

MSD plates were coated with antigen (30 μl per well). After washing the plates with PBS containing 0.05% (v/v) Tween20, equilibrated samples were transferred to these plates (30 μl per well) and incubated for 20 min. After washing, 30 µl per well of MSD-Sulfo-labeled detection antibody (anti-human (Fab) ₂ , typically at a final dilution of 1:2000) was added to the MSD plate and incubated for 30 min at RT on an Eppendorf shaker (700 rpm) .

After washing the MSD plates and adding 30 μl/well of MSD Read Buffer T with surfactant, electrochemiluminescent signals were detected using a Sector Imager 6000 (Meso Scale Discovery, Gaithersburg, MD, USA).

The data were evaluated using the XLfit program (IDBS) using suitable approximate models. The following approximate model was used to determine the K _D of Fab molecules (according to Haenel et al., 2005, modified according to Abraham et al., 1996):

where [Fab] _t : applied total concentration of Fab;

x: applied total concentration of soluble antigen (binding sites);

B _max : maximum Fab signal without antigen;

K _D : affinity.

The following approximate model for IgG was used to determine the K _D of IgG molecules (modified according to Piehler et al., 1997):

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where [IgG]: applied total concentration of IgG;

x: applied total concentration of soluble antigen (binding sites);

Bmax: maximum IgG signal without antigen;

KD: affinity.

Experiment parameters.

The determination of KD Fab fragments was basically performed as follows: MSD plates were coated with o/n at 4°C with 10 μl per well of hBMP6 at a concentration of 1-3 μg/ml in 10 mM Tris buffer, pH 8. After that, the plates were blocked for 1 h PBS containing 5% BSA. The hBMP6 antigen was used to titrate free Fab fragments. The antigen stock solution was pre-diluted 1:40 in 10 mM Tris buffer, pH 8.0, to 475 nM, before being adjusted to the intended starting concentration for titration with assay buffer.

Kinetic measurement of ForteBio Octet.

Affinity assessment by determining kinetic parameters was performed using biolayer interferometry technology.

Purified Fab samples were measured using Streptavidin Dip and Read biosensors. The plate was placed in an Octet QK instrument (ForteBio) and left to equilibrate at 25°C in a thermostated chamber. The cycle was started by placing the sensors in wells containing 15 μg/ml of biotinylated hBMP6 antigen for 600 s. The sensors were then placed in wells containing 0, 200, 400, 800, or 1600 nM of the purified Fab sample. The Fab concentration of 0 nM was used to determine the background. Fab association and dissociation were recorded by measuring the change in layer thickness (in nanometers, nm) over time for 800 s each, all under computer control. The data were processed automatically using the Octet User Software version 3.0.

Measuring the kinetics of BiaCore.

Measurements were made using purified Fab and IgG samples and human BMP6 and BMP7 as antigens.

Epitope sorting by ELISA.

Epitope sorting by competitive ELISA was performed to separate antibodies into groups of identical or significantly overlapping epitopes, ie. antibodies that could inhibit each other's binding.

Anti-BMP6 IgG antibodies were applied to a 384-well Maxisorp plate in a volume of 20 μl at a concentration of 66 nM in PBS. The plate was incubated o/n at 4°C. After washing twice with TBST, the plate was blocked with 100 μl per well of Superblock blocking buffer for 2.5 hours at RT, then washed twice with TBST.

When blocking the plate, Peprotech hBMP6 at 66 nM in PBST was premixed with purified anti-BMP6 Fab fragments (Flag-His labeled) at 300 nM in PBST in Eppendorf tubes (final concentrations in the mix indicated). After incubation for 2 h at RT, 20 µl of premixed hBMP6/Fab fragments were added to wells coated with blocked anti-BMP6 IgG, according to plate labeling, and incubated for no more than 20 min at RT.

The plate was washed four times with TBST and His6-POD antibody conjugate (His6 disclosed in SEQ ID NO: 96) diluted 1/1000 in PBST was added to the plate. After incubation for 1 hour at RT, the plate was washed 5 times with TBST. The ELISA chemiluminescent substrate solution was added to each well and luminescence was read without incubation using a Tecan plate spectrophotometer.

In vitro activity in reporter gene assay (RGA).

Antibody clones were tested in reporter gene assay (RGA) as purified samples of Fab fragments and/or IgG.

Briefly, the HEP3B hepatoma cell line was stably transfected with the pGL4-BRE2-Luc2 lentiviral vector, which contained the BMP-responsive BRE element in a promoter directing the expression of firefly luciferase. BMP proteins (R&D systems) were used to induce signaling. BrightGlo analysis was performed 24 hours after treatment.

Evaluation of off-target activity.

The Progen UNIchip® AV-VAR EP microarray contains 384 purified extracellular or secreted proteins expressed as an N-terminal His-tag fusion protein in E. coli.

After incubation with anti-hBMP6 antibodies at 5 μg/ml, bound antibodies were detected using DyLight649 F(ab') ₂ conjugated goat specific antibody.

- 64 039579 against the F(ab')2 hIgG fragment.

The hIgG internal control signal was set to 100%; off-target activity was normalized for hIgG; a hit is considered positive if the signal is equal to or greater than 4% (the threshold corresponds to m+3, measured relative to background).

In vivo efficacy in a mouse model.

Purified antibodies antibody 5, antibody 6 and antibody 7 were tested in a mouse model of ESP-resistant anemia.

Results.

Study of antibodies against BMP6.

Evaluation of the specificity of engineered IgGs.

engineered anti-hBMP6 IgG (germ-typed and PTM removed) were generated at the microscale and tested for specificity by ELISA at a concentration of 0.2 μM.

Engineered variants that retained limited cross-reactivity with other BMP proteins and strong affinity for hBMP6 compared to the unengineered mature clone were selected for further study.

All engineered IgG variants of NOV0429 showed greatly increased non-specific binding to BSA.

Engineered IgG variants of NOV04 41 showed slightly increased cross-reactivity with hBMP5 compared to their mature parental clone.

Engineered IgG variants of NOV04 42 retained their limited cross-reactivity with hBMP7 and with hBMP5.

table 2

ELISA specificity of engineered IgG antibody clones; 3 main antibodies are highlighted in gray

Engineered IgG IDs IgG (0.2 µM) Specificity in ELISA: Signal versus background LBMPb mBMP6_RD hBMP7 hBMP5_RD BSA NOVO 42 9 60 20 3 12 2 NOVO939 65 43 41 70 24 NOVO940 63 47 43 71 26 NOVO941 63 45 29 63 15 NOVO942 70 47 24 64 12

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NOV0441 70 27 3 5 1 NOV0943 68 26 2 7 1 NOV0944 68 27 3 6 6 NOVO945 68 thirty 4 10 4 NOV0946 65 31 7 21 1 NOVO947 65 37 17 52 7 NOVO442 46 7 2 2 1 NOV0442_VL[YGS] 42 10 2 4 1 NOV0442_VL[YGQ] 42 8 2 2 1 NOV0959 52 17 5 2 1 NOV0948 55 16 eleven 1 1 NOV0960 57 16 8 5 1 NOV0949 56 18 15 2 1 NOVO963 56 21 4 1 1 NOV0950 65 19 eleven 2 1 NOV0951 Λ 65 . 24 WEEEEE! .: 6 .. 1 NOV0964 61 20 4 2 1 NOVO952 72 25 9 2 1 NOVO965 thirty 23 2 2 1 NOVO953 39 20 4 3 1 NOV0954 . ^: i ³⁸ : 29 .'3.. 1 NOV0961 48 29 2 1 1 NOV0966 55 32 3 1 1 NOV0956 49 38 8 3 1 NOVO962 53 31 2 2 1 NOVO957 48 thirty 6 2 1 NOV0958 : '59. . 32 . 7. ' 1 1 Control IgG 61 18 14 4 1

Activity in reporter gene assay (RGA).

microscale engineered anti-hBMP6 IgG antibodies (germ-typed and PTM removed) were tested as described above. The results are shown in table. 3.

The engineered IgG variants of NOV0429 showed a 3-5-fold improved BMP6 activity, but acquired agonistic activity against all other BMP proteins.

Engineered IgG variants of NOV0441 acquired some cross-reactivity with all three other BMP proteins.

Engineered IgG variants of NOV0442 showed improved BMP activity, but also showed some inhibition of the BMP7-induced system at the highest concentration of 25 μg/ml.

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Table 3

Activity of engineered clones of IgG antibodies in RGA; IC ₅₀ values are given in [µg/mL].

NOV0951, NOV0954 and NOV0958 were selected as the main antibodies

IgG ID BMP6 BMP 7 BMP5 BMP2 min max IC50 min max IC50 min max IC50 min max IC50 NOV0429 0.03 1.0 0.9 0.8 1.2 >25 0.9 1.1 >25 0.8 1.1 >25 NOVO939 0.02 0.9 0.3 1.0 1.9 8 1.0 1.5 5 1.0 1.2 >25 NOVO940 0.01 0.9 0.3 1.0 1.9 7 0.9 1.5 5 0.9 1.2 >25 NOVO941 0.02 0.9 0.2 1.0 1.7 6 0.9 1.5 4 1.1 1.3 >25 NOVO942 0.02 1.0 0.2 1.0 1.7 >25 0.2 1.0 20 1.0 1.1 >25 NOV0441 0.02 1.0 0.6 0.9 1.1 >25 1.0 1.1 >25 1.0 0.9 >25 NOVO943 0.02 1.0 0.1 0.4 1.3 25 0.2 1.0 15 0.5 1.0 25 NOVO944 0.01 1.0 0.6 0.5 1.1 25 0.5 1.0 25 0.6 0.9 >25 NOVO945 0.01 1.0 0.2 0.4 1.1 25 0.4 1.0 25 0.5 0.9 22 NOVO946 0.01 1.0 0.2 0.4 1.2 25 0.5 1.0 25 0.7 0.9 >25 NOVO947 0.10 0.9 0.3 0.9 0.9 >25 0.6 1.0 >25 0.9 1.6 >2 5 NOV0442 0.02 1.0 0.24 0.8 1.0 >25 1.0 1.1 >25 0.8 0.9 >25 NOVO948 0.03 1.0 0.06 0.2 1.0 5 1.0 0.9 >25 1.0 1.1 >25 NOVO949 0.02 0.9 0.05 0.2 0.9 25 1.0 1.0 >25 0.9 0.9 >25 NOVO950 0.03 0.9 0.04 0.2 1.0 9 1.1 1.0 >25 0.9 0.9 >25

NOV0952 |0.021.0

NOV0953 \0.ϋ21.0

N. G/.K.'.L !>,(:,·{,T

0.05 0.3 ^oFToTs ι,ο

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1.0 >25 0.8 :_______l___ >25 10.9

0.8 >25

17θ |>25

NOVO955 0.03 0.9 0.04 0.4 0.9 25 1.0 , 1 >25 0.9 0.9 >25 NOVO956 0.02 0.9 0.05 0.5 0.9 25 1.0 1.0 >25 , 1.0 >25 NOVO957 0.03 0.9 0.08 0.4 0.9 12 1.0 1.0 >25 0.9 0.9 ^>25 NOVO958 0.02 0.9 0.04 0.5 0.9 25 1.1 1.1 >25 1.1 0.9 >25 NOVO959 0.02 1.0 0.20 0.5 0.9 25 0.8 0.9 >25 0.9 0.9 >25 NOVO960 0.01 0.9 0.07 0.5 1.0 25 0.8 1.0 >2 5 0.8 1.2 >25 NOVO961 0.02 0.9 0.08 0.8 1.0 > ^2r 0.8 0.9 >25 0.8 1.0 >25 NOVO962 0.01 0.9 0.26 0.7 1.0 >25 0.9 1.0 >25 0.8 1.0 >25 NOVO963 0.02 0.8 0.11 0.6 0.9 >25 0.8 1.1 >25' 0.8 1.0 >25 NOVO964 0.02 1.0 0.09 0.5 1.1 >25 0.7 1.0 >25 | 0.8 0.9 >25 NOVO965 0.02 1.0 0.11 0.8 1.1 >25 1.0 1.2 >25 1 0.8 0.9 >25 NOV0966 0.02 0.9 0.10 0.6 1.1 >25 0.9 1.2 >25 0.9 0.9 >25

S-DAS 3 development feasibility assessment.

After the germline sequences were changed and the PTM removed, the 23 anti-hBMP6 antibody variants from 18 mature clones were subjected to a third development evaluation.

The antibodies were found to have favorable risk profiles during development, with the exception of 2 clones that showed high risk due to high levels of aggregation (NOV0942, HCDR2 derivative of NOV0429; NOV0944, LCDR3 derivative of NOV0441).

other clones were noted with a medium risk profile due to their low productivity titer (NOV0957 and NOV0960, HCDR2 derivatives of the original NOV0442 antibody).

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Table 4

Review of S-DAS results of 3 anti-BMP6 antibodies (23 hIgGI engineered); NOV0951, NOV0954 and NOV0958 were selected as the main antibodies

NOV ID NOV936 NOV937 NOV938 NOV942 NOV943 (backup) NOV944 NOV945 (backup) NOV946 (backup) NOV951 (main) NOV953 NOV954 (main) NOV955 NOV956 NOV957 NOV958 (ultimate main) NOV959 NOV960 NOV961 (backup) NOV962 No. 8·! 50 0 about I I I I f £ and 0 S o £ X - to g * * £ § 2 in a o o 2 x < f * & no no no no no no no no no no no no no no no no no no no X 0 & I o low low low high short high short short low low low low low average low low average short short 3 in F and F I low low low high Low high low low low low low low low short low low short low low 4) В 0 0 X η X в X >> h 0 £ low low low short low low low low low low low low low average low low average low low x 0 £ in X φ -© φ X f 0 § c. f to 0 X f short short short short short short short short short short short short short short short short short short short J n F W F 0 0 o n 0 Ή V ⁸¹ 0 & n L 9.4 9.4 9.4 9.5 9.3 9.2 9.3 9.2 9.4 9.4 9.3 9.4 9.4 9.4 9.2 9.4 9.5 9.4 9.4 F Ya XX F h sr "-¾" ^N L i' in En 77.0 76.8 77.0 78.8 71.0 69.0 69.3 66.5 65.0 61.5 62.8 66 , 8 65.0 64.5 66.0 66.5 64.3 68.5 65.0 ABOUT 0 3 5 " 0 _ Θ „ „ o o & And I sch Y 0.87 0.82 0.86 0.88 0.97 0.78 0.78 0.97 1.03 1.00 1.01 1.05 1.03 0.88 1.03 0.80 1.06 1.05 0.90 I n B X U 0 l X o High tendency to aggregation High tendency to aggregation The title may not be relevant, if necessary, you can repeat it The title may not be relevant, if necessary, you can repeat it NOV963 - No short short short short 9.4 65.5 0.92 NOV964 - No short short short short 9.4 64.5 0.96 NOV965 - No short short short short 9.4 64.0 1.01 NOV966 - No short short short short 9.4 65.8 1.04 important parameters risk factors

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Critical RTM motifs yes RTM motifs recommended to be removed (NG, NS,

DG, N-Glyc, Cys, H-N30X) overall risk low low risk medium medium risk high high risk: recommendation not to continue with this candidate; this candidate does not meet the general DAS criteria aggregates low IgG monomer content is at least

95% average IgG monomer content is 90-95% high IgG monomer content is below 90%; this candidate does not meet the general DAS criteria (>90%) productivity low IgG productivity in transient expression systems is at least 10 mg/l average IgG productivity in transient expression systems is 5-10 mg/l high IgG productivity in transient expression systems is lower 5 mg/l; this candidate does not meet the general DAS criteria (>5 mg/l) pi >8.2 <8.2; indicates some risk; aggregation/difficulties may occur in the manufacture of dosage forms Tp (pH 7.4) >68°C <68°C; indicates some risk to stability; aggregation/formulation difficulties may occur hydrophobicity >0.8M (NH ₄ ) ₂ SO ₄ <0.8M (NH ₄ ) ₂ SO ₄ ; indicates some risk of aggregation/ may be difficult to formulate, high viscosity, precipitation

Evaluation of off-target activity.

prime candidates NOV0951, NOV0954 and NOV0958 were tested as purified hIgGI for off-target binding on a Protagen UNIchip microarray coated with 384 purified extracellular or secreted proteins as described above. All of them showed low non-target activity (<10 hits), which can be considered non-critical.

Table 5

Review of results obtained on protein arrays for 3 major antibodies

[Aaaaaaa' AB 5 AB b Protein gi number Protein LARP from 21707496 iiifg 3 Collagen XVIII - _17226298 1111¾¾¾ .· Protein 2810468K05Rik* 16741397 3 8|||D ? Rab40c; member of the RA3 oncogene family; RAR (RAS-like GTPase) similar; SOCS-box-containing protein RAR3* ! 21040231 3 Chromosome 4 open reading frame 14 13436155 ³ \ ||||^^ 1 Protein interacting with the plextrin homology domain 34996489 |||1I PTPRM protein Ribosomal protein kinase B (RSK-B) 3452409 Protein associated with thyroid hormone receptor 5; component of the protein complex associated with III" mm thyroid hormone receptor; a protein associated with the thyroid hormone receptor; subunit 95 kDa 38146094 VM-017 Human IGM -3-, 186200 3 Off-target activity (threshold >4%) (*) 8 10 3

(*): Off-target activity normalized for hsIgG (=100%). Positive hits are highlighted in orange.

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Selection of primary and reserve antibodies.

Based on protein binding data, activity and specificity data in RGA, and evaluation of development feasibility, it was decided to select engineered candidates NOV0951, NOV0954, NOV0958 as primary antibodies. In addition, they all showed an excellent window of specificity for hBMP6 compared to antibody 676. Engineered candidates NOV0961, NOV0943, NOV0945 were considered as reserve antibodies. In table. 6 shows the family tree of the final candidates.

Table 6

Family tree of selected primary and reserve anti-hBMP6 antibodies

Original clone, ID (skeleton) NOV0442 RTM Removal and Frame Replacement NOV0442_ Mature clone, ID (mature CDR) NOV0787 (HCDR2) Clone ID with Germ Type Change NOV0951 Number Antibody 5 (VH3, VL1) VL[Y49, G50, N51Q] NOV0798 (HCDR2) NOVO954 Antibody 6 NOV0806 (HCDR2) NOVO958 Antibody 7 NOVO44 2_ VL [Y49, G50, N51S] NOV0800 (HCDR2) NOVO961 Antibody 3 NOVO441 (VH3, VL3) Not applicable NOV0766 (LCDR3) NOVO943 LSR434 NOV0770 (LCDR3) NOVO945 LSR435 NOV0763 (HCDR2) NOVO946 LSR439

Conclusions and discussion.

The aim of this project was to obtain antibodies that inhibit BMP6 signaling and thus be suitable for the treatment of anemia in chronic diseases.

Thus, 3 different protein-based sorting strategies were applied. Primary ELISA hits were mainly found in solid phase sort pools, and after assaying for activity in RGA 12, the antibodies were shown to inhibit hBMP6 signaling. The 3 antibody clones (NOV0429, NOV0441 and NOV0442), obtained by sorting with subcode 2023.5, showed BMP6-specific activity and good development feasibility and were therefore selected for affinity maturation.

For each antibody clone, 2 libraries were generated, either with LCDR3 or randomized HCDR2. 3 different maturation sorting strategies were applied. After SET screening, ELISA specificity analysis and sequencing, 18 mature antibody clones were found to specifically inhibit BMP6 signaling in RGA and were selected for construction.

Mature clones derived from the NOV0442 family were subjected to removal of the potential deamidation site in LCDR2, framework replacement, and germline sequence changes. This resulted in NOV0951, NOV0954, NOV0958 and NOV0961 antibodies which were shown to have similar binding properties as their respective wild-type mature progenitors.

Mature clones derived from the NOV0441 family were subjected to germline sequence changes resulting in NOV0943, NOV0945 and NOV0946 antibodies, which were shown to have increased cross-reactivity with BMP5 compared to their corresponding wild-type mature progenitors.

Given their ability to inhibit BMP6 signaling with limited cross-reactivity to other BMP proteins and their favorable development profile, NOV0951, NOV0954 and NOV0958 were obtained at higher levels and submitted for subsequent evaluation of their utility as a therapeutic drug for the treatment of EPO. resistant (iron deficiency) anemia. A mouse model of ESP-resistant anemia was used to determine their efficacy in vivo.

the core antibodies NOV0951, NOV0954 and NOV0958 were subjected to a final development feasibility study. The in vivo fitness of the 3 major antibodies was assessed by determining their PK profiles in rats.

In table. 7 shows the properties of the final prime candidates that met the antibody requirements identified at the start of the project.

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Table 7

Properties of Selected Primary Anti-hBMP6 Antibodies Property CSP Criteria Basic antibodies Binding affinity K _D <1 nM to hBMP6 according to Biacore Antibody 5, Antibody 6, Antibody 7-®0.1 nM Specificity >30-fold selectivity for inhibition of hBMP6 activity compared to hBMP7 in a cellular assay (RGA) . Criteria met - all Ab ~1000x RGA selectivity Indistinguishable (undetectable) activity against BMP2 or 5 Activity in vitro Neutralizes hBMP6 in BRE-Luc RGA with 1C ₅₀ <10 nM. Antibody 5-0.26 nM Antibody 6-0.30 nM Antibody 7-0.26 nM Activity in vivo In the model of EPO-resistant anemia with inflammation, therapeutic treatment: - restores the response to EPO. - Significantly accelerates the recovery of hemoglobin (HGB) or hematocrit (HOT). For Antibody 5, Antibody 6, Antibody 7, a single dose of 2 mg/kg restores response to EPO and increases HGB by >2.0 g/dl at 1 week. Development feasibility assessment Successfully passed the final DAS assessment Antibody 5 and Antibody 7 meet the criteria for development Antibody 6 is not worth developing due to instability in cynomolgus monkey sera

Example 2 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, which were diluted in PBS prior to administration to animals. Male C56BL/6 mice or Sprague-Dawley rats were used (Table 9).

Table 8

Properties of antagonistic antibodies to BMP6

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

Table 9

Animal characteristics

View Line Category Provider Floor Age Mouse [Mus musculus] C57BL/6 WILD type Jackson Laboratory, Bar Harbor, ME Male 8-9 weeks Rat {Rattus norvegicus] SpragueDawley wild type Charles River Laboratory, Wilmington, MA Male 8-12 weeks

For assays with the BMP reporter gene, a lentiviral vector was constructed containing the BMP-responsive BRE element in the promoter [Korchynskyi et al. 2002. J. Biol. Chem. 277:4882-91], targeting firefly luciferase from pGL4-BRE2-Luc2. The lentiviral vector was used for stable transfection of the HEP3B hepatoma cell line. The cell line was maintained in EMEM with 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 vials was purchased from USDA, US Animal and Plant Health Inspection Service, National Veterinary Services Laboratories, Ames, Iowa. The brucellosis antigen for the ring test contains a suspension of killed stained cells of the strain B. abortus 1119-3 in a phenol-treated buffer. The concentration in each 60 ml vial is approximately 10 ⁹ particles/ml. Stock 5x10 ⁹ was washed and prepared as follows. First, the 60 ml vials were removed from the refrigerator and thoroughly mixed. Then 500 ml of BA was transferred into a 500 ml centrifuge flask. The flasks were then centrifuged at 10,000 rpm for 15 min in an ultracentrifuge. The supernatant was removed and resuspended in 100 ml PBS to give a stock of 5x10 ⁹ particles/ml, which was aliquoted and frozen at -80°C.

Conditions for keeping animals.

Animals were housed in shared Micro-Isolator hard-bottomed cages during periods of acclimatization and study. Animals were kept in the following standard light cycle: 12 hours dark, 12 hours light (lights on: 6:30, lights off: 18:30) at room temperature 2123°C and humidity 30-70%. During periods of acclimatization and study, animals were given access

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Experiment 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 minimal culture medium, except that the serum content was reduced 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 serum to give a final serum concentration of 1%. As intermediate assays, BMP2/4/7 activation was performed in parallel. A BrightGlo (Promega) assay was performed 24 hours after antibody addition according to the manufacturer's instructions using an Envision plate spectrophotometer (PerkinElmer). Data was calculated as percent inhibition for each antibody compared to complete reporter activation by control antibody.

Single dose pharmacokinetic study in rats.

The primary study of PK in rats is not intended to determine classical PK parameters with definite statistical significance, but rather to provide an estimate of the serum half-life of a test antibody. 3 animals received a single intravenous dose of antibody.

To study the dose dependence of PK/PD in mice, animals were equally divided into 2 separate cohorts. Each cohort included mice treated with both vehicle and compound. One cohort was tested on day 2.4 while the second cohort was tested on day 6.8 after antibody injection. The reason for dividing the cohorts was to reduce the need for intermittent blood draws so that the effects on serum iron levels were kept to a minimum. Groups of animals are shown in table. 10.

Table 10

Study Design, Animal Distribution, and Test Drug Doses

Experiment Group Qty Dose (mg/kg, IV) Frequency Mouse FC/FD hlgG control 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 Mouse VA anemia False (without VA) 6 0 0 BA, EPO+ control hlgG 6 2 once BA, EPO+ ANTIBODY 5 6 2 once BA, EPO+ ANTIBODY 6 7 2 once BA, EPO+ ANTIBODY 7 6 2 once

Induction of anemia by inflammation in mice and therapeutic treatment of 5×10 ⁸ particles BA for injection was prepared as follows (example for 10 mice). The initial concentration should be 2.5x10 ⁹ particles/ml, since 200 µl/mouse is administered. The stock is diluted 2-fold using PBS. For example, 10 mice per 0.200 ml=2 ml+20% total required=2.2 ml 2.5 x 10 ⁹ particles/ml. Stock 1.1 ml BA + 1.1 ml PBS. Administration of VA 1-8 days prior to ESP treatment has been shown to decrease HGB response after 6-7 days.

C57BL/6 mice were injected with BA (3x108 particles/mouse) and serum IL6 levels were measured 5 hours later using ELISA (KMC0061, Life Technologies) to determine the inflammatory response. Animals with IL6 concentrations below the 95% confidence interval of the mean for all 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 possibility of false positives when animals were included that did not have sufficient inflammation with an attenuated response to ESP. Following the exclusion process, mice were treated IV with antibodies as indicated on day 6 and were given EPO (subcutaneous injection of 100 g/kg darbepoetin alfa, Amgen) at 100 mg/kg on day 7 relative to BA treatment. Response to ESP and antibody therapy was measured after 6 days.

Pharmacokinetics, pharmacodynamics and end points of efficacy studies.

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

For the study of LA-induced anemia and antibody treatment, the last blood draw was made in EDTA-coated BD Microtainer tubes, completed by cardiac puncture. Whole blood was used for complete blood count XT-on an XT2000iV hematology analyzer. Efficacy results include HGB, HCT, RETA and RET-HE.

Statistical analyses.

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

Results.

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

All three anti-BMP6-5, 6 and 7 antagonist antibodies completely inhibit the bioactivity of recombinant human BMP6 BMP-induced reporter (BRE-luc) activity in the human hepatoma cell line Hep3B (IC ₅₀ = 0.4 nM versus 0.3 nM rhBMP6 ) and thus are active at an Ag/mAb mole ratio of 1:1 or better. The antibodies showed good selectivity for related proteins from the BMP family, including BMP 2, 5 and 7, with a window of 500 or more. See fig. 1.

Instantaneous pharmacokinetic and pharmacodynamic profiles of anti-BMP6 antagonistic antibodies in rats.

A primary single dose pharmacokinetic study in Sprague-Dawley rats was performed for BMP6 antibodies 5, 6 and 7 by intravenous injection through a jugular catheter at a dose level of 10 mg/kg body weight. Comparison of total serum antibody concentration versus time (particularly t _1/2 , TWC) for these three antibodies with a standard profile indicated features consistent with typical human IgG (see Figure 2 and Table 11). There was no evidence of target-mediated distribution of the drug. At this dose, all anti-BMP6 antibodies suppressed serum hepcidin to levels below the detection limit on day 1 post-injection. Long-term strong suppression of hepcidin expression was still observed at day 16, indicating a long-term maintenance of activity. Accordingly, a transient peak increase in circulating iron was observed on day 2 following antibody injection, with levels remaining elevated on day 16.

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

Table 11

Key Parameters in a Primary Single Dose PK Study 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 retention time (days) 8.6 7.0 6, 9

We also measured the total concentration of antibody 7 (free and BMP6-bound) in the serum of rats and cynomolgus monkeys after a single intravenous injection of Antibody 7 (in rats at doses of 10, 3, 1, 0.3, 0.1 and 0.03 mg /kg; in monkeys at a dose of 3 mg/kg) at the indicated time points using ELISA with 46 ng/ml LEL (dashed line) in rats and 0.2 μg/ml LEL (dashed line) in monkeys. The results are shown in FIG. 9 (rat) and 12 (monkey).

Dose-dependent response of serum iron values after anti-BMP6 antibody treatment in mice and cynomolgus monkey.

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

See fig. 3, dose-dependent effect of anti-BMP6 antibody on serum biomarkers of iron metabolism; top: the concentration of hIgG in serum over time after a single intravenous 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.

Similar experiments were performed with antibody 7. Dose- and time-dependent suppression of circulating hepcidin in 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 intravenous injection at a dose of 0.03 to 10 mg/kg . Serum hepcidin levels were measured by LC/MS at 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 BMP6 antibodies of the present invention are capable of causing a dose-dependent increase in serum iron. The action was persistent and persisted for at least 2 weeks after administration of the antibody.

The effect on serum iron levels in response to anti-BMP6 antibody was 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 tested for total serum iron (Fe) and hepcidin concentration. The results are shown in FIG. 13. Data from 3 individual animals are shown (plotted against pre-dose baseline levels). Mean values are indicated by an x line. Increases in serum iron and suppression of serum hepcidin were observed 24 hours after antibody administration, and the effect was maintained (relative to pre-dose levels) until the end of the 28-day study. These results indicate that the BMP6 antibodies of the invention potently induce hepcidin expression and reduce circulating iron concentrations in non-human primates.

Effect of anti-BMP6 antibodies on erythrocyte counts in inflammation-induced ESP-resistant anemia in mice.

Experiments were performed to evaluate the therapeutic applicability of anti-BMP6 antibodies in an inflammatory anemia model in mice. See fig. 4. Mice treated with Brucella abortus antigen (BA) developed anemia after 6 days. Anemic animals were treated with anti-BMP6 antibody plus recombinant erythropoietin (EPO) started one day later and the effect of antibody therapy on the development of anemia was monitored on day 13 against VA. HGB and HCT values decreased from start of treatment to day 13, with resistance on EPO treatment alone. Combined treatment with BMP6 antibody and EPO effectively restored the response to EPO and significantly increased HGB and HCT levels. This action was associated with a parallel stimulation of erythropoietin activity, which was reflected in a constant increase in RETA, as well as the restoration of hemoglobin content in reticulocytes, which suggests a correction of the synthesis of the theme due to a deficiency of functional iron in the erythropoiesis compartment.

See fig. 4, therapeutic treatment with anti-BMP6 antibody in a mouse model of ESP-resistant anemia in inflammation. Top: Experimental design of a model of VA-induced ESP-resistant anemia in inflammation. Bottom: Erythropoiesis scores 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 3 Clinical Plan for Anti-BMP6 Antibody Testing in Humans.

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

Patients with end-stage renal disease (ESRD) produce little, if any, amounts of erythropoietin (EPO), and they usually require intermittent exogenous EPO and intravenous (IV) infusions of iron supplements to provide.

EPO-induced Hgb synthesis. Up to one third of patients on chronic hemodialysis (HD) do not respond adequately to EPO, which is primarily due to intracellular iron sequestration. Hepcidin is initially excreted by the kidneys, but its removal by dialysis is not sufficient. Therefore, patients with chronic HD tend to have significantly elevated hepcidin levels, which block iron mobilization for erythropoiesis. IV iron therapy is no longer effective or recommended once the body's iron depot reaches a critical level (indicated by high serum ferritin levels). Current guidelines do not recommend giving IV iron to anemic dialysis patients with high ferritin levels, and therefore such patients may receive even higher doses of EPO, with an associated potential risk of EPO-hyporeactive anemia (Kidney Disease Improving Global Outcomes (KDIGO) Anemia). Work Group 2012). EPO-hyporeactive anemia is characterized by a significantly increased risk of all-cause mortality, which applies both to patients with anemia and to those with a higher dose of EPO on hemodialysis (Kilpatrick et al. 2008, Lopez-Gomez et al. 2008, Fukuma et al. 2012), and also to patients on peritoneal dialysis (Suttorp et al. 2013). Isolated antibodies or antigen-binding fragments thereof that bind human BMP6 as described herein may be effective in chronic kidney disease patients with iron deficiency anemia by increasing hemoglobin (Hgb) levels while reducing the need for EPO and IV iron supplementation. A lower EPO resistance index (the ratio of EPO dose to Hgb level) correlates with a lower risk of mortality.

In summary, the goal of therapy with isolated antibodies or antigen-binding fragments thereof that bind human BMP6 as described herein is to mobilize sequestered iron, which may then reduce the need for EPO and iron supplementation and increase Hgb levels, which in combination are expected to improve patient outcomes. This is the first single dose human study of therapy using isolated antibodies or antigen-binding fragments thereof that bind human BMP6. This study will evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics and efficacy in a population of patients on chronic hemodialysis. The purpose of this study is to evaluate whether therapy with isolated antibodies or antigen-binding fragments thereof that bind human BMP6 will warrant further clinical investigation in anemia associated with chronic kidney disease.

Research plan.

Study scheme.

This is the first human, two-stage, single-dose, non-confirmatory study of an isolated antibody or antigen-binding fragment thereof that binds human BMP6, designed to evaluate safety, tolerability, PK, PD, and efficacy in a chronic hemodialysis patient population. Part 1 is a first-time human, single-dose, open dose titration study. Part 2 is a randomized, double-blind, placebo-controlled, single dose study comparing two dose levels of an isolated antibody or antigen-binding fragment thereof that binds human BMP6.

Safety assessments will include physical examinations, ECG, vital signs, routine clinical laboratory tests (hematology, biochemistry, serum iron indices), monitoring of adverse events and serious adverse events.

Part 1.

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

During Part 1, a screening visit will be made to determine if the patient meets the inclusion criteria for the study (FIG. 6). Eligible patients are admitted to the study site and re-evaluated for eligibility at the first pre-study visit. All pre-study safety assessment results should be obtained and reviewed prior to dosing.

In FIG. 6 provides an overview of the design of Part 1 of the study. Patients are asked to report to the study site on Day 1, immediately following their usual visit for dialysis. Patients will then receive an infusion of an antibody or antigen-binding fragment that binds human BMP6 (the exact dose will depend on the cohort). If possible, dosing should preferably occur on the day of dialysis, two days before the next 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 dialysis day not preceding the two days prior to the next dialysis. After dosing, the first two patients in Part 1 are 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 assessments, and then weekly for a total of 29 days for PK assessments and a total of 12 weeks for safety assessments, with an end-of-study visit around day 85. Study visits, including all laboratory tests except post-dialysis PK assessment, should occur prior to the patient's scheduled dialysis visit.

Part 1 will be started at a dose that is not predicted to be pharmacologically active. The dose will be adjusted for each subsequent cohort in Part 1 based on the median change in Hgb in each cohort following a single dose of isolated antibody or antigen-binding fragment thereof that binds human BMP6, as well as transferrin saturation level (TSAT), as discussed in the section on statistical evaluations and shown in FIG. 7. The goal of this decision tree is to determine the lowest possible dose that causes iron mobilization (as indicated by transferrin saturation levels (TSAT) >50% observed in at least 4 patients in a cohort of 6 patients one week post-dose) and an increase in Hgb. If iron is mobilized but Hgb does not increase by at least 0.5 g/dl at 29 days post-dose, then clinical data will be analyzed to evaluate potential confounding factors (eg, blood loss due to off-study excess venotomy). Applicable Investigators and Sponsor representative(s) will review adverse events in each cohort and evaluate these events in the context of: (a) known medical problems associated with chronic renal failure and (b) findings from preclinical toxicology studies. Subsequent cohorts will not receive doses until Investigators and Sponsor indicate that the study may

- 75 039579 but it is safe to continue.

In FIG. 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 are assigned to one maximum of 6 unmasked dose cohorts, up to 6 patients each. The minimum FAD of an isolated antibody or antigen-binding fragment thereof that binds human BMP6 as defined above will be the lowest dose group selected for Part 2. The dose for each successive cohort can be increased or decreased as shown in FIG. 7. If the lowest possible dose (0.001 mg/kg) results in a median increase in Hgb >0.5 g/dL, this is the minimum FAD, and the lower dose adjusted for Part 2 and the next highest dose assessed in Part 1, will be the highest dose group for Part 2. If the highest dose (0.1 mg/kg) evaluated in Part 1 is the minimum FAD, Part 2 will continue the study with only 2 groups: placebo and minimum FAD. If additional dose cohorts are required in Part 1, these cohorts will be added as described in the present invention.

Each Part 1 cohort will include 6 patients. The first 2 patients in the first cohort of Part 1 will receive a dose at least 7 days apart. Timing of follow-up doses to patients in the Part 1 cohort will depend on availability, taking into account the respective center's schedule and service resources. 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 in response to a single dose of an antibody that binds human BMP6 compared to placebo. Part 2 will include up to three groups: up to two Ab dose groups and a placebo group (FIG. 8). The two Ab dose groups will be determined based on the data obtained in Part 1. Part 2 will include approximately 60 patients randomized 1:1:1 into three groups. If in Part 1 the trough FAD would also be the highest dose evaluated (0.1 mg/kg), then Part 2 would contain only two groups: trough FAD and placebo. In this case, 40 patients will be randomized into two groups with a randomization ratio of 1:1. The sample size in Part 2 may be adjusted based on the variability in change from pre-study Hgb levels in Part 1.

In FIG. 8 shows the design of the study for Part 2. During Part 2, a screening visit is made to determine if patients meet the inclusion criteria for the study. Eligible patients will be re-evaluated according to the inclusion criteria at the first pre-study visit. All pre-study safety assessment results should be obtained and reviewed prior to dosing.

Patients are asked to report to the study site on Day 1, immediately following their usual visit for dialysis. Patients then receive an infusion of Ab or placebo, as determined by randomization into groups. If possible, dosing should preferably occur on the day of dialysis two days before the next 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 dialysis day not preceding the two days prior to the next dialysis. Patients will return to the study center on days 4 and 6, then weekly for follow-up examinations. Patients undergo standard safety assessments at follow-up visits, and PK data will also be collected for 85 days. Study visits may be made after scheduled dialysis visits to patients in order to adhere to the patient's dialysis schedule. All patients enrolled in Part 2 will be followed up for 12 weeks after the dose of Ab (or placebo).

EPO dose selection (both parts).

Individual EPO dose adjustments during both parts will be made according to each dialysis center's standard treatment protocol. Center protocols will be reviewed as part of the center evaluation and will be reviewed against the recommendations of the standard of care (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 receive therapeutic venotomy, at the discretion of the investigator, in addition to the center's recommendations for monitoring Hgb values above target levels.

Selection of doses of intravenous iron preparations (both parts).

Patients receiving loading doses of intravenous iron (100 mg/week) will be excluded from the study. Patients receiving weekly maintenance doses of intravenous iron (<100 mg/week) may be included in this study. The weekly maintenance dose of IV iron will be maintained at the start of week 1 of the Ab dose.

Iron indices will be monitored during the first week post-dose of Ab, with mandatory iron therapy and IV iron maintenance therapy following the recommendations of the standard of care according to the operating protocol of the hemodialysis unit. Center protocols will be reviewed as part of the center evaluation and will be reviewed for compliance with the KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease.

Anemia Work Group 2012).

Justification of the research scheme.

Substantiation of the two-stage research scheme.

The rationale for the presence of two parts in the same patient population is the need to determine the minimum FAD safely and efficiently, while minimizing the number of patients and cohorts exposed to potentially subtherapeutic doses. Part 2 assesses the efficacy of the trough FAD and one dose level above the trough FAD (as defined in Part 1) compared to the placebo group.

Part 1 is designed to evaluate single dose safety, tolerability, PK/PD, and minimal FAD Ab in an open study. The minimum FAD will be determined based on each median change in Hgb in the dose cohort over 29 days post-dose of Ab. The rationale for defining FAD criteria is that clinically significant responses to EPO may take up to 4 weeks after a change in EPO dose. If Ab mobilizes iron in the target population, then this may allow the current dose of EPO in patients to have a more pronounced erythropoietic effect. The 29 day Hgb cutoffs listed in the FAD criteria are based on clinically relevant and safe rates of Hgb increase in response to EPO dose (~0.5 g/dL over 29 days). The rationale for looking for a minimal FAD rather than a maximal effect is that an excessive Hgb response poses a safety risk in this patient population, as reflected by the Hgb target limits in the current Kidney Disease Improving Global Outcomes (KDIGO) Anemia Work Group 2012 guidelines. ). The objectives of the safety and tolerability assessments in Part 1 are (a) to identify safety signals and (b) to communicate the decision to make dose adjustments, which ensures that the doses adjusted for Part 2 (minimum FAD + 1 higher dose) are appropriate. for further evaluation of safety and efficacy in comparison with placebo. While Part 1 is over-powered to provide an objective safety assessment, the placebo group and larger samples in Part 2 will provide an objective safety assessment at the minimum FAD and one higher dose. In addition to safety, tolerability, and PK/PD, Part 2 is designed to evaluate efficacy versus placebo in a double-blind study. Efficacy evaluation will be based primarily on Hgb with the EPO resistance index (ERI = weekly weight-adjusted EPO dose divided by Hgb) as a key secondary endpoint. The ERI index provides a quantitative indication of the amount of EPO required to achieve a given Hgb value and therefore provides clinically important information in addition to Hgb alone.

Rationale for FIH in dialysis patients.

This first human study (FIH) will be conducted in chronic hemodialysis (HD) patients and not in healthy volunteers (HV). Assessment of safety, tolerability and PK/PD response to anti-human BMP6 Ab in HV will probably not be translated into patients with chronic HD for several reasons:

In contrast to HV, chronic HD patients with anemia, high serum ferritin, and low TSAT chronically accumulate intracellular iron stores. Therefore, the safety, tolerability, and pharmacological effects associated with iron mobilization in response to low doses of Ab are best assessed in patients with chronic HD. In HV with normal renal function, hepcidin (of which BMP6 is the main regulator) is filtered by the kidneys and excreted efficiently in the urine, resulting in low circulating levels. In contrast, dialysis hepcidin is filtered less efficiently and temporarily, leading to higher circulating levels in patients with chronic HD (Zaritsky et al. 2010). In addition, normal kidneys regulate endogenous EPO levels over time, and changes in Hgb in response to Ab may not be apparent. Therefore, the safety and tolerability associated with the modulation of hepcidin through the BMP6 pathway and the action of Abs on Hgb is best assessed in patients with chronic HD.

Substantiation of the target population of patients.

This study is designed to evaluate Abs against human BMP6 in the setting of EPO-hyporeactive iron deficiency anemia. Established clinical practice guidelines (KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease Anemia Work Group 2012) define EPO hyposensitivity as the need for two dose increases of EPO, 50% above the stable dose, to maintain a stable Hgb concentration. The proposed inclusion criteria are designed to select stable patients on chronic HD with anemia and clinical indicators of iron limitation: elevated ferritin and low TSAT (TSAT=serum iron/total iron-binding capacity; TSAT is in close agreement with serum iron). In addition, when adjusting the doses of EPO and IV iron, a strict standard of care aimed at Hgb, TSAT and ferritin will be followed. This regimen reduces the risk of exceeding the required Hgb targets as changes in iron and haematological parameters will continue to be adjusted according to the standard of care. In addition, patients receiving loading doses of intravenous iron within 1 week prior to study entry will be excluded. Patients receiving IV iron maintenance may be included (if they meet all other inclusion criteria). The rationale for inclusion of these patients is that the current standard of care in the US dictates that Hgb and TSAT are required to be kept within narrow limits, and therefore the complete withdrawal of iron maintenance therapy in order to meet the inclusion criteria for the lower limit of TSAT would result in the inclusion of patients at risk. TSAT below 25% who require a course of intravenous iron supplements at saturating doses according to the standard of care. However, eligible patients on IV iron maintenance will maintain their weekly IV iron dose at the start of the week of Ab dosing and will resume IV iron maintenance therapy only when determined by center protocol according to the standard treatment, based on controlled iron indices.

Justification of the dose / regimen, duration of treatment.

Justification of the initial dose.

The maximum recommended initial dose (MRND) was calculated based on the no overt adverse effect dose level (NOAEL) in 13-week GLP toxicology studies (with 14 doses) 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 dose groups (only) were subsequently followed up for 16 weeks in rats or 24 weeks in cynomolgus monkeys after the last dose of isolated antibody or antigen-binding fragment thereof that binds human BMP6. The MRND was estimated first by calculating the human equivalent dose (HED) for the NOAEL from these studies (0.1 mg/kg) - a method considered appropriate for drugs with a molecular weight >100 kDa - and then applying a safety factor of 10 to account for differences between non-clinical species and patients, such as the amount of stored iron and the need for erythropoiesis. PK parameters for non-clinical species were derived from toxicokinetic (TK) data collected during toxicology studies for registration of IND status. The corresponding PK parameters in patients were then assessed using allometric scaling and these parameters were used to predict the concentration of free isolated antibody or antigen-binding fragment thereof that bind human BMP6 over time in patients for a given dose. When comparing TA data from toxicology studies with model-based studies, the Ab FC in patients indicated that a dose 10-fold lower than NOAEL/HED was predicted to give a (minimum) 10-fold excess based on Ab concentration.

The peak serum iron levels observed in response to Abs in animal studies may underestimate the predicted response of iron levels to human Abs, as HD patients who are treated with intravenous iron therapy are likely to have larger tissue depots of iron. than healthy animals. However, unlike healthy, non-anemic animals, patients with HD are expected to use released serum iron for erythropoiesis; therefore, animal models may lead to an overestimation of the duration of elevated iron levels. The liver pathology observed in the 13-week studies was not observed in the 4-week studies, suggesting that the toxicity was due to cumulative serum iron exposure rather than a response to a sudden release of iron.

In order to account for expected differences in stored iron between non-clinical species and patients, MRND was also predicted from an analysis based on modeling serum iron concentrations. The cumulative serum iron exposure that resulted in toxic events was presented as the area under the curve for iron (Fe AUC). In this method, the Fe AUC calculated for the NOAEL dose (0.1 mg/kg) was considered to be sufficiently safe. The Fe AUC at presumptive MRND in patients was then calculated and compared with the Fe AUC in non-clinical species at NOAEL. Because the model-based serum iron exposure for MRND in patients was >10-fold less than for non-clinical NOAEL, a reduction in NOAEL/HED with a safety factor of 10 was considered sufficient to evaluate MRND. The estimated MRND was thus 0.01 mg/kg.

Rationale for dose adjustment.

For this study, the maximum dose tested (xmax) will be the HED corresponding to the NOAEL in each of the 2 toxicology studies for IND status: 0.1 mg/kg. The minimum possible dose tested (xmin) is the lowest technically feasible dose based on compatibility studies: 0.001 mg/kg. MRND (xO) will be evaluated according to safety, TSAT and Hgb criteria (FIG. 7). If xO dose results in a median change in Hgb <0.5 g/dl from pre-dose levels, the choice of x+ (Figure 7) will be driven by linear extrapolation on a natural logarithmic scale (expecting a sigmoidal dose-response relationship) between xO and xmax. Preliminary doses for this dose escalation are given above. Such provisional doses may be adjusted based on a review of the data in an informal interim analysis in each cohort. This approach is used either until the minimum FAD is determined, or until xmax is reached. If xmax results in a median change in Hgb <0.5 g/dl, the safety of this

- 78 039579 doses and it will be decided whether the protocol should be refined to include additional cohorts with doses that exceed xmax based on safety, PK and PD data. If the highest dose tested in Part 1 results in a median increase in Hgb <0.5 g/dl, does not increase TSAT above 50%, and is below xmax, the protocol may be refined to include additional cohorts.

If xO instead results in a median change in Hgb >0.5 g/dl from pre-dose levels, the dose for the next cohort will be adjusted to xmin. If xmin also results in a median change in Hgb>0.5 g/dL, then xmin will be considered minimal FAD and xmin and xO will be assessed in Part 2. If xmin results in a median change in Hgb<0.5 g/dL and TSAT< 50% (FIG. 7), doses will be increased by linear extrapolation over the interval (xmin, xO) on a natural logarithmic scale until the minimum FAD is determined or until 6 doses (cohorts) are evaluated. Preliminary doses in the interval (xmin, xO) are given above. Such provisional doses may be adjusted based on a review of the data during the informal interim analysis in each cohort. The minimum FAD will be defined as the lowest dose tested that results in a median change in Hgb >0.5 g/dl from pre-dose levels.

Ab will be administered as a single dose IV infusion to ensure serum iron exposure (Fe AUC) is below the level associated with adverse events in preclinical toxicology studies. The Ab solution is infused immediately after the hemodialysis session on day 1 to minimize the potential effect of dialysis on PK or iron bioavailability immediately after dosing. An additional approximately 30 min post-dialysis dose infusion (on the day of dosing only) is not expected to pose significant risk or discomfort to patients.

Rationale for the choice of the comparator drug.

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

Purpose and timing of interim analyzes/design adaptations.

In Part 1, after completion of the post-dose evaluation at week 4 in each cohort of 6 patients, 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 panel, including applicable Investigators and representative(s) from the Sponsor. New cohorts will only be included if safety and tolerability are confirmed and if the PD conditions are met, as described in FIG. 7. Part 1 will have up to 5 informal midterm reviews. A formal interim review is scheduled after all patients in the final Part 1 cohort complete their post-dose evaluation at week 4 to evaluate the clinical effects of study doses and potentially initiate additional preclinical studies, and may report data on body temperature, arterial blood pressure in subsequent clinical studies. blood pressure, pulse rate, ECG results, blood biochemistry, hematological iron indices, EPO resistance index, and will include adverse events recorded up to day 29 in the last part 1 cohort.

For part 2, the minimum FAD and the dose one level higher than the minimum FAD will be selected. If the lowest tested dose causes an increase in Hgb>0.5 g/dL, the two lowest tested doses will be selected for Part 2.

Risk and benefit.

The intended benefit for patients participating in this study may include a reduction in EPO and IV iron requirements and an increase in Hgb levels during treatment and for some time after treatment.

The risk to patients in this study will be minimized by adherence to the inclusion criteria and careful clinical monitoring of all patients (as well as admission to the hospital of the first two patients in Part 1) during the first 48 hours after administration of Ab.

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) slightly increased susceptibility to bacterial infection, especially in patients with implanted vascular catheters. Some of the inclusion criteria reduce the risk of complications. Increased levels of liver function on tests may be noted due to redistribution of iron. Liver function will be monitored in parallel with iron and haematological parameters. Exceeding standard of care Hgb targets may lead to polycythemia. Hgb control, EPO therapy, and iron therapy may be attempted.

Patients with HD treated with intravenous iron may have larger tissue depots of iron than healthy animals; therefore, peak serum iron levels observed in patients treated with isolated antibody or antigen-binding fragment thereof that bind human BMP6 may exceed levels observed in animal studies. However, unlike healthy animals, patients with HD are expected to utilize released serum iron for erythropoiesis; therefore, animal models may overestimate the duration of elevated iron levels. Model-predicted Ab exposure (eg, Cmax, AUC) in MRND is expected to be 10-fold less than that observed with NOAEL in preclinical studies. This 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. Ab dose escalation to NOAEL will occur following the described safety assessments. Clinical experience with patients with chronic excess iron, as well as patients who receive parenteral iron, will probably not predict the effects that may occur as a result of sharp increases in intracellular iron caused by Ab. Therefore, 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. With the development of acute iron toxicity, patients may be subjected to emergency iron chelation therapy, 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 drawn over 115 days from each patient in the study. Additional samples to monitor any safety data may be collected in addition to this. It is not considered dangerous for this population.

To date, no reproductive toxicity studies have been performed with antibodies to human BMP6. Potential effects on the male or female reproductive organs were assessed in a rigorous standard histopathological examination of the ovaries and testicles and accessory reproductive organs in a 13-week toxicity study in cynomolgus monkeys. No treatment-related effects were observed. BMP6 knockout mice exhibited delayed sternum ossification and iron overload (Meynard et al. 2009).

Significant fetal and maternal morbidity and mortality are associated with chronic hemodialysis. In a retrospective cohort study comparing women on chronic hemodialysis (267 births) with women who had undergone a kidney transplant (264 births), women on hemodialysis had higher rates of placental abruption, blood transfusion, neonatal malnutrition, fetal death, and death. mothers (Saliem et al. 2015). Therefore, women of childbearing potential should use very 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.

Sample.

The sample will consist of patients with end-stage renal disease who require chronic hemodialysis therapy at least twice a week, and who have a clinical symptom of functional iron deficiency anemia, defined as anemia, in the presence of clinically sufficient iron depots, as determined by ferritin levels and transferrin saturation. . Part 1 includes an evaluation plan for up to 36 patients, initially in 6 cohorts (6 patients/cohort). If after 6 cohorts there are no effects on TSAT and Hgb, and there are no safety hazards (as determined by the applicable Investigators and Sponsor representative(s), up to 2 additional cohorts of 6 patients may be included (total 48 patients in Part 1) . Part 2 consists of a maximum of 3 groups (2 dose levels matched for further evaluation from Part 1 and a placebo group), with up to approximately 20 patients per group (total 60 patients in Part 2). Thus, a total of approximately 96 patients (up to a maximum of 108) are planned to be enrolled, of which approximately 60 patients 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 the study meet the following inclusion criteria. No additional criteria should be applied by the investigator to ensure that the sample is representative of all patients who meet the inclusion criteria.

Patient selection should be performed with all inclusion criteria checked at screening and at the first visit prior to treatment. An appropriate record (eg, control form) of the verification of the inclusion criteria should be kept with the original documentation at the study site. Deviation in any selection criterion leads to the exclusion of patients from enrollment in the study.

Inclusion criteria (both parts).

Patients eligible for inclusion in this study must meet all of the following criteria.

1. Written informed consent must be obtained prior to any assessment. If consent cannot be expressed in writing, it must be officially registered

- 80 039579 verified and certified, ideally by an independent trusted witness.

2. Age >18 years at the time of screening.

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

4. Carrying out appropriate hemodialysis at least 2 times a week in end-stage renal failure; appropriate is defined as Kt/V>1.2 at the last monthly assessment prior to screening.

5. Receipt of chronic erythropoietin (EPO) therapy, according to the dialysis center's anemia management protocol. The EPO dose was not increased by 50% or more within 14 days prior to study entry. EPO therapy should include only short-acting (not darbepoetin) and IV (not SC) drugs.

6. Hgb >8.5, including Hgb >8.5 and <11.5 g/dl, and not increased by >0.5 g/dl from pre-study levels in the previous 14 days.

7. Ferritin 200-2000 ng/mL (inclusive) for at least 28 days prior to study entry (may include screening).

8. TSAT<30%, at least once, within 90 days prior to study entry, and TSAT<30% at study entry.

Exclusion criteria (both parts).

Patients meeting any of the following criteria are not eligible for inclusion in this study. The investigator cannot make any further exceptions to ensure that the sample is representative of all eligible patients.

1. Use of other study drugs within 5 elimination half-lives prior to enrollment in the study, or until the expected pharmacodynamic effect returns to baseline, whichever is longer.

2. A case of hypersensitivity to the investigational drug or therapeutic antibodies.

3. Known diagnosis of hemochromatosis.

4. Known bone marrow malignancy, lymphatic malignancy, or myelodysplastic syndrome.

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

6. Severe comorbid liver disease/dysfunction (Child-Pugh score >6) or previous liver transplant 7.

Heart failure (functional class III or IV of the New York Heart Association (NYHA)).

8. Gastrointestinal bleeding requiring intervention within 2 months of screening. Patients with hepatitis C virus (HCV) infection may be included if all other inclusion criteria related to liver function are met.

9. ALT, ACT, or bilirubin >1.5x ULN within 4 weeks prior to study entry.

10. Uncontrolled renal osteodystrophy defined as intact PTH >750 pg/mL at screening.

11. Factors predisposing 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 within 4 weeks prior to study entry.

13. Receiving a loading dose (100 mg/week) of an intravenous iron preparation within 1 week prior to study entry.

14. History of drug or alcohol dependence within 12 months prior to dosing or confirmation of such dependence based on the results of laboratory tests conducted at the time of screening.

15. Positive test result for hepatitis B surface antigen.

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

17. Women of childbearing potential may be eligible for this study if high-efficiency 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 (if consistent with the patient's preferred and usual lifestyle; intermittent abstinence (eg, calendar, ovulation, symptothermal, postovulation methods) and coitus withdrawal are not acceptable methods of contraception), b. Sterilization of a man/woman, p. Use of oral, injectable or implantable hormonal methods of contraception or

- 81 039579 insertion of an intrauterine device (IUD) or intrauterine system (IUS), or other forms of hormonal contraception that have comparable efficacy (<1% failure rate), such as a hormone-containing vaginal ring or transdermal hormonal contraception.

Treatment.

treatment in the study.

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

Treatment groups.

In Part 1, patients are assigned to one of up to 6 dose 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 given in table. 12 (Hgb <0.5 g/dL in MRND) and Table. 13 (Hgb>0.5 g/dl at MRND).

Table 12

Predose Levels for Part 1

For Hgb less than 0.5 g/dl Predose Increase relatively at dose level MRND previous DOSES 1 (MRND) 0.010 mg/kg initial , dose 2 0.016 mg/kg 60%t 3 0.025 mg/kg bo%T 4 0.040 mg/kg 60%t 5 0.063 mg/kg 60%t 6 (NOAEL) 0.100 mg/kg 60%T

This table is intended as an example of dose adjustment in Part 1 for informational purposes only. Intermediate or higher dose levels may be used, and some dose levels may be omitted based on evaluation of data during informal interim analyzes in each cohort. Actual dose levels will be confirmed in writing by Novartis and submitted to all participating research centers prior to treatment of patients in a new cohort.

Table 13

Predose Levels for Part 1

For Hgb greater than or equal to 0.5 g/dL at MRND dose level Predose Increase previous regarding the DOSE 1 (MRND) 0.0100 mg/kg initial , dose 2 0.0010 mg/kg 90%F 3 0.0016 mg/kg 60%T 4 0.0025 mg/kg bo%T 5 0.0040 mg/kg bo%T 6 0.0063 mg/kg bo%T

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

Treatments in the study are defined as follows.

A: single dose placebo.

B: single dose of anti-human BMP6 Ab in minimal FAD as defined in part 1.

C: single dose of anti-human BMP6 Ab one dose level above the minimum FAD as 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 specified in the inclusion criteria, prior to and during the study, must be registered with the sections of the CRF Concomitant Treatment/Relevant Non-Drug Therapies. Therapy records should include brand name, single dose and unit, frequency and route of administration, dates of initiation and cessation, and reason for therapy.

Efficacy/pharmacodynamics.

Performance ratings are defined below. Samples for performance evaluations will be collected at various points in time. Hematological laboratories will be certified. Hgb and Fe indices will be considered during each intercohort informal interim analysis as part of the dose adjustment assessment during part 1 of the study. If the initial sampling schedule is considered sub-optimal for the study of the relationship between iron and FA, the sampling schedule may be changed in subsequent cohorts in Part 1.

Set of iron indexes.

Anti-human BMP6 Ab is expected to mobilize Fe from body stores resulting in changes in serum Fe parameters including: Serum Fe, Transferrin Saturation (TSAT), Unbound Fe Binding Capacity (UIBC), Total Fe Binding Capacity (TIBC), Ferritin and hemoglobin content in reticulocytes (CHr). These indicators will be measured in serum using approved assays.

Security safety scores are defined below.

Objective examination.

A complete physical examination will include general appearance, skin, neck (including thyroid), eyes, ears, nose, throat, lungs, heart, abdomen, back, lymph nodes, extremities, phlebological and neurological examination. If indicated based on past medical history and/or symptoms, examination of the rectum, external genitalia, breast, and/or gynecological examination may be performed.

Significant findings that are present prior to initiation of a drug study should be included in the screening for relevant past/current illness data in eRCI patients. Significant results obtained after initiation of a drug study that meet the definition of an adverse event should be reported when screening for adverse events in eRC patients.

Basic vital signs.

Body temperature.

Blood pressure (BP).

Pulse, height and weight.

Height.

Body mass.

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

Laboratory research.

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

Hematology.

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

Clinical chemistry.

Sodium, potassium, creatinine, urea, chloride, albumin, calcium, alkaline phosphatase, total bilirubin, LDH, GGT, ACT, and ALT will be monitored. If the concentration of total bilirubin is 1.5 times the upper limit of normal, it is necessary to differentiate directly and indirectly reacting bilirubin.

Electrocardiogram (ECG).

PR interval, QRS prolongation, heart rate, RR, QT, QTc. For clinical decision making, Fredericia's (QTcF) correction should be used.

Pregnancy and fertility assessments.

Pregnancy tests are required for all affected women, regardless of reported reproductive/menopausal status.

Serum pregnancy tests will be performed for this study. In the event of a positive result, the patient must discontinue participation in the study.

If performed at screening and study entry, the result of this test must be obtained before the patient can receive a dose.

Pharmacokinetics.

Samples will be collected for PK analysis. PK data will be reviewed during each intercohort informal interim analysis as part of the dose adjustment evaluation during Part 1.

- 83 039579 research. If the sampling schedule is initially considered suboptimal or inappropriate for PK profiling, the sampling schedule may be modified in subsequent cohorts.

The number of blood draws and the total volume of blood collected will not exceed the values specified in the protocol.

Samples for PK analysis will be collected and evaluated from all patients at all dose levels.

The concentration of free Ab against human BMP6 will be determined using an ELISA assay. The expected lower limit of quantitation (LLOQ) is 10 pg/mL.

Samples from untreated patients (in the placebo group) will not be analyzed.

Concentrations of free Ab against human.

BMP6 will be expressed in µg/ml. All concentrations below the LEL or missing data will be noted as such in the concentration data lists. Concentrations below the LEL will be treated as zero in the summary statistics only in the case of concentration data. They will not be taken into account when calculating the parameters of the FK.

PK samples remaining after determination of free anti-human BMP6 Abs may be used for exploratory evaluations or other bioanalytical purposes (eg, site-to-site cross-validation, stability assessment).

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

Other ratings.

Immunogenicity.

An ELISA assay will be used to detect antibodies against human BMP6. Ig samples remaining after immunogenicity analysis can be used for exploratory evaluation or other bioanalytical purposes (eg, cross-validation between different centers).

Research estimates.

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

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

Based on the biological process described above, exploratory biomarker assessments include, without limitation, hepcidin (as measured by LC-MS).

Additional exploratory evaluations may explore potential roles for markers of bone absorption as well as consider inflammation as a factor contributing to the mechanism of action.

The research objectives are as follows: to evaluate the relationship between hepcidin levels and several key indicators such as iron indices and ERI; explore dynamics between primary and secondary endpoints and research biomarkers in parallel; evaluate pharmacogenetics; assess immunogenicity.

The sample(s) will be collected at different time(s). Further information on sample collection, numbering, processing and transport will be provided in the central laboratory manual.

DNA.

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

In addition, recent advances in genotyping technologies have made genome-wide methods possible. Whole genome methods can also be applied in a limited area of the present research, 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.

Non-hypothetical platforms can be used to investigate disease heterogeneity, mechanism of action, and/or to potentially identify stratification markers. Samples for

- 84 039579 immunogenicity assays (IGs) will be collected at various time points. The immunogenicity of the anti-human BMP6 Ab will be assessed by measuring antibodies recognizing the anti-human BMP6 antibody.

Used sources

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): pp. 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 prior to publication) http://www.ncbi.nlm.nih.gov/pubmed/2 57192 92.

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

(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.

Unless otherwise defined, the technical and scientific terms used in this description have the same meaning as they are usually understood by a person familiar with the field to which this description relates.

Unless otherwise indicated, all methods, steps, techniques and manipulations that are not described in detail can be performed and have been performed in a manner known per se, as will be obvious to a person skilled in the art. Reference, for example, is again made to the standard manuals and the general state of the art cited herein and to other references cited therein. Unless otherwise indicated, each of the references cited herein is incorporated by reference in its entirety.

The claims are non-limiting and are presented below.

While specific aspects and claims have been detailed herein, this has been done by way of example, for purposes of illustration only, and should not limit the scope of the appended claims or the scope of the claimed subject matter of any related future application. In particular, the present inventors contemplate that various substitutions, changes and modifications may be made to the present specification without departing from the scope and spirit of the description as defined by the claims. The choice of the original nucleic acid, the desired clone or type of library, is believed to be a standard action for the average person skilled in the art, with knowledge of the aspects described herein. Other aspects, advantages and modifications are considered to be included within the scope of the following claims. Those skilled in the art will know, or be able to ascertain using no more than standard experimentation, many equivalents to the specific aspects of the invention described herein. Such equivalents are intended to be included in the following claims. Clarification of the claimed scope in the respective applications filed later may be due to the limitations of patent laws in various countries and should not be interpreted as a rejection of the claimed subject matter.

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Claims (31)

CLAIM 1. An isolated antibody or antigen-binding fragment thereof that binds human BMP6 and includes: (a) the sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 69, 70 and 71, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 79, 80 and 81, respectively; (b) the sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 72, 73 and 74, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 82, 83 and 84, respectively; (c) the sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 29, 30 and 31, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 39, 40 and 41, respectively; (d) the sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 32, 33 and 34, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 42, 43 and 44, respectively; (e) the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 49, 50 and 51, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 59, 60 and 61, respectively; (f) the sequences HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 52, 53 and 54, respectively, and the sequences LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 62, 63 and 64, respectively; (g) the sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 9, 10 and 11, respectively, and the sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NOs: 19, 20 and 21, respectively, or (h) the sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NOs: 12, 13, and 14, respectively, and the sequences LCDR1, LCDR2, and LCDR3 in SEQ ID NOs: 22, 23, and 24, respectively. 2. An isolated antibody or antigen-binding fragment thereof according to claim 1, including: (a) the VH sequence in SEQ ID NO: 75; (b) the VH sequence in SEQ ID NO: 35; (c) the VH sequence of SEQ ID NO: 55 or (d) the VH sequence of SEQ ID NO: 15. 3. The isolated antibody or its antigen-binding fragment according to claim 1, including: (a) the VL sequence in SEQ ID NO: 85; (b) the VL sequence in SEQ ID NO: 45; (c) the VL sequence of SEQ ID NO: 65 or (d) the VL sequence of SEQ ID NO: 25. 4. The isolated antibody or its antigen-binding fragment according to claim 1, including: (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. 5. An isolated antibody according to claim 1, including: (a) the sequence of the heavy chain in SEQ ID NO: 77; (b) the sequence of the heavy chain in 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. 6. An isolated antibody according to claim 1, including: (a) the light chain sequence of SEQ ID NO: 87; (b) the light chain sequence in SEQ ID NO: 47; (c) the light chain sequence of SEQ ID NO: 67 or (d) the light chain sequence of SEQ ID NO: 27. 7. An isolated antibody according to claim 1, including: (a) the heavy chain sequence in SEQ ID NO: 77 and the light chain sequence in 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) the heavy chain sequence of SEQ ID NO: 57 and the 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. 8. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof binds human BMP6 with a KD<1 nM. 9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof has at least about 100-fold greater affinity for human BMP6 than for human BMP2, human BMP5, or human BMP7. 10. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the isolated antibody or antigen-binding fragment thereof reduces BMP6-induced expression. - 86 039579 this hepcidin in liver cell lines or primary human liver cells in vitro. 11. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the isolated antibody or antigen-binding fragment thereof exhibits at least about 50% reduction in BMP6-induced hepcidin expression in liver cell lines or primary human liver cells in vitro. 12. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the isolated antibody or antigen-binding fragment comprises a scaffold selected from IgM and IgG. 13. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the isolated antibody or antigen-binding fragment is IgM and IgG, where the IgG is selected from IgG1, IgG2 and IgG3 or IgG4. 14. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-13, wherein the isolated antibody or antigen-binding fragment is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a single chain antibody, a Fab, and a scFv. 15. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1-14, wherein the isolated antibody or antigen-binding fragment is a component of an immunoconjugate. 16. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, which has an effector function altered by mutation of the Fc region. 17. A composition for reducing BMP6 activity in a cell, comprising an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-16 and a pharmaceutically acceptable carrier. 18. The composition according to claim 17, comprising an additional therapeutic agent, where the additional therapeutic agent reduces the activity of BMP6. 19. The composition of claim 18, wherein the additional therapeutic agent is an siRNA or an antibody or antigen-binding fragment thereof. 20. The composition of claim 18, wherein the therapeutic agent is an erythropoiesis-stimulating drug (ESD) or an iron preparation. 21. The composition of claim 20, wherein the therapeutic agent is an erythropoiesis-stimulating drug (ESD). 22. An isolated polynucleotide comprising a sequence encoding an isolated antibody or antigen-binding fragment thereof that binds human BMP6, according to any one of claims 1-16. 23. An isolated polynucleotide comprising a sequence encoding the VH sequence of an antibody or antigen-binding fragment thereof that binds human BMP6, according to any one of claims 1-16. 24. An isolated polynucleotide comprising a sequence encoding the VL sequence of an antibody or antigen-binding fragment thereof that binds human BMP6, according to any one of claims 1-16. 25. An isolated polynucleotide encoding the heavy chain of an antibody or its antigen-binding fragment to BMP6, comprising the sequence of any of: (a) the heavy chain sequences in SEQ ID NO: 78; (b) the VH sequence in SEQ ID NO: 76; (c) the heavy chain sequences in SEQ ID NO: 38; (d) the VH sequences in SEQ ID NO: 36; (e) the heavy chain sequences in SEQ ID NO: 58; (f) the VH sequences in SEQ ID NO: 56; (g) the heavy chain sequences in SEQ ID NO: 18; (h) VH sequences in SEQ ID NO: 16. 26. An isolated polynucleotide encoding the light chain of an antibody or its antigen-binding fragment to BMP6, comprising the sequence of any of: (a) the light chain sequences in SEQ ID NO: 88; (b) the VL sequences in SEQ ID NO: 86; (c) the light chain sequences in SEQ ID NO: 48; (d) the VL sequences in SEQ ID NO: 46; (e) the light chain sequences in SEQ ID NO: 68; (f) the VL sequences in SEQ ID NO: 66; (g) light chain sequences in SEQ ID NO: 28 or (h) VL sequences in SEQ ID NO: 26. 27. A vector for the expression of a polynucleotide in a cell, comprising a polynucleotide according to claims 22-26. 28. A host cell comprising the vector of claim 27 or the polynucleotide of claims 22-26. 29. The host cell of claim 28, wherein the cell is a Chinese hamster ovary (CHO) cell. 30. A method for reducing BMP6 activity in a cell, comprising the step of contacting the cell with an antibody or antigen-binding fragment thereof according to any one of claims 1-16 or a composition according to any one of claims 17-21. 31. A method for inhibiting BMP6 in a patient, comprising the step of administering a therapeutic agent to the patient. -