EA039579B1 - METHODS AND COMPOSITIONS OF ANTIBODIES DIRECTED AGAINST BMP6 - Google Patents

Abstract

The invention relates to antibodies and their antigen-binding fragments to human BMP6, as well as compositions and methods for their use.

Description

Related applications

This patent application claims the benefit of US Patent Application No. 62/094716, filed December 19, 2014, and US Patent Application No. 62/181803, filed June 19, 2015, the contents of which are incorporated herein by reference in their entirety.

List of sequences

The invention contains a sequence listing, which was provided in electronic form in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on December 16, 2015, is designated PAT056599-WO-РСТ_SL.txt and is 68,301 bytes in size.

Introduction

The present invention relates to antibodies and antigen-binding fragments thereof 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 outcomes, including cardiovascular disease and death. Several treatments for anemia in patients with CKD include erythropoiesis-stimulating drugs (ESDs), oral and intravenous iron supplements, and blood transfusions. However, many patients do not respond adequately to these treatments or require higher doses of ESDs and/or iron. High doses of iron can also cause toxicity associated with oxygen radical formation and allergic reactions. These treatments may be suboptimal because they do not fully address the underlying cause of anemia—impaired iron absorption and iron mobilization from stores.

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

In many patients with anemia, ferroportin-mediated iron export is suppressed by high hepcidin levels. Additional data suggest that elevated hepcidin levels correlate with a poor response to ESP during hemodialysis. Hepcidin-lowering agents may therefore be an effective strategy for reducing the severity of ESP-refractory anemia in this group of patients and in other forms of anemia of chronic disease (ACD) characterized by iron deficiency.

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

Despite existing treatment options for anemia associated with diseases and disorders, there remains a need for improved anemia treatment formulations 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 producing such molecules and compositions, and methods for using them in lowering hepcidin levels and in treating anemia.

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

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

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

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

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

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

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

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

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

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

- 1 039579 HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 29, 30 and 31, respectively, and LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 39, 40 and 41, respectively.

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

the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 32, 33 and 34, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 42, 43 and 44, respectively.

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

HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO:

49, 50 and 51, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 59, 60 and 61, respectively.

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

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

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

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

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

the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 72, 73 and 74, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 82, 83 and 84, respectively.

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

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 to one of the VH sequences described above.

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

the VL (variable domain of the light chain) sequence 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 to one of the VL sequences described above.

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

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

the VH sequence of SEQ ID NO: 35 and the VL sequence of 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%,

- 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 98%, or at least 99% sequence identity, but less than 100% sequence identity with one of the VL sequences described above. In an embodiment, the VH and VL are obtained from the same antibody listed in Table 1.

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

the heavy chain sequence of SEQ ID NO: 17;

the heavy chain sequence of 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 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, to 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 comprises:

the light chain sequence of SEQ ID NO: 27;

the light chain sequence of 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 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 to one of the light chain sequences described above.

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

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

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

the heavy chain sequence of SEQ ID NO: 57 and the light chain sequence of SEQ ID

NO: 67 or the heavy chain sequence of SEQ ID NO: 77 and 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 heavy 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, to one of the heavy chain sequences described above, and comprises 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, to one of the light chain sequences described above. In an embodiment, the heavy chain and light chain are derived from the same antibody listed in Table 1.

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

- 3,039,579 HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 9, 10 and 11, respectively, and LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 19, 20 and 21, respectively;

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

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

the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 32, 33 and 34, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 42, 43 and 44, respectively;

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

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

the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 79, 80 and 81, respectively, or the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 72, 73 and 74, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 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 comprises at least one complementarity determining sequence (CDR) identical to any one or more of:

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

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

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

the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 32, 33 and 34, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 42, 43 and 44, respectively;

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

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

the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 79, 80 and 81, respectively, or the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 72, 73 and 74, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 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:

antigen by less than the sequences of HCDR1, HCDR2 and HCDR3 sequences of HCDR1, HCDR2 and HCDR3 sequences of HCDR1, HCDR2 and HCDR3 sequences of HCDR1, HCDR2 and HCDR3 sequences of HCDR1, HCDR2 and HCDR3 sequences of HCDR1, HCDR2 and HCDR3 sequences of HCDR1, HCDR2 and HCDR3 sequences of HCDR1, HCDR2 and HCDR3 sequences of HCDR1, HCDR2 and HCDR3 in in in in in in

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

12, 13

29, 30

32, 33

49, 50

52, 53

69, 70

72, 73 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 by less than the sequences of LCDR1, LCDR2 and LCDR3 sequences of LCDR1, LCDR2 and LCDR3 sequences of LCDR1, LCDR2 and LCDR3 sequences of LCDR1, LCDR2 and LCDR3 sequences of LCDR1, LCDR2 and LCDR3 sequences of LCDR1, LCDR2 and LCDR3 sequences of LCDR1, LCDR2 and LCDR3 sequences of LCDR1, LCDR2 and LCDR3 in in in in in in

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

19, 20

22, 23

39, 40

42, 43

59, 60

62, 63

79, 80

82, 83 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 relates to 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 ^1x107 M ^-1 , ¹⁰⁸ M ^-1 , ¹⁰⁹ M ^-1 , ¹⁰¹⁰ M ^-1 , or ¹⁰¹¹ M ^-1 . In one embodiment, the present invention relates to isolated antibodies or antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibody has an affinity constant (KA) of at least ^1x107 M ^-1 , ¹⁰⁸ M ^-1 , ¹⁰⁹ M ^-1 , ¹⁰¹⁰ M ^-1 , or ¹⁰¹¹ 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 no more than about 1 nM or no more than about 0.1 nM. 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 no more than 1 nM or no more than 0.1 nM. 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<1 nM or <0.1 nM. In one embodiment, the Kd corresponds to measurement by BiaCore.

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

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

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

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

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

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof has no detectable binding to human BMP2 or BMP5 (e.g., in an ELISA).

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

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

In one embodiment, the antibodies and antigen-binding fragments thereof of the invention that specifically bind to BMP6 are isolated monoclonal antibodies. In one embodiment, the antibodies and antigen-binding fragments thereof of the invention that specifically bind to BMP6 are isolated human monoclonal antibodies. In one embodiment, the antibodies and antigen-binding fragments thereof of the invention that specifically bind to BMP6 are humanized monoclonal antibodies. In one embodiment, the antibodies and antigen-binding fragments thereof of 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 an 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 framework in which amino acids have been replaced with an antibody framework 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 the isolated antibody or antigen-binding fragment thereof and any of the following, but not limited to: an enzyme, a toxin, a hormone, a growth factor, or a drug.

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

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

In one embodiment of the present invention, the isolated antibody or antigen-binding fragment thereof inhibits BMP6-induced hepcidin expression in liver cells (e.g., 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 (e.g., liver cell lines and/or primary human liver cells in vitro) by at least about 50%. For example, the 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, but is not limited to, 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 (e.g., liver cell lines and/or primary human liver cells in vitro) by at least 50%. For example, the 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 the activity of human BMP6 in vitro as measured in a 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 an epitope of human BMP6 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 an epitope of human BMP6 consisting of the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 92 or amino acids 88-102 of 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 an epitope of human BMP6 consisting of the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 92 or amino acids 88-102 of SEQ ID NO: 89) than for: (a) an epitope of human BMP7 consisting of the sequence QTLVHFINPETVPKP (SEQ ID NO: 93 or amino acids 88102 of SEQ ID NO: 90) or (b) an epitope of human BMP5 consisting of the sequence QTLVHLMFPDHVPKP (SEQ ID NO: 94 or amino acids 87-101 of SEQ ID NO: 91). In one embodiment of the present invention, the isolated antibody or antigen-binding fragment thereof has at least a 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 for: (a) the human BMP7 epitope consisting of the sequence QTLVHFINPETVPKP (SEQ ID NO: 93 or amino acids 88-102 of SEQ ID NO: 90) or (b) the human BMP5 epitope consisting of the sequence QTLVHLMFPDHVPKP (SEQ ID NO: 94 or amino acids 87-101 of SEQ ID NO: 91). Such an isolated antibody or antigen-binding fragment thereof may include, but is not limited to: (a) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 69, 70, and 71, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 79, 80, and 81, respectively, or (b) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 72, 73, and 74, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of 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 of SEQ ID NO: 85. In an embodiment, the affinity is as measured by 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 the human BMP6 epitope consisting of the sequence QTLVHLMNPEYVPKP (SEQ ID NO: 92 or amino acids 88-102 of SEQ ID NO: 89) than for: (a) the human BMP7 epitope consisting of the sequence QTLVHFINPETVPKP (SEQ ID NO: 93 or amino acids 88-102 of SEQ ID NO: 90) or (b) the human BMP5 epitope consisting of the sequence QTLVHLMFPDHVPKP (SEQ ID NO: 94 or amino acids 87-101 of SEQ ID NO: 91). Such an isolated antibody or antigen-binding fragment thereof may include, but are not limited to: (a) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 69, 70, and 71, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 79, 80, and 81, respectively, or (b) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 72, 73, and 74, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of 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 of SEQ ID NO: 85. In an embodiment, the affinity is as measured by BiaCore.

In another aspect, the invention relates to a composition, such as a pharmaceutical composition, comprising an isolated antibody or antigen-binding fragment thereof 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, the additional therapeutic agent reduces BMP6 activity.

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

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 preparation (e.g., an iron supplement or an IV iron preparation).

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

In one embodiment, the isolated antibody or antigen-binding fragment thereof described in Table 1 can be administered to a patient in combination with a therapeutic method or procedure, such as those described herein or known in the art. The isolated antibody or antigen-binding fragment thereof described in Table 1 can be administered before, after, or concurrently with 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 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 procedure

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

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 an isolated antibody or antigen-binding fragment thereof described in Table 1, comprising any one or more of:

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

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

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

the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 32, 33 and 34, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 42, 43 and 44, respectively;

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

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

the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 79, 80 and 81, respectively, or the HCDR1, HCDR2 and HCDR3 sequences in SEQ ID NO: 72, 73 and 74, respectively, and the LCDR1, LCDR2 and LCDR3 sequences in SEQ ID NO: 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 thereof described in Table 1, comprising an amino acid sequence comprising a sequence selected from the group consisting of:

the heavy chain sequences of SEQ ID NO: 17;

VH sequences in SEQ ID NO: 15;

the light chain sequences of SEQ ID NO: 27;

VL sequences in SEQ ID NO: 25;

the heavy chain sequences of SEQ ID NO: 37;

VH sequences in SEQ ID NO: 35;

the light chain sequences of SEQ ID NO: 47;

VL sequences in SEQ ID NO: 45;

the heavy chain sequence of SEQ ID NO: 57;

VH sequences in SEQ ID NO: 55;

the light chain sequences of SEQ ID NO: 67;

VL sequences in SEQ ID NO: 65;

the heavy chain sequence of SEQ ID NO: 77;

VH sequences in SEQ ID NO: 75;

the light chain sequence of SEQ ID NO: 87 and the VL sequence of 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 thereof described in Table 1, comprising an amino acid sequence having at least 90, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from the group consisting of:

the heavy chain sequences of SEQ ID NO: 17;

VH sequences in SEQ ID NO: 15;

the light chain sequences of SEQ ID NO: 27;

VL sequences in SEQ ID NO: 25;

the heavy chain sequences of SEQ ID NO: 37;

VH sequences in SEQ ID NO: 35;

the light chain sequences of SEQ ID NO: 47;

VL sequences in SEQ ID NO: 45;

the heavy chain sequence of SEQ ID NO: 57;

VH sequences in SEQ ID NO: 55;

the light chain sequences of SEQ ID NO: 67;

VL sequences in SEQ ID NO: 65;

the heavy chain sequence of SEQ ID NO: 77;

VH sequences in SEQ ID NO: 75;

the light chain sequence of SEQ ID NO: 87 and

- 8 039579 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 thereof described in Table 1, wherein the nucleic acid comprises a sequence selected from the group consisting of:

the heavy chain sequences of SEQ ID NO: 18;

heavy chain sequences of SEQ ID NO: 38;

the heavy chain sequences of SEQ ID NO: 58;

the heavy chain sequence of SEQ ID NO: 78;

the light chain sequences of SEQ ID NO: 28;

the light chain sequences of SEQ ID NO: 48;

the light chain sequences of SEQ ID NO: 68;

the light chain sequences of SEQ ID NO: 88;

VH sequences in SEQ ID NO: 16;

VH sequences in SEQ ID NO: 36;

VH sequences in SEQ ID NO: 56;

VH sequences in SEQ ID NO: 76;

VL sequences in SEQ ID NO: 26;

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, 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 a first and a second promoter and are capable of being expressed in said host cell. In another embodiment, the isolated host cells comprise a recombinant DNA segment encoding the heavy chain and the 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 antigen-binding fragment thereof is a human monoclonal antibody or antigen-binding fragment thereof.

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

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

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

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

In another embodiment, the present invention relates to 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 of the invention. In embodiments, the patient suffers from anemia. In embodiments, the anemia is anemia of chronic disease (ACD), such as anemia in chronic kidney disease (CKD), anemia in cancer, or anemia in 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 (ESD), such as erythropoietin (EPO). In embodiments, the anemia is EPO-hyporesponsive anemia. In embodiments, the anemia is iron deficiency anemia. In embodiments, the patient is a patient on chronic hemodialysis.

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 thereof according to the invention.

The present invention further relates to a method for increasing serum iron levels, transferrin saturation (TSAT), reticulocyte hemoglobin content (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 fragment thereof according to the invention.

The present invention further relates to a method for increasing or maintaining hemoglobin levels in a patient, comprising administering to the patient an antibody or antigen-binding fragment thereof described in the present invention. In embodiments, the patient suffers from anemia. In embodiments, the anemia is anemia of chronic disease (ACD), such as anemia in chronic kidney disease (CKD), anemia in cancer, or anemia in 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 (ESD), such as erythropoietin (EPO). In embodiments, the anemia is EPO-hyporesponsive anemia. In embodiments, the anemia is iron deficiency anemia. In embodiments, the patient is a patient on chronic hemodialysis. In embodiments, the method further comprises decreasing the patient's required iron dose, decreasing the patient's required EPO dose, or decreasing the patient's required iron dose and the required EPO dose, compared to said required EPO dose and/or required iron dose in the absence of treatment with the antibody or antigen-binding fragment described in the present invention. In embodiments, the hemoglobin level increases or is maintained at a level of 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 is maintained 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 the patient an antibody or antigen-binding fragment thereof described in the present invention comprises the step of administering to the patient a composition that includes the antibody or antigen-binding fragment thereof described in the present invention.

In any of the above methods, the antibody or antigen-binding fragment thereof 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 can be administered at a dose of from about 0.001 to about 0.1 mg/kg, such as 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 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 approximately 30 to approximately 60 min.

In another aspect, the present invention relates to an antibody or antigen-binding fragment thereof to BMP6 comprising the CDRs listed in Table 1. The present invention relates to an antibody or antigen-binding fragment thereof to BMP6 listed in Table 1. The present invention relates to an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof to BMP6 comprising the CDRs 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 defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

BMP6, as used herein, refers to bone morphogenetic protein 6 (BMP6) or the gene or nucleic acid encoding BMP6. Hahn et al. 1992 Genomics 14:759-62; Sauermann et al. 1993 J. Neurosci. Res. 33:142-7; NCBI Gene ID: 654. BMP6 is also known as: BMP-6; VGR; VGR1; External IDs: OMIM: 112266 MGI: 88182; HomoloGene: 1300; GeneCards: BMP6 gene. Orthologs: Species: Human: Entrez: 654; Ensembl: ENSG00000153162; UniProt: P22004; RefSeq (mRNA): NM_001718; RefSeq (protein): NP_001709; localization (UCSC): Xp 6: 7.73-7.88 Mb; species: mouse: Entrez: 12161; Ensembl: 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, refers to bone morphogenetic protein 2 (BMP2) or the gene or nucleic acid encoding BMP2. BMP2 is also known as: BDA2; and BMP2A; External IDs OMIM: 112261 MGI: 88177 HomoloGene: 926 GeneCards: BMP2 gene. Species: human; Entrez: 650; Ensembl: ENSG00000125845; UniProt: P12643; RefSeq (mRNA): NM_001200; RefSeq (protein): NP_001191; Localization (UCSC): Xp 20: 6.75-6.76 Mb. Species: mouse; Entrez: 12156; Ensembl: 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, refers to bone morphogenetic protein 5 (BMP5) or the gene or nucleic acid encoding BMP5. BMP5 is also known as: MGC34244; External IDs OMIM: 112265 MGI: 88181 HomoloGene: 22412 GeneCards: BMP5 gene. Species: human; Entrez: 653; Ensembl: ENSG00000112175; UniProt: P22003; RefSeq (mRNA): NM_021073; RefSeq (protein): NP_066551; localization (UCSC): Xp 6: 55.62-55.74 Mb. Species: mouse; Entrez: 12160; Ensembl: 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, refers to bone morphogenetic protein 7 (BMP7) or the gene or nucleic acid encoding BMP7. BMP7 is also known as: osteogenic protein 1; OP-1; External IDs OMIM: 112267 MGI: 103302 HomoloGene: 20410 GeneCards: BMP7 gene. Species: human; Entrez: 655; Ensembl: ENSG00000101144; UniProt: P18075; RefSeq (mRNA): NM_001719; RefSeq (protein): NP_001710; localization (UCSC): Xp 20: 55.74-55.84 Mb. Species: mouse; Entrez: 12162; Ensembl: 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 stands for hepcidin gene or hepcidin protein, a peptide hormone. Hepcidin is also known as: HAMP (hepcidin antimicrobial protein or peptide); HPC; 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; Major 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 decreased 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 approximately 130-140 g/L (13-14 g/dL) and in women below approximately 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 antigen-binding fragments thereof) include antibodies (and antigen-binding fragments thereof) that bind to the BMP6 protein.

The terms "antibody," "antigen-binding fragment," "antigen-binding portion," and the like, when used herein, include complete antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chains thereof. A natural antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists 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 one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called 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 the binding domain that interacts with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to self-tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

The terms antigen-binding fragment, its antigen-binding fragment, antigen-binding portion of an antibody, etc. as used herein, refer to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., BMP6. The antigen-binding functions of an antibody can be provided by fragments of an intact antibody. Examples of binding fragments encompassed by the term antigen-binding portion of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; an F(ab) ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment, consisting of the VH and CH1 domains; an Fv 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 the VH domain; and an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, encode separate genes, they can be joined recombinantly by an artificial peptide linker that allows for their synthesis 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, e.g., Bird et al., 1988 Science 242:423–426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879–5883). Such single-chain antibodies include one or more antigen-binding portions of the antibody. Such antibody fragments are prepared using standard techniques known to those skilled in the art and are tested for suitability in the same manner as intact antibodies.

Antigen-binding portions can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., 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 fibronectin type III (Fn3) (see U.S. Patent 6,703,199, which describes monobodies based on fibronectin polypeptides).

Antigen-binding moieties can be incorporated into single-chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10):10571062; and U.S. Patent 5,641,870).

As used herein, the term affinity refers to the strength of interaction between an antibody and an antigen at single antigenic sites. At each antigenic site, the variable region of the antibody's arm interacts with the antigen at multiple sites through weak noncovalent forces; 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; the valence of the antigen and antibody; and the structural arrangement of the interacting moieties. Ultimately, these factors determine the specificity of the antibody, that is, the probability that a particular antibody will bind to a particular epitope of the antigen.

- 12 039579

The term amino acid refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those amino acids that undergo subsequent modification, such as hydroxyproline, gammacarboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as a natural amino acid, i.e., an alpha carbon atom bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones but retain the same basic chemical structure as the natural amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function similarly to the natural amino acid.

The term binding specificity, as used herein, refers to the ability of an individual antibody paratope to react with only one antigenic determinant. The antibody paratope is located in the Fab portion of the molecule and is formed by the hypervariable regions of the heavy and light chains. Antibody binding affinity is the strength of interaction between one antigenic determinant and one paratope on the antibody. It 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 1 x 10 ⁷ M' ¹ , 10 ⁸ M' ¹ , 10 ⁹ M' ¹ , 10 ¹⁰ M ^-1 or 10 ¹¹ M' ¹ . The phrase "specifically (or selectively) binds to an antibody (e.g., a BMP6-binding antibody)" refers to a binding reaction that detects the presence of a cognate antigen (e.g., human BMP6 protein) in a heterogeneous population of proteins and other biologicals. In addition to the equilibrium constant (KA) noted above, the 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 lower, and binds to BMP6 with an affinity that is at least twofold greater than its affinity for binding to a non-specific antigen (e.g., BMP2, BMP5, or BMP7). The phrases "antibody recognizing an antigen" and "antigen-specific antibody" 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 10 5 M ^-1 , at least 5x10 5 M ^-1 , at least 10 6 M ^-1 , at least 5x10 6 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 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: (a) the constant region or a portion thereof is altered, replaced, or substituted such that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and/or biological species, or an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof is altered, replaced, or substituted with a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. As a result of the replacement with a human constant region, the chimeric antibody can maintain its specificity in recognizing the antigen while simultaneously having a decrease in antigenicity in humans compared to the original mouse antibody.

The term conservatively modified variant applies to both amino acid sequences and nucleic acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to nucleic acids that encode identical or substantially identical amino acid sequences, or, in the case where a nucleic acid does not encode an amino acid sequence, to substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode a given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified 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 type of conservatively modified variant.

- 13,039,579 identified variations. Each nucleic acid sequence in the present disclosure that encodes a polypeptide also describes every possible silent variation of the nucleic acid. It will be apparent to those skilled in the art that every codon in the nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be altered to produce a functionally identical molecule. Thus, every silent variation of the nucleic acid that encodes a polypeptide is implicitly implied in each described sequence.

In the case of polypeptide sequences, conservatively modified variants include individual 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 list functionally similar amino acids are known in the art. Such conservatively modified variants are provided in addition 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, e.g., 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 comprising the 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 locates and interacts with (e.g., binds to) its conformational epitope.

The terms cross-blocks, cross-blocked, cross-blocking, competes, cross-competes, and related terms are used interchangeably herein to refer to the ability of an antibody or other binding agent to disrupt 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 can disrupt the binding of another antibody or binding molecule to BMP6, and thus can 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 (e.g., using the BIAcore 3000 instrument (BiaCore, Uppsala, Sweden)), which measures the degree of interactions 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, thereby reducing, at least partially, the activity, level, or stability of BMP6, such as signaling or its role in hepcidin levels and anemia.

The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge properties. 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 encompasses any protein determinant capable of specific binding to immunoglobulin or other interaction with the molecule. Epitope determinants typically consist of chemically active 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 properties. An epitope can be linear or conformational.

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

As used herein, the term "high affinity" with respect to 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, such as BMP6. However, high binding affinity may vary for other antibody isotypes. For example, high binding affinity for the 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. Furthermore, if the antibody comprises a constant region, the constant region is also derived from such human sequences, such as human germline sequences or mutant variants of human germline sequences. The human antibodies and antigen-binding fragments thereof of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo).

The phrases "monoclonal antibody" or "monoclonal antibody composition" (or antigen-binding fragment thereof), as used herein, refer 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 a single binding specificity and affinity for a particular epitope.

The term "human monoclonal antibody" (or antigen-binding fragment thereof) refers to antibodies (and their antigen-binding fragments) 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, created, or isolated by genetic engineering techniques, such as antibodies isolated from an animal (e.g., a mouse) 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, created, or isolated by any other means that involve splicing all or part of a 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 human germline immunoglobulin sequences. In one embodiment, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, in the case of using 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 that, while they are derived from and related to human germline VH and VL sequences, may not naturally exist within the human germline antibody repertoire in vivo.

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 exhibiting reduced immunogenicity 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 (i.e., 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, but are not limited to, the Hoth technology disclosed in U.S. Patent 5,766,886.

The terms "identical" or "percent identity" in relation to two or more nucleic acid sequences or polypeptide sequences refer to two or more sequences or subsequences that are the same. Two sequences are substantially identical if the 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 a specified region, or, if not specified, over the entire sequence), when compared and aligned with the maximum match in the comparison window or specified region, as measured using one of the following sequence comparison algorithms or manual alignment and visual inspection. Non- 15 039579 necessarily, the identity is present in a region that has a length of at least about 50 nucleotides (or 10 amino acids), or more preferably in a region that has a length of 100-500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids). Optionally, the identity is present in a region that has a length of at least 50 nucleotides (or 10 amino acids), or more preferably in a region that has a length of 100-500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids).

To compare sequences, one sequence typically serves as a reference sequence, against which the analyzed sequences are compared. When using a sequence comparison algorithm, the analyzed and reference sequences are entered into the computer, subsequence coordinates are determined if necessary, and the program parameters of the sequence analysis algorithm are set. The default program parameters can be used, or alternative parameters can be set. The sequence comparison algorithm then calculates the percent sequence identity of the analyzed sequences relative to the reference sequence, based on the program parameters.

A comparison window, as used herein, includes a reference to a segment of any number of contiguous positions selected from a group consisting of 20 to 600, typically from about 50 to about 200, more often from about 100 to about 150, in which a sequence can be compared to a 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 (1970) Adv. Appl. Math. 2:482c), the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol. 48:443, 1970), the similarity search method of Pearson and Lipman (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 inspection (see, for example, Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389–3402, 1977; and Altschul et al., J. Mol. Biol. 215:403–410, 1990, respectively. A program for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by defining short words of length W in the query sequence that match or satisfy some threshold score T with a positive value when aligned with a word of the same length in a database sequence. T is called the nearest word score threshold (Altschul et al., supra). Such initial nearest word matches act as similarity points to initiate searches to find longer HSP sequences containing them. Word matches are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a substitution matrix is used to calculate the cumulative score. Extension of word matches in each direction is stopped if: the cumulative alignment score decreases by X from its maximum achieved value; the cumulative score drops to zero or below due to the accumulation of one or more negatively scoring residue alignments; or the end of any sequence is 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, the BLASTP program uses a wordlength of 3 and an expectation (E) of 10 as default parameters, as well as 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 a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., 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 with which a match between two nucleotide or amino acid sequences would 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 to 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 approximately 0.001.

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

In addition to the percentage of sequence identity noted above, another indicator 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, a polypeptide is typically substantially identical to the second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indicator 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. Yet another indicator 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 antigen specificities (e.g., an isolated antibody that specifically binds BMP6 is substantially free of antibodies that specifically bind antigens other than BMP6). Furthermore, an isolated antibody may be substantially free of other cellular material and/or chemical compounds.

The term "isotype" refers to the antibody class (e.g., IgM, IgE, IgG, such as IgG1 or IgG4) specified by the heavy chain constant region genes. An 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 intended to refer to the rate of dissociation in a particular antibody-antigen interaction. In one embodiment, the term K _D as used herein is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and expressed in molar concentration (M). K _D values for antibodies can be determined using methods well known in the art. The method for determining 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, refer to the production of an antibody molecule (or antigen-binding fragment thereof) as a single-molecule composition. A monoclonal antibody composition exhibits monospecific binding and affinity for a specific epitope.

The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof, either in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone or linkage residues, which are synthetic, natural, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which 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, and peptide nucleic acids (PNA).

Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses corresponding conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly stated sequence. In particular, as detailed below, degenerate codon substitutions can be obtained by creating 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 "functionally linked" refers to a functional relationship between two or more polynucleotide segments (e.g., DNA). Typically, this refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is functionally linked to a coding sequence if it stimulates or modulates transcription of the coding sequence in the corresponding host cell or other expression system. Typically, transcriptional regulatory promoter sequences that are functionally linked to a transcribed sequence are in physical contact with the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be in physical contact or in close proximity to the coding sequences whose transcription they enhance.

As used herein, the term "optimized" means that a nucleotide sequence has been altered 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. An optimized nucleotide sequence is designed to fully or as closely 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 polymers of amino acids in which one or more amino acid residues are artificial chemical mimetics of the corresponding natural amino acid, as well as to natural polymers of amino acids and non-naturally occurring polymers of amino acids. Unless otherwise specified, a specific polypeptide sequence also implicitly encompasses the 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, created, or isolated using recombinant techniques, such as antibodies isolated from an animal (e.g., a mouse) 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, created, or isolated using any other methods that involve splicing all or part of a human immunoglobulin gene sequence with other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, however, such recombinant human antibodies may 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 that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human germline antibody repertoire in vivo.

The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific cell in question but also to the progeny of such a cell. Because some modifications may occur in subsequent generations, either as a result of 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 humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise noted, the terms "patient" and "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 (e.g., anemia), alleviating symptoms, or halting or inhibiting the further progression of a disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of a disease or to prevent the manifestation of its clinical or subclinical symptoms) or therapeutic suppression or alleviation 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 circular loop of double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell and thus replicate along with the host organism's genome. Additionally, some vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). Expression vectors useful in recombinant DNA techniques are typically in the form of plasmids. In the present description, plasmid and vector may be used interchangeably, as plasmids are the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

The term hematocrit, as used herein, is also known as the sedimented cell volume (SV) or red blood cell volume fraction (EVF) and is the volume (%) of red blood cells in the blood. It typically accounts for approximately 45% in men and approximately 50% in women. It is considered an integral part of a person's complete blood count (CBC) results, along with hemoglobin concentration, white blood cell count, and platelet count. In one embodiment, anemia refers to an abnormally low hematocrit, as opposed to polycythemia, which is an abnormally high hematocrit.

Brief description of the figures

Figure 1A shows the inhibition of BMP activity by antagonist antibodies 5, 6, and 7 in a reporter gene assay. Activity against BMP2, BMP5, BMP6, and BMP7 is shown. Figure 1B shows an ELISA binding assay in which 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 herein: Ab 5=Antibody 5; Ab 6=Antibody 6; and Ab 7=Antibody 7.

Figure 2 shows the pharmacodynamic profiles in a preliminary PK study in rats following a single dose. Antibodies 5, 6, and 7 were used. Serum hepcidin and iron levels were measured at 1 and 6 h, 1, 2, 4, 8, and 16 days post-dose (10 mg/kg, IV).

Figure 3 shows the dose-dependent effect of antibody BMP6 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.

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

Figure 5 shows HDxMS (hydrogen/deuterium exchange coupled to mass spectrometry) linear epitope mapping. The BMP6 epitope bound by antibody 7 (residues 88-102 of human BMP6 (QTLVHLMNPEYVPKP (SEQ ID NO: 92))) is shown. 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)).

Fig. 6 shows the protocol for Part 1 of the clinical program to study the safety and efficacy of antibodies to BMP6.

Figure 7 shows a dose selection decision tree for a clinical trial program to study the safety and efficacy of antibodies to BMP6.

Fig. 8 shows the protocol for Part 2 of the clinical program for studying the safety and efficacy of antibodies to BMP6.

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Fig. 9 shows the pharmacokinetic profiles following a single dose of antibody 7 in male rats.

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

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

Figure 12 shows the concentration-time profile of a single intravenous injection of antibody 7 (3 mg/kg) in cynomolgus monkeys. The total concentration of antibody 7 (free and BMP6-bound) is indicated on the graph.

Figure 13 shows the serum concentrations of hepcidin and Fe in male cynomolgus macaques 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 mean.

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 producing and methods for 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 being bound by any particular theory, this disclosure proposes an antagonist antibody to BMP6 as a hepcidin-lowering therapy that is expected to be effective in patients with iron deficiency anemia by overcoming erythropoiesis-stimulating drug (ESD) resistance, which significantly contributes to the severity of the underlying disease and is often a prognostic factor for 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 further evidence that targeting this pathway can lead to improved functional endpoints, we investigated the ability of BMP6-specific antibodies, Antibodies 5-7, to modulate serum biomarkers of iron metabolism in normal mice and rats and to reverse ESP-resistant anemia in a mouse model of inflammatory anemia. A single injection of the BMP6 antibody to animals was found to result in a sustained increase in serum iron levels, accompanied by potent suppression of circulating hepcidin. Furthermore, therapeutic treatment of mice subjected to inflammation-induced anemia significantly improved erythropoietic parameters in response to concomitant erythropoietin treatment.

Here, inhibition of BMP6 signaling in a mouse model of inflammatory anemia significantly improved iron-dependent red blood cell parameters.

The BMP6 antagonist antibodies disclosed herein represent a novel therapeutic approach for safely improving the condition of erythropoietin-resistant anemia. Without wishing to be bound by any particular theory, it is proposed that this may occur through the mobilization and availability of iron stores 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 1,000-fold higher affinity to human BMP6 than to either human BMP5 or human BMP7. Specificity for BMP6 without binding to BMP7 is important because knockout of BMP6 is not lethal in mice. However, BMP7 knockout mice die after birth with kidney, eye, and bone defects. Single knockouts of either gene do not cause alterations in cardiogenesis, but double knockouts of BMP6 and BMP7 showed several defects and delays in cardiac development; embryos died of 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 undesirable. The antibodies of the present invention are specific for BMP6 compared to BMP7; see, for example, Table 4A. Fig. 1B also shows confirmation of the binding specificity to human BMP6 compared to human BMP2, BMP5, and BMP7 proteins.

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

Antibodies of the invention include, but are not limited to, 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 generated by germline PTM deletion from NOV0442_VL(YGQ), which is derived from the NOV0442 (VH3_3-15, VΠ_1e) parental IgG hit. Antibody 7 binds with high affinity to human BMP6 in an ELISA binding assay, with 500-fold selectivity over human BMP7 (i.e., 500-fold greater affinity for human BMP6 than for human BMP7). This antibody also has no detectable activity against human BMP2 or BMP5. The BMP6 peptide recognized by parental IgG NOV0442 and antibody 7 is shown in Fig. 5. The peptide includes 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 VSSASDYNSSELK sequence (SEQ ID NO: 95) of human BMP6. Thus, the epitope recognized by IgG NOV0442 and antibody 7 is a novel epitope of BMP6. Antibody 7 also inhibits BMP6 binding to receptors in vitro. BMP6 binding to BMPR1A is maximally inhibited by 59%; binding to BMPR1B is maximally inhibited by 85%; and binding to RGM-c is maximally inhibited by 72%. A single treatment of rats at a dose of 10 mg/kg resulted in long-term suppression of circulating hepcidin. The calculated minimum effective dose in mice is less than or equal to 0.1 mg/kg. Serum iron also increased, and hepcidin decreased after a single 3 mg/kg dose of antibody in monkeys. In mice, using Brucella abortus antigen to model anemia, the result of antibody 7 treatment (2 mg/kg) was 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 mutating N51Q in LCDR2 to increase the homogeneity of the subsequent product. The antibody, derived from the VH3/lambda1 framework, was engineered to match the closest human germline sequence: VH with a V40A mutation, VL with D1Q and I2S mutations, and the introduction of amino acids Y49 and G50 to restore the framework originally lacking these two 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 exhibit high specificity for the human BMP6 protein compared to human BMP2, BMP5, or BMP7 proteins. The epitope of all these antibodies, as predicted, is the same, since they are all derived from the same initial Fab before affinity maturation. Antibody 3, for example, shares a common Fab clone with both antibodies 5 and 7 before HCDR2 affinity maturation. Antibody 5 is derived from NOV0442 (VH3_3-15, VI1_1e)^NOV0442_VL (YGQ)^(HCDR2 affinity maturation)^Antibody 5. Antibody 3 is derived from NOV0442 (VH3_3-15, VI1_1e)^NOV0442_VL (YGS)^(HCDR2 affinity maturation)^Antibody 3. Further detailed information regarding the production of the antibodies described in the present invention is provided in the Examples.

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

The invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibodies or antigen-binding fragments thereof comprise (or, alternatively, consist of) a VH amino acid sequence listed in Table 1, wherein no more than about 10 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein a mutation, as various non-limiting examples, is an addition, substitution, or deletion). The invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibodies or antigen-binding fragments thereof comprise (or, alternatively, consist of) a VH amino acid sequence listed in Table 1, wherein no more than 10 amino acids in the framework sequence (e.g., a sequence that is not

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

The invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibodies or antigen-binding fragments thereof comprise (or, alternatively, consist of) a VH amino acid sequence listed in Table 1, wherein no more than about 20 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein a mutation, as various non-limiting examples, is an addition, substitution, or deletion). The invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibodies or antigen-binding fragments thereof comprise (or, alternatively, consist of) a VH amino acid sequence listed in Table 1, wherein no more than 20 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein a mutation, as various non-limiting examples, is an addition, substitution, or deletion).

The invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibodies or antigen-binding fragments thereof comprise (or, alternatively, consist of) the VL amino acid sequence listed in Table 1, wherein no more than about 10 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein a mutation, as various non-limiting examples, is an addition, substitution, or deletion). The invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibodies or antigen-binding fragments thereof comprise (or, alternatively, consist of) the VL amino acid sequence listed in Table 1, wherein no more than 10 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein a mutation, as various non-limiting examples, is an addition, substitution, or deletion).

The invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibodies or antigen-binding fragments thereof comprise (or, alternatively, consist of) the VL amino acid sequence listed in Table 1, wherein no more than about 20 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein a mutation, as various non-limiting examples, is an addition, a substitution, or a deletion). The invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to BMP6, wherein said antibodies or antigen-binding fragments thereof comprise (or, alternatively, consist of) the VL amino acid sequence listed in Table 1, wherein no more than 20 amino acids in the framework sequence (e.g., a sequence that is not a CDR) have been mutated (wherein a mutation, as various non-limiting examples, is an addition, a substitution, or a deletion).

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to the BMP6 protein, wherein said antibodies or antigen-binding fragments thereof comprise a VL domain listed in Table 1. The present invention also relates to antibodies and antigen-binding fragments thereof that specifically bind to the BMP6 protein, wherein said antibodies or antigen-binding fragments thereof comprise 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 antigen-binding fragments thereof that specifically bind to the BMP6 protein, wherein said antibodies or antigen-binding fragments thereof comprise (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 antigen-binding fragments thereof of the invention include amino acids that have been mutated and have at least 60, 70, 80, 90, or 95% identity in the CDR regions with the CDR regions shown 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 (e.g., Table 1 shows exemplary nucleic acid sequences of the heavy chain and light chain of antibodies 3, 5, 6, and 7).

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Table 1

Examples of antibodies to BMP6 of the present invention

ANTIBODY 3 SEQ ID NO: Kebat HCDR1 SYWH 9 Kebat HCDR2 RIKDHKQGYTTAYAASVKG 10 Kebat HCDR3 VERSKSGFDN 11 Chotia HCDR1 GFTFSSY 12 Chotia HCDR2 KDHKQGYT 13 Chotia HCDR3 VERSKSGFDN 14 VH QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAPGKGLEWVGRIKDHKQGYTTAYAASVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSS 15 VH DNA 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 ctggggggcacagcggccctgggctgcctggtcaaggac 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 Kebat LCDR1 TGSSSNIGAGYSVH 19 Kebat LCDR2 GSSERPS 20 Kebat LCDR3 QSWDSSQTLW 21 Chotia LCDR1 SSSNIGAGYS 22 Chotia LCDR2 GSS 23 Chotia LCDR3 WDSSQTLV 24 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGSSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVL 25 VL DNA CAGAGCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGC ACCGGGCCAGCGCTGTGACCATTAGCTGTACCGGCAGCA GCAGCAACATTGGTGCTGGTTACTCTGTGCATTGGTAC CAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTA TGGTAGCTCTGAACGCCCGAGCGGCGTGCCGGATCGCT TTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCG ATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTA CTGCCAGTCTTGGGACTCTTCTCAGACTCTGGTTGTGT TTGGCGGCGGCACGAAGTTAACCGTCCTA 26 Light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGSSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECS 27 Light chain DNA CAGAGCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGC ACCGGGCCAGCGCTGTGACCATTAGCTGTACCGGCAGCA GCAGCAACATTGGTGCTGGTTACTCTGTGCATTGGTAC CAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTA TGGTAGCTCTGAACGCCCGAGCGGCGTGCCGGATCGCT TTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCG ATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTA CTGCCAGTCTTGGGACTCTTCTCAGACTCTGGTTGTGT TTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCAGCCC AAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTC TGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTC TCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCC TGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGA GAG CAC CACAC C C T C CAAACAAAG CAACAACAAGTAC G CGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGG AAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGA AGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAT GTTCA 28 ANTIBODY 5 Kebat HCDR1 SYWH 29 Kebat HCDR2 RIKRESSSYTTMYAAPVKG 30 Kebat HCDR3 VERSKSGFDN 31

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Chotia HCDR1 GFTFSSY 32 Chotia HCDR2 KRESSSYT 33 Chotia HCDR3 VERSKSGFDN 34 VH QVQLVESGGGLVKPGGSLRLSCAASGFFSSYWHWVR QAPGKGLEWVGRIKRES S SYTTMYAAPVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSS 35 VH DNA 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 AGACCAAGCCCAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTG GCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGT CCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGAT ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACG GCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT 38

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GATGCACGAGGCCCTGCACAACCACTACACCAGAAGT CCCTGAGCCTGAGCCCCGGCAAG Kebat LCDR1 TGSSSNIGAGYSVH 39 Kebat LCDR2 GQSERPS 40 Kebat LCDR3 QSWDSSQTLW 41 Chotia LCDR1 SSSNIGAGYS 42 Chotia LCDR2 GQS 43 Chotia LCDR3 WDSSQTLV 44 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVL 45 VL DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTG 46 Light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECS 47 Light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCT AAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAG CGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCC TGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGA GAG CAC CAC C C C CAG CAAG CAGAG CAACAACAAGTAC G CCGCCAGCAGCTACCTGAGCCTGACCCCGAGCAGTGG AAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGA GGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGT GCAGC 48 ANTIBODY 6 Kebat HCDR1 SYWH 49 Kebat HCDR2 RT RH S DMGYAT S YAAPVKG 50 Kebat HCDR3 VERSKSGFDN 51 Chotia HCDR1 GFTFSSY 52 Chotia HCDR2 RHSDMGYA 53 Chotia HCDR3 VERSKSGFDN 54 VH QVQLVESGGGLVKPGGSLRLSCAASGFFSSYWHWVR QAP GKGLEWVGRT RH S DMGYAT S YAAPVKGRFTIS RDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSS 55 VH DNA CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAA GCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTG GCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGA CAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGAC TAGACACTCAGATATGGGCTACGCTACTAGCTACGCCG 56

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CTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGAC TCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAA AACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG AACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGC Heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFFSSYWHWVR QAP GKGLEWVGRTRH S DMGYAT S YAAPVKGRFTIS RDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WD VS N E D R E 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 AGACCAAGCCCAGAGGAGCAGTACAACAGCACCTAC AGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTG GOTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGT CCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGAT ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACG GCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT GATGCACGAGGCCCTGCACAACCACTACACCCCAGAAGT CCCTGAGCCTGAGCCCCGGCAAG 58 Kebat LCDR1 TGSSSNIGAGYSVH 59 Kebat LCDR2 GQSERPS 60 Kebat LCDR3 QSWDSSQTLW 61 Chotia LCDR1 SSSNIGAGYS 62 Chotia LCDR2 GQS 63 Chotia LCDR3 WDSSQTLV 64 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVL 65

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VL DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTG 66 Light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVLGQP KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECS 67 Light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCT AAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAG CGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCC TGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGA GAG CAC CAC C C C CAG CAAG CAGAG CAACAACAAGTAC G CCGCCAGCAGCTACCTGAGCCTGACCCCGAGCAGTGG AAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGA GGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGT GCAGC 68 ANTIBODY 7 Kebat HCDR1 SYWH 69 Kebat HCDR2 RIRLETHGYAAEYAASVKG 70 Kebat HCDR3 VERSKSGFDN 71 Chotia HCDR1 GFTFSSY 72 Chotia HCDR2 RLETHGYA 73 Chotia HCDR3 VERSKSGFDN 74 VH QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWHWVR QAPGKGLEWVGRIRLETHGYAAEYAASVKGRFTISRDD SKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDNWGQG TLVTVSS 75 VH DNA CAGGTGCAGCTGGTGGAATCAGGCGGCGGACTGGTCAA GCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTG GCTTCACCTTCTCTAGCTACGTGGTGCACTGGGTCAGA CAGGCCCCTGGTAAAGGCCTGGAGTGGGTCGGACGGAT TAGACTGGAAACTCACGGCTACGCCGCCGAGTACGCCG CTAGTGTGAAGGGCCGGTTCACTATCTCTAGGGACGAC ТCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAA AACCGAGGACACCGCCGTCTACTACTGCGCTAGAGTGG AACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGC 76 Heavy chain QVQLVESGGGLVKPGGSLRLSCAASGFFSSYWHWVR 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 SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 GCTGAACGGCAAAGAATACAAGTGCAAAGTCTSSAACA AGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAG GCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCT GCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGT CCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGAT ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA CAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACG GCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT GATGCACGAGGCCCTGCACAACCACTACACCCCAGAAGT CCCTGAGCCTGAGCCCCGGCAAG 78 Kebat LCDR1 TGSSSNIGAGYSVH 79 Kebat LCDR2 GQSERPS 80 Kebat LCDR3 QSWDSSQTLW 81 Chotia LCDR1 SSSNIGAGYS 82 Chotia LCDR2 GQS 83 Chotia LCDR3 WDSSQTLV 84 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVL 85 VL DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTG 86 Light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYSVHWY QQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLA ITGLQAEDEADYYCQSWDSSQTLWFGGGTKLTVLGQP 87 KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECS Light chain DNA CAGTCAGTCCTGACTCAGCCCCCTAGCGTCAGCGGCGC TCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTA GCTCTAATATCGGCGCTGGCTATAGCGTGCACTGGTAT CAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTA CGGTCAGTCAGAGCGGCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCT ATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTA CTGTCAGTCCTGGGACTCTAGTCAGACCCTGGTGGTGT TCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAGCCT AAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAG CGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCC TGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGA GAC CAC CAC C C C CAG CAAG CAGAG CAACAACAAGTAC G CCGCCAGCAGCTACCTGAGCCTGACCCCGAGCAGTGG AAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGA GGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGT GCAGC 88

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

- 29 039579 amino acids or nucleic acids encoding amino acids have been mutated and yet have at least 60, 70, 80, 90 or 95% identity with the sequences described in Table 1. In one embodiment, these 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, while maintaining substantially the same therapeutic activity.

In another specific embodiment, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds human BMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 9, 10, and 11, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 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 HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 12, 13, and 14, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 22, 23, and 24, 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 HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 29, 30, and 31, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 39, 40, and 41, 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 HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 32, 33, and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 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 HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 49, 50, and 51, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 59, 60, and 61, 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 HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 52, 53, and 54, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 62, 63, and 64, 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 HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 69, 70, and 71, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 79, 80, and 81, 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 HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 72, 73, and 74, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 82, 83, and 84, respectively.

Because each of these antibodies can bind BMP6, the VH, VL, full-length light chain, and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding the amino acid sequences) can be mixed and combined to produce other BMP6-binding antibodies and antigen-binding fragments thereof according to the invention. Such mixed and combined BMP6-binding antibodies can be tested using binding assays known in the art (e.g., ELISA and other assays described in the examples section). When mixing and combining these chains, the VH sequence from a particular VH/VL pair should be replaced with a structurally similar VH sequence. Likewise, the 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. Likewise, the VL sequence from a particular VH/VL pair should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence of 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 that comprise the CDR1, CDR2, and CDR3 regions of the heavy chain and light chain as described in Table 1, or combinations thereof. The CDR regions are defined 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 antigen-binding specificity is primarily provided by the CDR1, 2, and 3 regions, the VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be mixed and matched (i.e., CDR regions from different

- 30 039579 individual antibodies may be mixed and combined, although each antibody must contain VH CDR1, 2 and 3 and VL CDR1, 2 and 3) to produce other BMP6-binding molecules of the invention. Such mixed and combined BMP6-binding antibodies can be tested using binding assays known in the art and described in the examples (e.g., ELISA assays). When VH CDR sequences are mixed and combined, the CDR1, CDR2 and/or CDR3 sequence from a particular VH sequence should be replaced with a 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 should be replaced with a 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 VH and/or VL CDR sequences with structurally similar sequences from the CDR sequences shown herein for the monoclonal antibodies of the present invention.

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

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

As used herein, a human antibody includes variable regions of a heavy or light chain, or full-length heavy or light chains, that are the product of or derived from a specific germline sequence if the variable regions or full-length chains of the antibody are obtained from a system that utilizes human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest or screening a library of human immunoglobulin genes presented on a bacteriophage with the antigen of interest. A human antibody that is the product of or derived from a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., has the highest % identity) to the human antibody sequence. A human antibody that is a product of or derived from a specific human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, such as those caused by naturally occurring somatic mutations or the intentional introduction of site-directed mutations. However, in the VH or VL framework regions, a selected human antibody typically shares at least 90% amino acid sequence identity with 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 of germline immunoglobulins from other species (e.g., mouse germline sequences). In some cases, a 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 a germline immunoglobulin gene. Typically, a recombinant human antibody will exhibit no more than 10 amino acid differences in the VH or VL framework regions of the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a human antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequences encoded by the germline immunoglobulin gene.

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BMP family members and hepcidin

In one embodiment, the invention relates to an antibody or a binding fragment thereof that specifically binds to BMP6. In one embodiment, the antibody or binding fragment thereof 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 capable of inducing all osteogenic markers in mesenchymal stem cells.

Bone morphogenetic proteins (BMPs) are a family of secreted signaling molecules that can induce ectopic bone growth. BMPs are part of the transforming growth factor beta (TGF-beta) superfamily. BMPs were initially identified by the ability of demineralized bone extract to induce endochondral osteogenesis in vivo at an extraskeletal site. Because of its expression early in embryogenesis, the BMP encoded by this gene is thought to play a role in early development. Furthermore, the fact that this BMP is closely related to BMP5 and BMP7 has led to the suggestion of possible bone growth-inducing activity. An additional function for BMP6 has been identified as described in Nature Genetics April; 41[4]:386–8.

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

BMP6 is the primary regulator of hepcidin, a small peptide secreted in the liver that is the primary regulator of iron metabolism in mammals. Hepcidin regulates both the amount of iron absorbed from food in the duodenum and the iron released by reticuloendothelial cells. Hepcidin levels are elevated by a variety of stimulating factors, including inflammation and iron overload, and decreased by anemia, hypoxia, and iron deficiency.

Without being bound by any particular theory, it is hypothesized herein that an antagonist antibody to BMP6 would be effective as a hepcidin-lowering therapy in patients with iron deficiency anemia, overcoming erythropoiesis-stimulating drug (ESD) resistance, which significantly contributes to the severity of the underlying disease and is often a prognostic factor for poor outcome. By binding to BMPR1 and BMPR2 receptors, it induces receptor dimerization and hepcidin transcription. BMP6 also binds to the HJV coreceptor in liver and muscle cells.

Thus, BMP6 is known to increase hepcidin expression. Hepcidin is a key hormone involved in iron homeostasis. High hepcidin levels are associated with iron-deficient erythropoiesis in ankylosing spondylitis.

WO 2010/056981 discloses that administration of an antibody to BMP6 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 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 by Kishimoto et al. 1997 Dev. 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 preferable that the anti-BMP6 antibody does not bind to BMP2.

BMP2 is also described, among others, 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 promote 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 on BMP5 is known in the art, e.g., 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 conversion of mesenchymal cells into bone and cartilage.

It induces phosphorylation of SMAD1 and SMAD5, which in turn induce transcription of numerous osteogenic genes.

As noted above, BMP6 knockout mice are viable and fertile, and exhibit normal bone and cartilage development. However, BMP7 knockout mice die after birth with kidney, eye, and bone defects. Individual knockouts of either gene do not cause alterations in cardiogenesis, but double knockout of BMP6 and BMP7 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 the anti-BMP6 antibody with BMP7 is undesirable.

Additional information related to BMP7 is provided in the prior art, such as 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 the emergence of two similar hepcidin genes in mice, hepcidin 1 and hepcidin 2, Ilyin et al. 2003 FEBS Lett. 542: 22-26. Murine 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 maintaining iron homeostasis, and is essential for regulating iron accumulation in macrophages and 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-aa peptide is secreted primarily in the liver and is considered the main regulator of iron metabolism. The 20- and 22-aa metabolites are present in urine. The N-terminal region of hepcidin is essential for function; deletion of the 5 N-terminal amino acids results in loss of function.

Active hepcidin peptides are rich in cysteines, which form intramolecular bonds that stabilize their beta-sheet structures.

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

The 25-amino-acid peptide hepcidin is secreted primarily in the liver and is considered a key 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 iron secretion from intestinal enterocytes into the hepatic portal system, thereby functionally reducing iron absorption. Inhibition of ferroportin also prevents iron release from macrophages; therefore, hepcidin maintains iron homeostasis. Hepcidin activity is also partially responsible for the iron sequestration observed in anemia secondary to chronic inflammation, such as inflammatory bowel disease, chronic heart failure, carcinomas, rheumatoid arthritis, and renal failure.

Mutations in the hepcidin gene cause hemochromatosis type 2B, also known as juvenile hemochromatosis, a disease caused by severe iron overload that leads to cardiomyopathy, cirrhosis, and endocrine failure.

Most cases of juvenile hemochromatosis result from mutations in hemojuvelin, a regulator of hepcidin production.

Genetically modified mice created to overexpress hepcidin die shortly after birth with severe iron deficiency, indicating its central and essential role in the regulation of iron metabolism. The first evidence linking hepcidin to anemia during inflammation was obtained when researchers analyzed tissue from two patients with liver tumors and severe microcytic anemia that was unresponsive to iron supplements. The tumor tissue produced increased hepcidin, and surgical removal of the tumors cured the anemia.

There are many conditions in which impaired iron absorption 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, administering an antibody or a binding fragment thereof to BMP6 reduces the activity and/or level of hepcidin and can thus be used in the treatment of anemia. In one embodiment, the invention relates to a method for reducing the activity or level of hepcidin in a patient, comprising the step of administering to the patient an antibody or an antigen-binding fragment thereof.

- 33 039579 fragment 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 diseases. This includes any disease associated with hepcidin and/or mutations and/or overexpression of wild-type and/or mutant hepcidin, and/or diseases in which disease progression is enhanced or prognosis is worsened by the presence of hepcidin and/or mutations and/or overexpression of wild-type and/or mutant hepcidin, and/or decreased renal elimination of hepcidin in urine.

Non-limiting examples of hepcidin-associated diseases include: anemia, iron deficiency erythropoiesis, hypoferremia, impaired dietary iron absorption, iron sequestration, anemia of inflammation (AI), atherosclerosis, diabetes and many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich ataxia, heart failure, chronic kidney disease, cardiorenal anemia 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 individuals 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 (ESD)-resistant anemia, and/or iron deficiency anemia.

Anemia in CKD is a common and early complication of chronic kidney disease. Cancer-associated anemia is caused by hematological malignancies and some solid tumors. As defined herein, this term also includes chemotherapy-induced anemia, which is anemia caused by chemotherapeutic agents. Anemia in chronic kidney disease can cause progression of diabetic neuropathy, cardiovascular disease, retinopathy, and other disorders. Cancer-associated anemia is associated with an increased risk of mortality.

Some chronic diseases, such as cancer, kidney disease, and autoimmune disorders, can lead to anemia. Excessive inflammatory cytokines can cause dysregulation of iron homeostasis, decreased erythropoiesis, and a shortened red blood cell lifespan. Some treatments for anemia include ESA, erythropoietin, iron supplementation, or blood transfusions.

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

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

Homologous antibodies

In another embodiment, the present invention relates to an antibody or antigen-binding fragment thereof comprising amino acid sequences that are homologous to the sequences described in Table 1, wherein said antibody binds to BMP6 and retains the desired functional properties of those antibodies 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 an 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 can inhibit erythrocyte lysis in a hemolytic assay, wherein the hemolytic assay is known in the art. In a specific example, such antibodies have an IC ₅₀ value in a hemolytic assay of 20-200 pM using human BMP6-depleted serum reconstituted with 100 pM human BMP6.

In one embodiment, the VH and/or VL amino acid sequences may be 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% identical to the sequences set forth in Table 1. In one embodiment, the VH and/or VL amino acid sequences 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 having high (i.e., 80% or greater) identity to the VH and VL regions described in Table 1 can be produced 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 herein.

In one embodiment, the amino acid sequences of the full-length heavy chain and/or the full-length light chain may be 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% identical to the sequences set forth in Table 1. An antibody having a full-length heavy chain and a full-length light chain having high (i.e., 80% or greater) identity to the full-length heavy chains of any of SEQ ID NOs: 18; 38; 58, or 78 and the full-length light chains of any of SEQ ID NOs: 28; 48; 68 or 88, respectively, can be obtained 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 herein.

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

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

As used herein, the percent identity between two sequences depends on the number of identical positions shared by the sequences (i.e., the percent identity is equal to the number of identical positions/the total number of positions x 100), taking into account the number of gaps and the length of each gap required to optimally align the two sequences. Sequence comparison and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

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

Antibodies with conservative modifications.

In one embodiment, an antibody of the invention has a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of such CDR sequences have specific amino acid sequences based on the antibodies described in the present invention, or conservative modifications thereof, and wherein the antibodies retain the required functional properties of the BMP6-binding antibodies and antigen-binding fragments thereof of 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 comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: the heavy chain variable region CDR1 comprises an amino acid sequence selected from any of SEQ ID NOs: 29, 49, 69, 12, 32, 52, 72, or 9, or conservative variants thereof; the heavy chain variable region CDR2 comprises an amino acid sequence selected from any of SEQ ID NOs: 10, 30, 50, 70, 13, 33, 53, or 73, or conservative variants thereof; the heavy chain variable region CDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 11, 31, 51, 71, 14, 34, 54, or 74, or conservative variants thereof; the light chain variable region CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 19, 39, 59, 79, 22, 42, 62, or 82, or conservative variants thereof; the light chain variable region CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 20, 40, 60, 80, 23, 43, 63, or 83, or conservative variants thereof; and the light chain variable region CDR3 comprises an amino acid sequence selected from any of SEQ ID NO: 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 defined amino acid sequences based on the antibodies described in the present invention, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the BMP6-binding antibodies and antigen-binding fragments thereof of the invention. Accordingly, 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, wherein: the full-length heavy chain has amino acid sequences selected from the group of SEQ ID NOs: 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 NOs: 28; 48; 68 or 88, and conservative modifications thereof; the antibody specifically binds to BMP6; and the antibody inhibits erythrocyte lysis in a hemolytic assay as described in the present invention. In a specific embodiment, such antibodies have an IC ₅₀ value in a hemolytic assay of 20-200 pM using human BMP6-depleted serum that is reduced with 100 pM human BMP6.

Antibodies that bind to the same epitope

The present invention provides antibodies that bind to the same epitope as the BMP6-binding antibodies listed in Table 1. The epitope bound by antibody 7 is shown in Fig. 5. Additional antibodies can thus be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies and antigen-binding fragments thereof of the invention in BMP6 binding assays. The ability of a test antibody to inhibit binding of the antibodies and antigen-binding fragments thereof of the present invention to the BMP6 protein demonstrates that the test antibody can compete with such antibody for binding to BMP6; such antibody, according to a non-limiting theory, can bind to the same or a related (e.g., structurally similar or spatially proximate) epitope on BMP6 as the antibody with which it competes. In a specific embodiment, an antibody that binds to the same epitope on BMP6 as the antibodies and antigen-binding fragments thereof of the present invention is a human monoclonal antibody. Such human monoclonal antibodies can be produced and isolated as described herein.

Once a desired epitope on an antigen has been identified, antibodies to that epitope can be produced, for example, using the methods described herein. Alternatively, information about desired epitopes can be obtained during the process of antibody discovery, production, and analysis. Based on this information, antibodies can then be competitively screened for binding to the same epitope. An approach to achieving this is to conduct cross-competition studies to find antibodies that competitively bind to each other, for example, antibodies that compete for binding to the 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 apparent to one of skill in the art, virtually anything to which an antibody can specifically bind can be an epitope. An epitope can include those residues to which the antibody binds.

Typically, antibodies specific to a particular target antigen preferentially recognize an epitope on the target antigen within a complex mixture of proteins and/or macromolecules.

The regions of a given polypeptide that include the 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 U.S. Patent 4,708,871; 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 readily identified by determining the spatial structure of BMP6 amino acids, for example, by hydrogen/deuterium exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, above. Antigenic regions of proteins can also be identified using standard antigenicity and hydrophobicity curves, such as those calculated using, for example, the Omiga version 1.0 program available from the Oxford Molecular Group. This computer program uses the Hopp/Woods method, Hopp et al. (1981) Proc. Natl. Acad. Sci USA 78:3824–3828; for determining antigenicity profiles, and the Kyte-Doolittle technique, 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 VH and/or VL sequences as shown in the present invention as a starting material for constructing a modified antibody, wherein the modified antibody may have altered properties compared to the original antibody. The antibody can be engineered by modifying one or more residues in one or both variable regions (i.e., 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 altering residues in the constant region(s), for example, to alter the effector function(s) of the antibody.

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One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. Therefore, amino acid sequences within the CDRs are more diverse in individual antibodies than sequences outside the CDRs. Because CDR sequences are responsible for much of the antibody-antigen interaction, it is possible to express recombinant antibodies that recapitulate the properties of certain natural antibodies by constructing expression vectors that include CDR sequences from a particular natural 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 publicly available DNA databases or published sources that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available online at www.mrc-cpe.cam.ac.uk/vbase) and 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 the antibodies and antigen-binding fragments thereof according to the invention include framework sequences that are structurally similar to the framework sequences used in the selected antibodies and antigen-binding fragments thereof according to the present invention, for example, consensus sequences and/or framework sequences used in the monoclonal antibodies according to the invention. The VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be grafted onto framework regions that have an identical 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 the germline sequences. For example, it has been found that in some cases it is advantageous to mutate framework residues while maintaining or increasing the antigen-binding ability of the antibody (see, e.g., U.S. Patents 5,530,101; 5,585,089; 5,693,762; and 6,180,370 (Queen et al.)).

Another type of variable region modification involves mutating amino acid residues in the CDR1, CDR2, and/or CDR3 regions of the VH and/or VL to thereby improve 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), and the effect on antibody binding or another functional property of interest can be assessed in vitro or in vivo assays, as described herein and provided in the examples. Conservative modifications (as discussed above) can be introduced. Mutations can be amino acid substitutions, additions, or deletions. Furthermore, typically no more than one, two, three, four, or five residues in the CDR region are changed.

Grafting of antigen-binding domains into alternative frameworks or scaffolds

A variety of antibody/immunoglobulin scaffolds or frameworks can be used, provided the resulting polypeptide includes at least one binding region that specifically binds to BMP6. Such scaffolds or frameworks include the five major human immunoglobulin idiotypes, their antigen-binding fragments, and include immunoglobulins from other animal species, preferably with humanized aspects. Single-heavy-chain antibodies, such as those found in camelids, are particularly interesting in this regard. Scientists continue to discover and create new scaffolds, scaffolds, and fragments.

In one aspect, the invention relates to a method for generating non-immunoglobulin-based 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 to the target protein BMP6. Known non-immunoglobulin scaffolds or frameworks 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.), maxibodies (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 the fibronectin type III domain (e.g., the tenth module of fibronectin type III (Fn3 domain 10)). The fibronectin type III domain has 7 or 8 beta strands distributed between two beta sheets that fold 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 sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see U.S. Patent 6,818,418). Such fibronectin-based scaffolds are not related to immunoglobulin, although the overall fold is very similar to the smallest functional fragment of antibodies, the heavy chain variable region, which comprises the entire antigen-recognition unit in camel and llama IgG. This structure allows the non-immunoglobulin antibody to mimic antigen-binding properties similar in nature and affinity to those of antibodies. Such scaffolds can be used in a randomization and loop shuffling strategy in vitro, which is similar to the affinity maturation process of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds, where the loop regions of the molecule can be replaced with the CDR regions of the invention using standard cloning methods.

Ankyrin technology is based on the use of proteins with ankyrin-based repeat modules as scaffolds carrying variable regions that can be used for binding to various targets. The ankyrin repeat module is a 33-amino acid-long polypeptide consisting of two antiparallel alpha helices and a beta turn. Binding of the variable regions is typically optimized using ribosome display.

Avimers are derived from a naturally occurring A-domain-containing protein such as LRP-1. These domains are exploited due to their nature of protein-protein interactions, with over 250 human proteins structurally based on A-domains. Avimers consist 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 techniques described, for example, in U.S. Patent Application Publications 20040175756; 20050053973; 20050048512; and 20060008844.

Affibodies, affinity ligands, are small, simple proteins consisting of a three-helix strand based on a scaffold of one of the IgG-binding domains of protein A, a surface protein of Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to produce affibody libraries with a large number of ligand variants (see, for example, U.S. Patent 5,831,012). Affibodies are similar to antibodies, with a molecular mass of 6 kDa, compared to the 150 kDa of antibodies. Despite their small size, the binding site of affibodies is similar to that of antibodies.

Anticalins are products developed by Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small, rigid proteins typically involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins are present in human body tissues and fluids. The protein architecture resembles that of immunoglobulins, with hypervariable loops atop a rigid framework. 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 flexibility and allows for multiple side chains. The shape of the binding site can thus be modified using a proprietary method to recognize desired target molecules of varying shapes with high affinity and specificity. One protein of the lipocalin family, bilin-binding protein (BBP) from Pieris brassicae, was used to create anticalins by mutagenesis of a set of four loops. One example of a patent application describing anticalins is PCT publication WO199916873.

Affiliate molecules are small non-immunoglobulin proteins engineered with specific affinities for proteins and small molecules. New affinity molecules can be rapidly selected from two libraries, each based on a distinct human-derived scaffold protein. Affiliate molecules exhibit no structural homology to immunoglobulin proteins. Two affinity scaffolds are currently used: one is gamma-crystallin, a human structural protein of the lens, and the other is a protein from the ubiquitin superfamily. Both human scaffolds are very small, exhibit thermal stability, and are virtually resistant to changes in pH and denaturing agents. This high stability is primarily 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 (MW 1–2 kDa) that mimic the beta-hairpin secondary structures of proteins, the major secondary structure involved in protein–protein interactions.

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Antibodies that bind human BMP6 can be produced using methods known in the art. For example, humanizing technology is used to convert non-human antibodies into engineered human antibodies. U.S. Patent Publication 20050008625 describes an in vivo method for replacing the variable region of a non-human antibody with a human variable region in an antibody while maintaining or improving binding properties compared to the non-human antibody. The method is based on epitope-directed replacement of the variable regions of a non-human reference antibody with a fully human antibody. The resulting human antibody typically differs structurally from the reference non-human antibody but binds to the same epitope on the same antigen as the reference antibody. Briefly, the serial epitope-directed complementarity exchange method is performed by inducing competition in cells between a competitor and a library of diverse reference antibody hybrids (test antibodies) for binding to limited amounts of antigen in the presence of a reporter system that responds to the binding of the test antibody to the antigen. The competitor may be the reference antibody or its derivative, such as a single-chain Fv fragment. The competitor may also be a natural or artificial antigen ligand that binds to the same epitope as the reference antibody. The only requirement for the competitor is that it binds to the same epitope as the reference antibody and competes with the reference antibody for binding to the antigen.

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

Various types of reporter systems can be used to detect the desired interactions between test antibodies and antigens. For example, reporter complementary fragments can be linked to the antigen and the test antibody, respectively, with reporter activation resulting from fragment complementation occurring only when the test antibody binds to the antigen. When fusion constructs of the test antibody and the antigen-reporter fragment are coexpressed 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 reactivator of an autoinhibitory reporter (RAIR) system disclosed in U.S. Patent Application Ser. No. 10/208,730 (Publication No. 20030198971) or the competitive activation system disclosed in U.S. Patent Application Ser. No. 10/076,845 (Publication No. 20030157579).

In the case of a serial epitope-directed complementarity exchange system, selection is performed to detect cells that express a single test antibody along with a competitor, antigen, and reporter components. In these cells, each test antibody competes one-on-one with its competitor for binding to a limited amount of antigen. Reporter activity is proportional to the amount of antigen bound by the test antibody, which in turn is proportional to the affinity of the test antibody for the antigen and the stability of the test antibody. Test antibodies are initially selected based on their activity relative to that of a reference antibody when expressed as the 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 a 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 comparable to or greater than the affinity of the reference antibody.

In the second round of V region replacement, the human V regions selected in the first round 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. The 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 structurally from the reference antibody but compete with the reference antibody for binding to the same antigen. Some of the selected human antibodies bind to the same epitope on the same antigen as the reference antibody. Of these selected human antibodies, one or more bind to the same epitope with an affinity comparable to or higher 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 be readily applied to the production of human antibodies that bind to human BMP6 with the same binding specificity and the same or better binding affinity. Furthermore, such antibodies binding to human BMP6 can also be purchased from companies that commonly manufacture human antibodies, such as KaloBios, Inc. (Mountain View, Calif.).

Camelid antibodies

Antibody proteins obtained from members of the camel and dromedary family (Camelus bactrianus and Calelus dromaderius), which includes New World representatives such as llama species (Lama paccos, Lama glama, and Lama vicugna), have been characterized for size, structural complexity, and antigenicity to humans. Some IgG antibodies from this mammalian family have been found in nature to lack light chains and thus differ structurally from the typical four-chain quaternary structure containing two heavy and two light chains found in antibodies from other animals. See PCT/EP93/02214 (WO 94/04678, published 3 March 1994).

The region of a camelid antibody that is the small single variable domain, designated VHH, can be genetically engineered to be a small protein with high affinity for a target, resulting in a low molecular weight antibody protein known as a camel nanobody. See U.S. Patent 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. Constructed libraries of camel antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies and their antigen-binding fragments of non-human origin, the amino acid sequence of the camel antibody can be recombinantly altered to produce a sequence that is more similar to the human sequence, i.e., the nanobody can be humanized. In this way, the naturally occurring low antigenicity of camel 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 nanometers. One consequence of its small size is the ability of camel nanobodies to bind to antigenic sites that are functionally undetectable by larger antibody proteins. This means that camel nanobodies can be used as reagents for detecting antigens that are otherwise hidden using classical immunological methods, as well as potential therapeutic agents. Thus, another consequence of its small size is that the camel nanobody can inhibit by binding to a specific site within a groove or narrow cavity of the target protein, and thus may serve a function more reminiscent of a classical small molecule drug than a natural antibody.

The low molecular weight and compact size also result in camel nanobodies that are extremely heat-stable, resistant to extreme pH and proteolytic degradation, and have low antigenicity. Another consequence is that camel nanobodies readily migrate from the cardiovascular system into tissues and even penetrate the blood-brain barrier, potentially treating disorders that affect nervous tissue. Nanobodies can also facilitate drug transport across the blood-brain barrier. See U.S. Patent Application 20040161738, published August 19, 2004. These properties, combined with low antigenicity in humans, indicate great therapeutic potential. Furthermore, these molecules can be fully expressed in prokaryotic cells such as E. coli and expressed as fusion proteins with bacteriophage, yet still be functional.

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

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Bispecific molecules and multivalent antibodies

In another aspect, the present invention provides bispecific or multispecific molecules comprising a BMP6-binding antibody or fragment thereof according to the invention. The antibody of the invention or its antigen-binding regions can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the invention can actually be derivatized or linked to more than one functional molecule to produce 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 according to the invention, an antibody of the invention may be operably linked (e.g., by chemical crosslinking, genetic fusion, non-covalent linkage, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, thereby producing 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.

Furthermore, in an invention in which the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity, in addition to the first and second target epitopes.

In one embodiment, the bispecific molecules of the invention comprise, as a 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 dimer of a light chain or a heavy chain, or any minimal fragment thereof, such as Fv or the single-chain construct described in U.S. Patent 4,946,778 (Ladner et al.).

Diabodies are bivalent, bispecific molecules in which the VH and VL domains are expressed on a single polypeptide chain, linked by a linker that is too short to permit 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 produced by expressing two polypeptide chains with either the VHA-VLB and VHB-VLA structure (VH-VL configuration) or the VLA-VHB and VLB-VHA (VL-VH configuration) in the same cell. Most of them can be expressed in soluble form in bacteria. Single-chain diabodies (scDb) are formed by joining two diabody-forming polypeptide chains with a linker of approximately 15 amino acid residues in length (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45 (3-4):128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36). scDb can be expressed in bacteria in a soluble, active monomeric form (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(34):128–30; Wu et al., 1996 Immunotechnology, 2(1):21–36; Pluckthun and Pack, 1997 Immunotechnology, 3(2):83–105; Ridgway et al., 1996 Protein Eng., 9(7):617–21). The diabody can be fused with Fc to produce a di-diabody (see Lu et al., 2004 J. Biol. Chem., 279(4):2856–65).

Other antibodies that may be used in the bispecific molecules of the invention include 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 the bispecific molecule can be created separately and then conjugated to one another. When 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).

When the binding specificities are antibodies, they can be conjugated via sulfhydryl linkage between the C-terminal hinge regions of 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 may be encoded in a single vector and expressed and assembled in a single host cell. This method is particularly convenient if 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 comprise at least two single-chain molecules. Methods for producing bispecific molecules are described, for example, in U.S. Patent 5,260,203; U.S. Patent 5,455,030; U.S. Patent 4,881,175; U.S. Patent 5,132,405; U.S. Patent 5,091,513; US patent 5476786; US patent 5013653; US patent 5258498; and US patent 5482858.

Binding of bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., 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 (e.g., an antibody) specific to the complex of interest.

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

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

Antibodies with an extended half-life

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

Many factors can influence the half-life of a protein in vivo. Examples include renal filtration, hepatic metabolism, degradation by proteolytic enzymes (proteases), and immunogenic responses (e.g., 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 coupling to polyethylene glycol (PEG), reCODE PEG, antibody backbone, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate shields; genetic fusion to proteins that bind to serum proteins such as albumin, IgG, FcRn, and transferrin; cross-linking (genetically or chemically) with other binding molecules that bind to serum proteins, such as nanobodies, Fab fragments, DARPins, avimers, affibodies, 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 antibody circulation in vivo in serum, inert polymeric molecules such as high-molecular-weight PEG can be attached to antibodies or antibody fragments through or without a multifunctional linker, or by site-specific conjugation of PEG to the N- or C-terminus of antibodies, or via epsilon-amino groups present on lysine residues. To PEGylate an antibody, the antibody or its antigen-binding fragment is typically reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that bind one or more PEG groups to the antibody or antibody fragment. PEGylation can be accomplished via an acylation reaction or an alkylation reaction using a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" encompasses any of the forms of PEG 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. Derivatization of the linear or branched polymer that results in minimal loss of biological activity will be used. The degree of conjugation can be precisely monitored using SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to antibodies. Unreacted PEG can be separated from the antibody-PEG conjugates using size-exclusion or ion-exchange chromatography. PEGylated antibodies can be analyzed for binding activity and in vivo efficacy using methods known in the art, such as the immunoassays described herein. Methods for PEGylation of proteins are known in the art and can be applied to the antibodies and antigen-binding fragments thereof according to the invention. See, for example, EP 0 154 316 (Nishimura et al.) and EP 0 401 384 (Ishikawa et al.).

Other modified PEGylation technologies include reconstitutive chemical orthogonal directed engineering (ReCODE PEG), which enables the introduction of chemically specific side chains into biosynthetic proteins using a reconstituting system that includes tRNA synthetase and tRNA. This technology enables the incorporation of over 30 new amino acids into biosynthetic proteins in E. coli, yeast, and mammalian cells. tRNA incorporates the target amino acid into any position containing an amber codon, converting the amber from a termination codon to a codon that signals the incorporation of the chemically specified amino acid.

Recombinant PEGylation (rPEG) technology can also be used to extend serum half-life. This technology involves genetically fusing a terminal non-structural protein segment of 300-600 amino acids to an existing pharmaceutical protein. Because the apparent molecular weight of this non-structural protein chain is approximately 15 times greater than its actual molecular weight, the serum half-life of the protein is significantly increased. Unlike traditional PEGylation, which requires chemical conjugation and repeated purification, the manufacturing process is significantly simplified, and the product is homogeneous.

Polysialylation is another technology that utilizes a natural polymer, polysialic acid (PSA), to extend the duration of action and enhance the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a 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 its recognition by the immune system. The polymer PSA is naturally present in the human body. It is used by certain bacteria that evolved millions of years ago to coat their cell walls. These naturally polysialic bacteria are able to evade the body's immune system through molecular mimicry. PSA, the most advanced camouflage technology in nature, can be easily obtained from such bacteria in large quantities and with tailored physical properties. Bacterial PSC is completely non-immunogenic, even when bound to proteins, since it is chemically identical to PSC in the human body.

Another technology involves the use of hydroxyethyl starch (HES) derivatives bound to antibodies. HES is a modified natural polymer derived from waxy maize starch and can be degraded by the body's enzymes. HES solutions are typically used to replace depleted blood volume and improve blood rheology. HES acylation of the antibody increases the circulation half-life, enhances molecule stability, and reduces renal clearance, leading to increased biological activity. By varying various parameters, such as the molecular weight of HES, a wide range of different HES-antibody conjugates can be produced.

Antibodies having an increased half-life in vivo can also be obtained by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the constant domain of IgG or its FcRn binding fragment (preferably into the Fc fragment or the hinge region of the Fc domain). See, for example, International Patent Publication No. WO 98/23289; International Patent Publication No. WO 97/34631; and U.S. Patent 6,277,375.

Additionally, antibodies can be conjugated to albumin to make the antibody or antibody fragment more stable in vivo or to have a longer half-life in vivo. Such methods are known in the art; see, for example, International Patent Applications WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent EP 413622.

Half-life extension strategies are particularly useful for nanobodies, fibronectin-based binders, and other antibodies or proteins that require an extended half-life in vivo.

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 an antigen-binding fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to produce fusion proteins. In particular, the invention relates to fusion proteins comprising an antigen-binding fragment of an antibody as described herein (e.g., a Fab fragment, an Fd-43 039579 fragment, an Fv fragment, an F(ab) ₂ fragment, a VH domain, a VH CDR, a VL domain, or a VL CDR) and a heterologous protein, polypeptide, or peptide. Methods of fusing or conjugating proteins, polypeptides, or peptides to an antibody or antibody fragment are known in the art. See, for example, U.S. Patents 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patents EP 307,434 and EP 367,166; International publications 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 Vil 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 techniques (collectively referred to as DNA shuffling). DNA shuffling can be used to alter the activity of the antibodies and antigen-binding fragments thereof of the invention (e.g., antibodies and antigen-binding fragments thereof with higher affinity and lower off-rate). See, generally, U.S. Patents 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; 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, may be altered by random mutagenesis in low-fidelity PCR, random nucleotide insertion, or other methods prior to recombination. A polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to BMP6 may be recombinant with one or more components, motifs, sections, portions, domains, fragments, etc. of one or more heterologous molecules.

Additionally, 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 are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine (SEQ ID NO: 96) allows for convenient purification of the fusion protein. Other peptide tags used for purification include, but are not limited to, the hemagglutinin (HA) tag, which corresponds to an epitope derived from the hemagglutinin protein of influenza virus (Wilson et al., 1984, Cell 37:767), and the flag tag.

In one embodiment, the antibodies and antigen-binding fragments thereof of the present invention are conjugated to a diagnostic or detectable agent. Such antibodies may be used to monitor or predict the onset, development, progression, and/or severity of a disease or disorder as part of a clinical investigation, such as determining the effectiveness of a particular therapy. Such diagnosis and detection may be accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or 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. An antibody or its antigen-binding fragment can be conjugated to a therapeutic molecule, such as a cytotoxin, e.g., a cytostatic or cell-destroying agent, a therapeutic agent, or a radioactive metal ion, e.g., an alpha-radiation source. A cytotoxin or cytotoxic agent includes any substance that has a destructive effect on cells.

In addition, the antibody or its antigen-binding fragment may be conjugated to a therapeutic molecule or drug molecule that modifies a given biological response. Therapeutic molecules or drug molecules should not be considered limited to classical chemical therapeutic agents. For example, a drug molecule may be a protein, peptide, or polypeptide possessing 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 alpha interferon, beta interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agent, antiangiogenic agent; or a biological response modifier such as, for example, a lymphokine.

In addition, the antibody can be conjugated to therapeutic molecules such as a radioactive metal ion, such as alpha emitters 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, to polypeptides. In one embodiment, the macrocyclic chelating agent is 1,4,7,10-tetraααsαcyclododekαN-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 entireties.

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

Antibodies can also be attached to solid supports, which are particularly useful for immunoassays or target antigen purification. Such solid supports 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 provides substantially purified nucleic acid molecules that encode polypeptides comprising segments or domains of the chains of the BMP6-binding antibodies described above. Some nucleic acids of the invention include a nucleotide sequence encoding a heavy chain variable region shown in any of SEQ ID NOs: 16; 36; 56, or 76, and/or a nucleotide sequence encoding a light chain variable region shown in any of SEQ ID NOs: 26; 46; 66, or 86. In a particular embodiment, the nucleic acid molecules are identified in Table 1. Some other nucleic acid molecules of the invention include nucleotide sequences that are substantially identical (e.g., at least 65%, 80%, 95%, or 99%) to the nucleotide sequences identified in Table 1. 1. When expressed from suitable expression vectors, polypeptides encoded by such polynucleotides are capable of demonstrating BMP6 antigen-binding ability.

The invention also relates to polynucleotides that encode at least one CDR region, and typically all three CDR regions, of the heavy or light chain of the BMP6-binding antibody shown in Table 1. Some other polynucleotides encode all or substantially all of the variable region sequence 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 a variable region and a constant region of an antibody. Some 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 a mature heavy chain variable region sequence shown in any of SEQ ID NOs: 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 a mature light chain variable region sequence shown in any of SEQ ID NOs: 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 (e.g., the sequence described in the Examples below) encoding a BMP6-binding antibody or a binding fragment thereof. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method (Narang et al., 1979, Meth. Enzymol. 68:90); the phosphodiester method (Brown et al., Meth. Enzymol. 68:109, 1979); the diethyl phosphoramidite method (Beaucage et al., Tetra. Lett., 22:1859, 1981); and the solid support synthesis method of US Patent 4,458,066. The introduction of mutations into polynucleotide sequences by PCR can be performed as described, for example, in PCR Technology: Principles and Applications for DNA Amplification,

H.A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

The invention also relates to expression vectors and host cells for the production of the 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 retrovirus, adenovirus, adeno-associated virus, herpesvirus, SV40, papillomavirus, Epstein-Barr virus HBP, vaccinia virus, and Semliki Forest virus (SFV) vectors. See Brent et al., supra; Smith, Annu. 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. Expression vectors typically contain a promoter and other regulatory sequences (e.g., 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 promoters. Cultures of transformed organisms can be propagated under non-stimulating conditions without shifting the population composition toward coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or may be necessary for efficient expression of the antigen-binding fragment of the BMP6-binding antibody chain. Such elements typically include an ATG initiation codon and an adjacent ribosome binding site or other sequences. In addition, expression efficiency can be increased by incorporating enhancers appropriate for the cell system used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

Expression vectors can also provide the position of a secretion signal sequence to produce a fusion protein with polypeptides encoded by inserted BMP6-binding antibody sequences. Typically, inserted BMP6-binding antibody sequences are linked to signal sequences before incorporation into the vector. Vectors used to insert sequences encoding the variable domains of the light and heavy chains of the BMP6-binding antibody sometimes also encode constant regions or portions thereof. Such vectors allow expression of the variable regions as fusion proteins with the 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 may be prokaryotic or eukaryotic. E. coli is one prokaryotic host suitable for cloning and expressing the polynucleotides of the present invention. Other suitable host microorganisms include bacilli, such as Bacillus subtilis, and other enterobacteria, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can also be created for such prokaryotic hosts, which typically contain expression regulation sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of different known promoters will be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. Promoters typically regulate expression, optionally via an operator sequence, and contain ribosome binding site sequences, etc., 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 in combination with baculovirus vectors can also be used.

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 (e.g., the myeloma hybridoma clone 1D6.C9, as described in the examples) or a mammalian cell line carrying an exogenous expression vector (e.g., the SP2/0 myeloma cells presented below). They include any normal mortal or normal or abnormal immortal cell of an animal or human. 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 polypeptide expression is discussed generally, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells may include expression control sequences such as an origin of replication, 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 constitutive adenovirus large late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP poIIII 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 the polynucleotide sequences of interest vary depending on the cellular host type. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cellular 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 herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), enhanced DNA uptake by specialized agents, and ex vivo transduction. Stable expression is often required for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express BMP6-binding antibody chains or binding fragments can be generated using expression vectors of the invention, which contain viral origins of replication 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 expanded using tissue culture techniques appropriate for the particular cell type.

Obtaining monoclonal antibodies according to the invention

Monoclonal antibodies (mAbs) can be produced by a variety of methods, including standard monoclonal antibody production techniques, such as the standard somatic cell hybridization method of Kohler and Milstein (1975 Nature 256:495). A variety of methods can be used to produce monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes.

The mouse system is a system in which animals are used to produce hybridomas. Hybridoma production in mice is a well-established technique. Immunization protocols and methods for isolating immunized splenocytes for fusion are known in the art. Fusion agents (e.g., mouse myeloma cells) and fusion techniques are also known.

Chimeric or humanized antibodies and antigen-binding fragments thereof according to the present invention can be prepared based on the sequence of a murine monoclonal antibody obtained as described above. DNA encoding the immunoglobulin heavy and light chains can be obtained from the murine hybridoma of interest and genetically engineered to include non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Patent 4,816,567 (Cabilly et al.)). To create a humanized antibody, murine CDR regions can be inserted into a human framework sequence using methods known in the art. See, e.g., U.S. Patent 5,225,539 (Winter) and U.S. Patents 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 produced using transgenic or transchromosomal mice carrying parts of the human immune system, rather than the murine 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 miniloci of the human immunoglobulin genes that encode unrearranged sequences of the heavy (mu and gamma) and kappa light chains of human immunoglobulins 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 exhibit reduced expression of mouse IgM or K, and, in response to immunization, introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity monoclonal human IgG kappa (Lonberg, N. et al., 1994, supra; reviewed in Lonberg, N., 1994 Handbook of Experimental Pharmacology 113:49–101; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol. 13: 65–93, and Harding, F. and Lonberg, N., 1995 Ann. N.Y. Acad. Sci. 764:536–546). The production and use of HuMAb mice and the genomic modifications they carry 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. 152:2912-2920; 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 U.S. Patents 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429 (Lonberg and Kay); U.S. Patent 5,545,807 (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, the human antibodies of the invention can be raised 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.).

Furthermore, 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 U.S. Patents 5,939,598; 6,075,181; 6,114,598; 6,150,584; and 6,162,963 (Kucherlapati et al.).

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

The human monoclonal antibodies of the invention can also be prepared by using phage display methods to screen libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are known in the art or are described in the Examples below. See, for example, U.S. Patents 5,223,409; 5,403,484; and 5,571,698 (Ladner et al.); U.S. Patents 5,427,908 and 5,580,717 (Dower et al.); U.S. Patents 5,969,108 and 6,172,197 (McCafferty et al.); and U.S. Patents 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081 (Griffiths et al.).

The human monoclonal antibodies of the invention can also be produced using SCID mice in which human immune cells have been reconstituted so that immunization with them can elicit a human humoral immune response. Such mice are described, for example, in U.S. Patents 5,476,996 and 5,698,767 (Wilson et al.).

Framework 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 framework residues in VH and/or VL, for example, to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one method involves back-mutating one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has been somatically mutated may contain framework residues that differ from the germline sequence from which it was derived.

- 48 039579 is the antibody's origin. Such residues can be determined by comparing the antibody's framework sequences with the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can be reverse-mutated to the germline sequence, for example, using site-directed mutagenesis. Such reverse-mutated antibodies are also contemplated as being included in the invention.

Another type of framework modification involves mutating one or more residues in the framework region 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 in the framework or CDR regions, the antibodies of the invention may be genetically engineered to incorporate modifications in 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. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, again to alter 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 Kabat EU index numbering.

In one embodiment, the CH1 hinge region is altered such that the number of cysteine residues in the hinge region is altered, for example, increased or decreased. This approach is also described in U.S. Patent 5,677,425 (Bodmer et al.). The number of cysteine residues in the CH1 hinge region is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease antibody stability.

In another embodiment, the hinge of the Fc region of the antibody is mutated to reduce the biological half-life of the antibody. Specifically, one or more amino acid mutations are introduced into the CH2-CH3 interface region of the Fc hinge domains such that the antibody exhibits impaired binding to staphylococcal protein A (SpA) compared to the SpA binding of the native Fc hinge domain. This approach is described in more detail in U.S. Patent 6,165,745 (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 can be introduced: T252L, T254S, T256F, as described in U.S. Patent 6,277,375 (Ward et al.). Alternatively, to increase the biological half-life, the antibody can be altered in the CH1 or CL region to include a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents 5,869,046 and 6,121,022 (Presta et al.).

In one embodiment, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter 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 altered affinity for an effector ligand but retains the antigen-binding capacity of the original antibody. The effector ligand whose affinity is altered can be, for example, an Fc receptor or complement component C1. This approach is described in more detail in U.S. Patents 5,624,821 and 5,648,260 (Winter et al.).

In another embodiment, one or more amino acids selected from the amino acid residues may be replaced with another amino acid residue such that the antibody has altered binding to C1q and/or reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent 6,194,551 (Idusogie et al.).

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

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

In another embodiment, the glycosylation of the antibody is altered. For example, an aglycosylated antibody can be created (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid sequences can be modified.

- 49,039,579 substitutions that result in the elimination of one or more glycosylation sites within the variable region framework, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is described in more detail in U.S. patents.

5714350 and 6350861 (Co et al.).

Additionally or alternatively, an antibody can be created that has an altered glycosylation pattern, such as a hypofucosylated antibody with a reduced number of 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 the antibody in a host cell with an altered glycosylation apparatus. Cells with an altered glycosylation apparatus have been described in the prior art and can be used as host cells in which to express the recombinant antibodies of the invention, resulting in an antibody with altered glycosylation. For example, EP 1 176 195 by Hang et al. describes a cell line with a functionally interrupted FUT8 gene, which encodes fucosyltransferase, wherein antibodies expressed in such a cell line exhibit 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 results in hypofucosylation of antibodies expressed in this host cell (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 (Umana et al.) describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta (1,4)-N-acetylglucosaminyltransferase III (GnTIII)), resulting in antibodies expressed by the engineered cell lines exhibiting increased GlcNac branching structures, leading to increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Methods for constructing modified antibodies

As discussed above, BMP6-binding antibodies having the VH and VL sequences or the full-length heavy and light chain sequences shown herein can be used to generate new BMP6-binding antibodies by modifying the full-length heavy chain and/or light chain sequences, the VH and/or VL sequences, or the constant region(s) linked thereto. Thus, in another aspect of the invention, the structural features of the 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 thereof of the invention, such as binding to human BMP6, as well as inhibition of one or more functional properties of BMP6 (e.g., inhibition of red blood cell lysis in a hemolytic assay).

For example, one or more CDR regions of the antibodies and antigen-binding fragments thereof of the present invention, or mutations thereof, can be recombinantly combined with known framework regions and/or other CDR regions to produce additional, recombinantly engineered BMP6-binding antibodies and antigen-binding fragments thereof of the invention, as discussed above. Other types of modifications include those described in the previous section. The starting material for the engineering method is one or more VH and/or VL sequences provided herein, or one or more CDR regions thereof. To create an engineered antibody, it is not necessary to actually produce (i.e., express as a protein) an antibody having one or more VH and/or VL sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as starting material to create second-generation sequence(s) derived from the original sequence(s), and the second-generation sequence(s) are then produced 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 different CDR1 and CDR2 sequences. Screening can be performed using any screening technology suitable for screening antibodies in antibody libraries, such as phage display technology.

Standard molecular biology techniques can be used to produce and express the altered antibody sequence. The 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 in the present invention, wherein the functional properties include, but are not limited to, specific binding to the human BMP6 protein; and the antibody inhibits erythrocyte lysis in a hemolytic assay.

The functional properties of the altered antibodies can be examined using standard assays available in the art and/or described herein, such as the assays provided in the examples (e.g., ELISA assays).

- 50 039579

In one embodiment of the methods for constructing antibodies and antigen-binding fragments thereof according to the invention, mutations can be introduced randomly or selectively throughout all or part of the coding sequence of BMP6-binding antibodies, and the resulting modified BMP6-binding antibodies can be screened for binding activity and/or other functional properties as described herein. Mutational methods have been described in the art. For example, PCT Publication WO 02/092780 by Short describes methods for generating and screening 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 computational screening methods to optimize the physiochemical properties of antibodies.

Analysis of antibodies according to the invention

The antibodies and their antigen-binding fragments according to the invention can be tested using various functional assays. For example, they can be tested for their ability to inhibit BMP6.

The ability of an antibody to bind to BMP6 may be detected by directly labeling the antibody of interest, or the antibody may not be labeled, with binding detected indirectly using various sandwich assay formats known in the art.

In one embodiment, the BMP6-binding antibodies and antigen-binding fragments thereof of the invention block or compete with the binding of a reference BMP6-binding antibody to a BMP6 polypeptide. They may be fully human BMP6-binding antibodies as 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 a reference antibody indicates that the BMP6-binding antibody, when assayed, binds to the same or a similar epitope as a given reference antibody, or to an epitope that is located sufficiently close to the epitope bound by the reference BMP6-binding antibody. Such antibodies are particularly likely to possess 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 competitive 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 the BMP6 polypeptide. A test antibody competes with the reference antibody for specific binding to the antigen if an excess of the test antibody significantly inhibits binding of the reference antibody. Substantial inhibition means that the test antibody reduces specific binding of the reference antibody, typically by at least 10, 25, 50, 75, or 90%.

There are a number of known competitive binding assays that can be used to assess the competition of an antibody with 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-linked immunosorbent assay (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 label-assisted assay, solid-phase direct label-assisted sandwich assay (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 label-using direct RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82, 1990). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either, unlabeled test BMP6-binding antibody, and labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of test antibody. Typically, the test antibody is present in excess. Antibodies identified by competitive analysis (competing antibodies) involve binding of antibodies to the same epitope as that bound by the reference antibody and binding of antibodies to an adjacent epitope that is close enough to the epitope bound by the reference antibody to cause steric hindrance.

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

- Isotype-specific ELISAs can be performed to detect binding antibodies. For example, wells of a microtiter plate can be coated with 1 μg/ml of 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 to determine the isotype of the purified antibody.

Flow cytometry can be used to demonstrate the binding of monoclonal BMP6-binding antibodies to live cells expressing the BMP6 polypeptide. Briefly, BMP6-expressing cell lines (grown under standard growth conditions) can be mixed with varying concentrations of BMP6-binding antibody in PBS containing 0.1% BSA and 10% fetal bovine 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 for primary antibody staining. Samples can be analyzed on a FACScan cytometer using forward and side scatter parameters for single-cell sorting. Alternatively, fluorescence microscopy can be used (in addition to or instead of flow cytometric analysis). Cells can be stained in the same manner as described above and examined using fluorescence microscopy. This method allows for visualization of individual cells, but may have reduced sensitivity depending on the antigen density.

The BMP6-binding antibodies and antigen-binding fragments thereof of the invention can also be tested for reactivity with a BMP6 polypeptide or antigen fragment by 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. Following electrophoresis, the separated antigens are transferred to nitrocellulose membranes blocked with 10% fetal calf serum and treated with the test monoclonal antibodies. Binding of human IgG can be detected using an alkaline phosphatase conjugate of anti-human IgG antibody and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

Examples of functional analyses are also described in the examples section below.

Prophylactic and therapeutic uses

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 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.

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

In one embodiment, the present invention relates to methods of 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 in (e.g., associated with) chronic kidney disease (CKD), anemia in cancer, anemia in inflammation, erythropoiesis-stimulating drug (ESD)-resistant anemia (e.g., erythropoietin (EPO)-resistant anemia, ESD-hyporesponsive anemia (e.g., EPO-hyporesponsive 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 the 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 (e.g., anemia of chronic disease) is ESP (e.g., EPO) resistant anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is ESP (e.g., EPO) hyporeactive anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a patient on chronic hemodialysis (HD). 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 relates to 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 according to the invention, wherein said disease or disorder is functional iron deficiency anemia. In an embodiment, the patient is a chronic hemodialysis patient treated with ESP (e.g., EPO). In an embodiment, the patient is a chronic hemodialysis patient with chronic kidney disease treated with ESP (e.g., EPO).

In a particular embodiment, the present invention relates to methods of 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 of 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 anemia in chronic kidney disease. In an embodiment, the anemia is ESP (e.g., EPO) resistant anemia. In an embodiment, the anemia is ESP (e.g., 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 patient on chronic hemodialysis (HD). 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 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 chronic hemodialysis patient treated with ESP (e.g., EPO). In an embodiment, the patient is a chronic hemodialysis patient with chronic kidney disease treated with ESP (e.g., EPO).

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 administration of ESP (e.g., EPO) doses to 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 (e.g., anemia of chronic disease) is ESP (e.g., EPO) resistant anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is ESP (e.g., EPO) hyporesponsive anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a patient on chronic hemodialysis (HD). 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 ESP (e.g., EPO) administration to a patient by administering to the patient an effective amount of an antibody or antigen-binding fragment thereof according to the invention, wherein said patient has functional iron deficiency anemia. In this embodiment, the patient is a chronic hemodialysis patient treated with ESP (e.g., EPO). In this embodiment, the patient is a chronic hemodialysis patient with chronic kidney disease treated with ESP (e.g., EPO).

In a particular embodiment, the present invention relates to a method for reducing the need for administration of ESP (e.g., EPO) dosages to 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 suffers from 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 (e.g., anemia of chronic disease) is ESP (e.g., EPO)-resistant anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is ESP (e.g., EPO)-hyperreactive anemia. In an embodiment, the anemia (e.g., 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 failure.

In a particular embodiment, the present invention relates to methods for reducing the need for ESP (e.g., EPO) administration to 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 chronic hemodialysis patient treated with ESP (e.g., EPO). In an embodiment, the patient is a chronic hemodialysis patient with chronic kidney disease treated with ESP (e.g., EPO).

In a particular embodiment, the present invention relates to a method for reducing the need for administration of iron doses (e.g., 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 suffers from 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 (e.g., anemia of chronic disease) is ESP (e.g., EPO) resistant anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is ESP (e.g., EPO) hyporeactive anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a patient on chronic hemodialysis (HD). 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 iron administration (e.g., intravenous iron) in a patient by administering to the patient an effective amount of antibodies and antigen-binding fragments thereof according to the invention, wherein said patient has functional iron deficiency anemia. In this embodiment, the patient is a chronic hemodialysis patient treated with ESP (e.g., EPO). In this embodiment, the patient is a chronic hemodialysis patient treated with ESP (e.g., EPO) with chronic kidney disease.

In a particular embodiment, the present invention relates to a method for reducing the need for iron administration doses (e.g., 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 suffers from 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 (e.g., anemia of chronic disease) is ESP (e.g., EPO) resistant anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is ESP (e.g., EPO) hyporeactive anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a patient on chronic hemodialysis (HD). 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 iron administration (e.g., intravenous iron) 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 chronic hemodialysis patient treated with ESP (e.g., EPO). In an embodiment, the patient is a chronic hemodialysis patient treated with ESP (e.g., EPO) with chronic kidney disease.

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

- 54 039579 (e.g., EPO) is an ESP (e.g., EPO) hyporeactive anemia. In an embodiment, the anemia (e.g., anemia of chronic disease) is iron deficiency anemia. In embodiments, including any of the above embodiments, the patient is a patient on chronic hemodialysis (HD). 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 of mobilizing sequestered iron by administering to a patient an effective amount of antibodies and antigen-binding fragments thereof according to the invention.

In an embodiment, the present invention relates to 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 for improving (e.g., increasing) the hemoglobin level in a patient with anemia, while reducing the need for administration of doses of erythropoietin and/or an iron preparation (e.g., an intravenous iron preparation), comprising administering to the patient an antibody or an 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 comprises improving the level to a level established in a clinical practice guideline, for example, Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidney inter., Suppl. 2012; 2: 279-335, the contents of which are hereby incorporated by reference in their entirety. In an embodiment, improving the hemoglobin level comprises improving the hemoglobin level to at least about 11.0 g/dL, such as to a level of from about 11.0 to about 12.5 g/dL. In an embodiment, improving the hemoglobin level comprises improving the hemoglobin level to at least 11.0 g/dL, such as to a level of from 11.0 to 12.5 g/dL.

In an embodiment, the present invention relates to a method for improving (e.g., increasing) the hemoglobin level in a patient with anemia, while reducing the need for administration of doses of erythropoietin and/or an iron preparation (e.g., an intravenous iron preparation), 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 comprises improving the level to a level established in a clinical practice guideline, such as the 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 comprises improving the hemoglobin level to at least about 11.0 g/dL, such as to a level of about 11.0 to about 12.5 g/dL. In an embodiment, improving the hemoglobin level comprises improving the hemoglobin level to at least 11.0 g/dL, such as to a level of 11.0 to 12.5 g/dL.

In an embodiment, the present invention relates to a method for maintaining hemoglobin levels in a patient with anemia by reducing the need for administration of doses of erythropoietin and/or an iron preparation (e.g., an intravenous iron preparation), comprising administering to the patient an antibody or an 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 comprises improving the level to a level established in a clinical practice guideline, for example, Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidney inter., Suppl. 2012; 2:279-335, the contents of which are hereby incorporated by reference in their entirety. In an embodiment, improving the hemoglobin level comprises improving the hemoglobin level to at least about 11.0 g/dL, such as to a level from about 11.0 to about 12.5 g/dL. In an embodiment, improving the hemoglobin level comprises improving the hemoglobin level to at least 11.0 g/dL, such as to a level of 11.0 to 12.5 g/dL.

In an embodiment, the present invention relates to a method for maintaining hemoglobin levels in a patient with anemia by reducing the need for erythropoietin and/or iron (e.g., IV iron) dosages, 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 comprises improving the level to a level established 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 comprises improving the hemoglobin level to at least about 11.0 g/dL, such as to a level of from about 11.0 to about 12.5 g/dL. In an embodiment, improving

- 55 039579 hemoglobin level includes an improvement in the hemoglobin level to at least 11.0 g/dL, such as to a level of 11.0 to 12.5 g/dL.

In one embodiment, the isolated antibody or antigen-binding fragment thereof described in Table 1 may be administered to a patient in combination with a therapeutic method or procedure, such as those described herein or known in the art. Such a method or procedure includes, but is not limited to, administration of a therapeutically effective amount of ESP (e.g., EPO), erythropoietin, or an iron preparation, and blood transfusion. Treatment is typically continued intermittently for a week, a month, three months, six months, or a year. In some patients, treatment is administered for the remainder of the patient's life.

When administering the therapeutic agents of the present invention together with another agent, the two agents can 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 (e.g., ESP, erythropoietin, iron supplementation, blood transfusion). In other aspects, the binding molecule is administered in combination with surgical treatment.

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

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

Andriopoulos et al. 2009 Nat. Genet. 41: 482-487;

Arndt et al. 2010 Gastroent. 138: 372-382;

Barnes et al. 1995 World J Urol. 13: 337-343;

Camaschella et al. 2009 Nat. Genet. 41: 386-388;

Celement et al. 1999 Int. J Cancer 80: 250-256;

Corradini et al. 2011 Hepatol. 54: 273-284;

Crews et al. 2010 J Neuro. 30: 12252-12262;

Dai et al. 2005 Cancer Res. 65: 8274;

Darby et al. 2007 J. Pathol. 214: 394-404;

Hadziahmetovic et al. 2011 179: 335-348;

Handy et al. 1997 Cancer Res. 57: 4427;

Haudenschild et al. 2004 Cancer Res. 64: 8276;

Hee et al. 2008 J. Orth. Res. 27: 162-168;

Herrera et al. 2009 BMC Cell Biol. 10:20;

Inagaki et al. 2005 Endocrin. 147: 2681-2689;

Jung et al. 2008 Stem Cells 26: 2042-2051;

Kaiser et al. 1998 J. Invest. Derm. Ill: 1145-1152;

Kautz et al. 2011 Haematol. 96: 199-203;

Khalaf et al. 2012 Euro. J. Endocrin. 168: 437-444;

Kochanowska et al. 2002 Exp. Biol. Med. 227: 57-62;

Li et al. 2006 Int. J. Med. Sci. 3: 97-105;

Maynard et al. 2011 Blood 118: 747-756;

Pederson et al. 2008 Proc. Natl. Acad. Sci. USA

105: 20764-69;

Plant et al. 2002 J. Bone Min. Res. 17: 782-790;

Schluesener et al. 1994 Atheroscl. 113: 153-156;

Schluesener et al. 2004 GLIA 12: 161-164;

Shi et al. 2009 Fert. Steril. 92: 1794-1798;

Varley et al. 1996 Exp. Neuro. 140: 84-94;

Wang et al. 2007 Mol. Cell. Neurosci. 34: 653-661;

Zhang et al. 2006 Neurosci. 138: 47-53;

US Patent 8,795,665; And

WO 2010/056981;

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

Various siRNAs to BMP6 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 result in synergistic results (e.g., a greater reduction in BMP6 activity than 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 another BMP6-associated disease, as described above, using a BMP6-binding antibody of the invention and an anti-anemia 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) from either a person suffering from a disease or disorder, or at risk of developing a disorder associated with anemia.

Diagnostic assays, such as competition assays, rely on the ability of a labeled analogue (radioisotope tracer) to compete with the analyte in the test sample for a limited number of binding sites on a common binding partner. The binding partner is typically insoluble before or after competition, after which the radioisotope tracer and analyte bound to the binding partner are separated from the unbound radioisotope tracer and analyte. This separation is accomplished by siphoning (where the binding partner has been previously insoluble) or centrifugation (where the binding partner has been precipitated after the competitive reaction). The amount of analyte in the test sample is inversely proportional to the amount of bound radioactive tracer, as measured by the amount of tracer substance. Dose-response curves with known amounts of analyte are constructed and compared with test results to quantify the amount of analyte present in the test sample. These assays are called ELISA systems when enzymes are used as detectable markers. In the assay format of this invention, competitive binding between antibodies and BMP6-binding antibodies results in binding of BMP6, preferably BMP6 epitopes according to the invention, which corresponds to the level of antibodies in the serum sample, more particularly neutralizing antibodies in the serum sample.

A significant advantage of this assay is that it directly measures neutralizing antibodies (i.e., antibodies that interfere with BMP6 binding, particularly to its epitopes). This assay, particularly in the form of an ELISA test, has significant applications in clinical settings and routine blood testing.

In clinical diagnosis or monitoring of patients with anemia-related disorders, detection of BMP6 proteins compared to levels in a corresponding biological sample from a healthy patient indicates a patient with anemia-related disorders.

In vivo diagnostics or imaging are described in US2006/0067935. Briefly, these methods typically involve administering to a patient a diagnostically effective amount of a BMP6-binding molecule operably linked to a marker or label that can be detected noninvasively. The antibody-marker conjugate is allowed sufficient time to localize and bind to BMP6. The patient is then imaged with a detection device to detect 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 elevated levels of BMP6 proteins or protein combinations compared to a healthy individual without anemia indicates a predisposition to and/or the occurrence of anemia-related disorders. These aspects of the invention are also intended for use in tissue imaging methods 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 prediction), in order to thereby carry out preventive 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 dysregulated 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, leading to prophylactic treatment of an individual before 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 to thereby select suitable therapeutic agents.

- 57 039579 or prophylactic agents for an individual (referred to herein as pharmacogenomic studies). Pharmacogenomics allows for the selection of agents (e.g., medications) for the therapeutic or prophylactic treatment of an individual based on the individual's genotype (e.g., the genotype of an individual being tested to determine the individual's ability to respond to a specific agent).

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

Pharmaceutical compositions

The invention relates to pharmaceutical compositions comprising a BMP6-binding antibody or a binding fragment thereof together with a pharmaceutically acceptable carrier. The compositions may additionally contain one or more other therapeutic agents that are suitable for the treatment or prevention of a BMP6-associated disease (e.g., 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 substances and absorption delaying substances, and the like substances that are physiologically compatible.

The pharmaceutical composition according to the present invention can be administered by a variety of routes known in the art. The route and/or method of administration vary depending on the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administration can be performed near the target site.

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

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

The pharmaceutical compositions of the invention can be prepared according to methods 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. The pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or an effective dose of BMP6-binding antibody is used in the pharmaceutical compositions of the invention. BMP6-binding antibodies are formulated into pharmaceutically acceptable dosage forms by standard methods known to those skilled in the art. Dosage regimens are adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally reduced or increased according to the exigencies of the therapeutic situation. Parenteral compositions are most preferably formulated in unitary dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable for use as single doses for patients undergoing treatment; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

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

The physician or veterinarian may begin administering doses of the antibodies and antigen-binding fragments thereof according to the invention, used in the 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 doses of the compositions of the present invention for the treatment of an allergic inflammatory disease described in the present invention vary depending on many different factors, including the route of administration, the target site, the psychological state of the patient, whether the patient is a human or an animal, other drugs used, and whether the treatment is prophylactic or therapeutic. Doses used in treatment must be titrated to optimize safety and effectiveness. When administering the antibody 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. When administering the antibody intravitreal, the dose varies from about 0.0001 to about 10 mg. An example of a treatment regimen includes systemic administration once every two weeks or once a month, or once every 3-6 months.

The antibody is typically administered multiple times. Intervals between doses may be weekly, monthly, or yearly. Intervals may also be irregular, depending on measurements of BMP6-binding antibody levels in the patient's blood. In some systemic administration routes, the dose is adjusted to achieve plasma antibody concentrations of 1-1000 μg/mL, and in some routes, 25-500 μg/mL. Alternatively, the antibody may be administered as a sustained-release formulation, which requires less frequent administration. The dose and frequency of administration vary depending on the half-life of the antibody in the patient's body. Generally, 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. For prophylactic use, a relatively low dose is administered at relatively long intervals over an extended period of time. Some patients continue treatment for life. Therapeutic applications sometimes require relatively high dosages at relatively short intervals until disease progression is slowed or halted, and preferably until the patient demonstrates a partial or complete reduction in the severity of symptoms. After this, the patient may be prescribed 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 from 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 from about 0.001 to about 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 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 certain 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 intravenous infusion. In embodiments, the infusion is carried out for 30-60 minutes. In embodiments, the infusion is carried out for about 30-60 minutes.

Examples

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

Example 1.

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

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

Original clone NOV0442 (VH3_3-15, Vll_le) RTM removal + FW-LCDR2 recovery NOV0442_VL[Y49,G50,N51Q] NOV0442_VL [Y49,G50,N51S] A mature clone with a 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 nucleotide base pair BRE BMP-sensitive element BSA Bovine serum albumin Cam Chloramphenicol CDR Complementarity-determining region CKD Chronic kidney disease su Jovanese macaque Yes Dalton DMEM Dulbecco's Modified Eagle's Medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTF Deoxyribonucleoside triphosphate DTT DL-Dithiothreitol es ₅₀ Median effective concentration ECL Enhanced chemiluminescence E. coli Escherichia coli ED TA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay ERO Erythropoietin ESP Erythropoiesis-stimulating drug Fab Antigen-binding fragment of an antibody Yes Crystallizable fragment F-DAS Final assessment of 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 K _D 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 for the night 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 pei Polyethyleneimine REM Protein expression medium Pen/Strep Penicillin/streptomycin PK/PD Pharmacokinetics/pharmacodynamics POD Peroxidase RTM Post-translational modification site RE Restriction enzyme RGA Reporter gene assay rpm Revolutions per minute CT Room temperature RU resonant units With seconds S-DAS Evaluation of the feasibility of development during selection DSN-PAAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SET Equilibrium titration of a solution SP Solid phase SPR Surface plasmon resonance Tp Melting point UV Ultraviolet VH Heavy chain variable domain fragments VL Light chain variable domain fragments

Brief overview.

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 lower quality of life and 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 simultaneously increasing hemoglobin and reducing the need for erythropoiesis-stimulating drugs, such as erythropoietin and intravenous iron supplements. Hepcidin-lowering agents may be an effective strategy for reducing the severity of ESP-hyporesponsive anemia in this group of patients and in other forms of anemia of chronic disease (ACD) characterized by iron deficiency.

The overall goal of the project is to identify and develop anti-BMP6 antibodies capable of reducing hepcidin levels and thereby improving the condition of patients with iron deficiency anemia by reducing the need for erythropoiesis-stimulating drugs (ESDs). In this study, phage display was used to identify the BMP6-specific binding agent.

Preferred BMP6 antibodies meet most or all of the criteria listed below.

The dissociation constants (KD values) of Fab fragments for human BMP6 are below 1 nM.

KD values for the cynomolgus macaque BMP6 antigen are no more than 5 times weaker than those for human BMP6.

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

Selectivity for human BMP6 over human BMP5 and human BMP7 is greater than 100-fold.

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. The BrightGlo assay was performed 24 hours after treatment.

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

Low to moderate development risks. One of the final antibody formats is human IgG1.

Antibodies were generated using a commercially available phage display library as described previously (Knappik et al. 2000 J. Mol. Biol. 296:57–86, Prassler et al. 2011 J. Mol. Biol. 413:261–278), which utilizes phage surface Fab display technology (Rothe et al. 2008 J. Mol. Biol. 376:1182–1200). Sorting and initial ELISA screens were performed on hBMP6 (R&D Systems). ELISA screening of protein-binding hits resulted in the identification of hBMP6-specific binders. Fab antibody fragments were then tested for interspecies 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. BMP6-specific activity of Fab fragments was also assessed 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 results of this functional analysis, combined with the profiles obtained during the feasibility study, led to the selection of three clones for affinity maturation (NOV0429, NOV0441, and NOV0442).

Clones were randomized in either their LCDR3 or HCDR2, generating two new Fab libraries for each original clone. Solid-phase sorts were performed using hBMP6 (Peprotech), as well as liquid-phase sorts using randomly biotinylated hBMP6 (Peprotech). MSD-SET screening of E. coli Fab lysates, combined with a specific ELISA for hBMP5 and hBMP7 proteins, led to 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 assess their hBMP6-specific activity.

Mature antibody clones with the desired properties and good development feasibility profiles were selected for engineering (removal of potential PTM regions and sequence modification according to germline). The 28 resulting variants were generated as hIgG1 by microscale expression and characterized as described previously.

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

ELISA screening on directly antigen-coated plates.

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

Primary screening was performed using 384-well Maxisorp plates 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 the top hits was performed using 96-well Maxisorp plates coated with hBMP6 RD (1.5 μg/ml in 50 mM citrate buffer, pH 4.7) and 96-well Maxisorb 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 hours with 5% skim milk in PBS. Fab-containing E. coli lysates were added, and binding was allowed to proceed for 2 hours at RT. To detect bound Fab fragments, the plates were washed five times with TBST, and an AP-conjugated anti-human F(ab') ₂ antibody was added at a 1/5000 dilution. After incubation for 2 hours at RT, the plates were washed five times with TBST, and AttoPhos substrate was added according to the manufacturer's instructions. The plates were scanned in an ELISA spectrophotometer 10 minutes after substrate addition.

SET screening after affinity maturation.

Affinity comparisons were performed in principle as described below. To evaluate 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.

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

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

In vitro tests.

Evaluation of selectivity and cross-reactivity in ELISA.

To determine the interspecies cross-reactivity of antibodies against BMP6, recombinant human and mouse BMP6 proteins were coupled on a plate, and the antibodies' ability to bind the recombinant proteins was determined using ELISA. To assess selectivity, binding to the closest human homologues, BMP5 and BMP7, was also assessed.

Antigen reagents were coated onto ELISA plates by direct immobilization at a concentration of 3-5 μg/ml o/n at 4°C. For coating, 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 coated 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. Subsequent experiments were 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 purified Fab or IgG samples at a concentration of 1 μM and 0.2 mM, respectively (in PBST buffer).

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

Affinity assessment.

ELISA binding curves.

The hBMP6 antigen (Peprotech) was applied to 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 following day, the antigen solution was removed, and the plates were washed three times with 100 μl of TBST. Each well was then blocked with 100 μl of 5% milk in TBST for 2 h 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 dilution series 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 h at RT, the plates were washed three times again, and bound Fabs/IgGs were detected by adding 30 μl of secondary AP-conjugated anti-human IgG antibody F(ab2) at a 1:5000 dilution. After 1 h at RT, the signal was developed by adding 30 μl of AttoPhos substrate according to the manufacturer's protocol, and the plates were immediately analyzed using an excitation wavelength of 430 nm and an emission wavelength of 535 nm on an ELISA plate spectrophotometer.

Solution equilibrium titration (SET) method for determining K _D using the Sector Imager 6000 (MSD).

Affinity determination in solution was performed essentially as described in the literature (Friquet et al., 1985). To improve the sensitivity and accuracy of the SET method, it was adapted from classical ELISA to ECL-based technology (Haenel et al., 2005).

mg/mL of goat anti-human IgG fragment ₂ specific antibody (Fab) 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 PBS containing 0.5% (w/v) BSA and 0.02% (v/v) Tween20 as the assay buffer. Unlabeled antigen was diluted in 2n increments, starting with a concentration at least 10-fold higher than the expected KD. Wells without antigen were used to determine Bmax values; wells containing only assay buffer were used to determine background. After adding the appropriate amount of binding agent (antibody concentration corresponding to or lower than 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 the 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 appropriate 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 : total Fab concentration applied;

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

B _max : maximum Fab signal without antigen;

K _D : affinity.

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

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where [IgG]: total IgG concentration applied;

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

Bmax: maximum IgG signal without antigen;

KD: affinity.

Experimental parameters.

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

Measuring ForteBio Octet kinetics.

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 equilibrated at 25°C in a thermostatted chamber. The cycle was initiated 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 purified Fab sample. A Fab concentration of 0 nM was used to determine 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. Data were processed automatically using Octet User Software version 3.0.

BiaCore kinetics measurement.

Measurements were performed using purified Fab and IgG samples, as well as human BMP6 and BMP7 as antigens.

Epitope sorting using ELISA.

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

IgG antibodies against BMP6 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 h at RT, then washed twice with TBST.

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

The plate was washed four times with TBST, and His6-POD antibody conjugate (His6 is 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 five times with TBST. Chemiluminescent ELISA substrate solution was added to each well, and luminescence was read without incubation using a Tecan plate spectrophotometer.

In vitro activity in the reporter gene assay (RGA).

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

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

Assessment of non-target activity.

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

After incubation with anti-hBMP6 antibodies at a concentration of 5 μg/ml, bound antibodies were detected using DyLight649 F(ab') _2- conjugated goat specific antibody.

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

The internal control hIgG signal was set to 100%; non-target activity was normalized to hIgG; a hit was considered positive if the signal was equal to or greater than 4% (the cutoff value corresponded 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 constructed IgG.

Engineered anti-hBMP6 IgGs (with germline sequence modification and PTM deletion) were produced at 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 parent clone.

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

Table 2

ELISA specificity of engineered IgG antibody clones; the 3 primary antibodies are highlighted in gray.

Constructed IgG IDs IgG (0.2 μM) Specificity in ELISA: Signal vs. background ЬВМРб 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 30 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 11 1 1 NOV0960 57 16 8 5 1 NOV0949 56 18 15 2 1 NOVO963 56 21 4 1 1 NOV0950 65 19 11 2 1 NOV0951 Λ 65 .24 WEEEEEEEE! .: 6 .. 1 NOV0964 61 20 4 2 1 NOVO952 72 25 9 2 1 NOVO965 30 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 30 6 2 1 NOV0958: ' 59 . .32 . 7 . ' 1 1 Control IgG 61 18 14 4 1

Activity in a reporter gene assay (RGA).

Microscale engineered IgG antibodies against hBMP6 (with germline sequence modification and PTM deletion) were tested as described above. The results are shown in Table 3.

Engineered IgG variants of NOV0429 showed 3-5-fold improved BMP6 activity, but also acquired agonistic activity to all other BMP proteins.

Engineered IgG variants of NOV0441 acquired some cross-reactivity to 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 constructed IgG antibody clones in RGA; IC ₅₀ values are given in [μg/ml].

NOV0951, NOV0954 and NOV0958 were selected as primary 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 \0.21.0

N. G/.K.'.L !>,(:,·{,Ц

0, 05 0,3 ^oFToTs ι,ο

1, o

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 >2 ^r 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 germline modification and PTM removal, 23 engineered anti-hBMP6 antibody variants from 18 mature clones were subjected to a third feasibility study.

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

Other clones were noted to have an intermediate risk profile due to their low titer productivity (NOV0957 and NOV0960, HCDR2 derivatives of the parent antibody NOV0442).

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Table 4

Overview of S-DAS results of 3 anti-BMP6 antibodies (23 engineered hIgG1s); NOV0951, NOV0954, and NOV0958 were selected as primary antibodies

NOV ID NOV936 NOV937 NOV938 NOV942 NOV943 (backup) NOV944 NOV945 (backup) NOV946 (backup) NOV951 (primary) NOV953 NOV954 (primary) NOV955 NOV956 NOV957 NOV958 (final primary) NOV959 NOV960 NOV961 (backup) NOV962 No. 8·! 5 & 0 0 o I I I I ф £ и 0 S о £ X - к g * * £ § 2 в а о о 2 х < ф * & no no 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 low high low low low low low low medium low low medium low low 3 in F y F I low low low high low high low low low low low low low low low low low low low low low 4) In 0 0 X η X in X >> h 0 £ low low low low low low low low low low low low low medium low low medium low low x 0 £ in X φ -© φ X f 0 § c. f in 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 V 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 Ф Я XX Ф ч sr „ -¾ « ^Н Л и' в Ен 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 O 0 3 5 « 0 _ Θ· „ o o & and Я сч Й 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 Ya n V X U 0 l X o High tendency to aggregation High tendency to aggregation The titer may not be relevant; a repeat may be performed if necessary. The titer may not be relevant; a repeat may be performed if necessary. 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 that are recommended to be removed (NG, NS,

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

95% average IgG monomer content is 90-95% high IgG monomer content is below 90%; the candidate does not meet the general DAS criteria (>90%) low productivity 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 below 5 mg/L; the candidate does not meet the general DAS criteria (>5 mg/L) pi >8.2 <8.2; indicates some risk; aggregation/difficulties in dosage form manufacture may occur Tp (pH 7.4) >68°C <68°C; indicates some stability risk; aggregation/difficulties in dosage form manufacture may occur hydrophobicity >0.8 M (NH ₄ ) ₂ SO ₄ <0.8 M (NH ₄ ) ₂ SO ₄ ; indicates some risk of aggregation/may cause difficulties in manufacturing dosage forms, high viscosity, precipitation

Assessment of non-target activity.

The lead candidates NOV0951, NOV0954, and NOV0958 were tested as purified hIgG1s for off-target binding on a Protagen UNIchip microarray coated with 384 purified extracellular or secreted proteins, as described above. All showed low off-target activity (<10 hits), which can be considered non-critical.

Table 5

A review of protein chip results for 3 major antibodies

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

(*): Non-target activity normalized to 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 a feasibility study, it was decided to select the engineered candidates NOV0951, NOV0954, and NOV0958 as the primary antibodies. Furthermore, all of them demonstrated an excellent specificity window for hBMP6 compared to antibody 676. Engineered candidates NOV0961, NOV0943, and NOV0945 were considered as backup antibodies. Table 6 presents the family tree of the final candidates.

Table 6

Family tree of selected primary and reserve antibodies against hBMP6

Original clone, ID (skeleton) NOV0442 Removing the RTM and replacing the frame NOV0442_ Mature clone, ID (mature CDR) NOV0787 (HCDR2) Clone ID with Germline Variation 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 generate antibodies that inhibit BMP6 signaling and thus be suitable for the treatment of anemia of chronic diseases.

Thus, three different protein-based sorting strategies were employed. Primary ELISA hits were primarily detected in solid-phase sorting pools, and after RGA activity analysis, 12 antibodies were shown to inhibit hBMP6 signaling. Three antibody clones (NOV0429, NOV0441, and NOV0442) obtained from sorting with subcode 2023.5 demonstrated BMP6-specific activity and good developmental feasibility and were therefore selected for affinity maturation.

For each antibody clone, two libraries were generated, either with LCDR3 or with randomized HCDR2. Three different maturation sorting strategies were used. 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 deletion of a potential deamidation site in LCDR2, scaffold replacement, and germline sequence alteration. This resulted in the production of antibodies NOV0951, NOV0954, NOV0958, and NOV0961, which were shown to possess similar binding properties as their corresponding non-mutated mature precursors.

Mature clones derived from the NOV0441 family were germlined, resulting in antibodies NOV0943, NOV0945, and NOV0946, which were shown to have increased cross-reactivity with BMP5 compared to their respective non-mutated mature precursors.

Given their ability to inhibit BMP6 signaling with limited cross-reactivity to other BMP proteins and their favorable developmental feasibility profile, NOV0951, NOV0954, and NOV0958 were produced in higher quantities and subsequently evaluated for their utility as a therapeutic agent for the treatment of EPO-resistant (iron deficiency) anemia. A mouse model of EPO-resistant anemia was used to determine their in vivo efficacy.

The primary antibodies NOV0951, NOV0954, and NOV0958 underwent final feasibility assessment. The in vivo suitability of the three primary antibodies was assessed by determining their PK profiles in rats.

Table 7 presents the properties of the final lead candidates that met the antibody requirements defined at the initial stage of the project.

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Table 7

Properties of selected primary antibodies against hBMP6 Property CSP Criteria Major 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 cell-based assay (RGA). The criteria are met - all antibodies ~1000x RGA selectivity Indistinguishable (undetectable) activity against BMP2 or 5 In vitro activity 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 In vivo activity In the EPO-resistant anemia model of inflammation, therapeutic treatment: - restores the response to EPO. - significantly accelerates the recovery of hemoglobin (HGB) or hematocrit (HCT). For Antibody 5, Antibody 6, and Antibody 7, a single dose of 2 mg/kg restores the EPO response and increases HGB by >2.0 g/dL at 1 week. Evaluation of the feasibility of development The final DAS assessment was successfully completed. Antibody 5 and Antibody 7 meet the development criteria. Antibody 6 is not feasible for development due to instability in cynomolgus macaque serum.

Example 2. In vitro and in vivo activity and PK/PD of antibodies against BMP6.

Materials.

The tested compounds were antibodies 5, 6, and 7 (Table 8) at a concentration of ~8 mg/ml in 50 mM citrate buffer, pH 7.0, 150 mM NaCl, which were diluted in PBS before administration to animals. Male C56BL/6 mice or Sprague-Dawley rats were used (Table 9).

Table 8

Properties of antagonist antibodies to BMP6

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

Table 9

Animal characteristics

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

For BMP reporter gene assays, a lentiviral vector containing the BMP-responsive element BRE [Korchynskyi et al. 2002. J. Biol. Chem. 277:4882–91] in the promoter, which directs firefly luciferase from pGL4-BRE2-Luc2, was constructed. The lentiviral vector was used to stably transfect 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 the United States Department of Agriculture, Animal and Plant Health Inspection Service, National Veterinary Services Laboratories, Ames, Iowa. Brucellosis ring test antigen contains a suspension of killed, stained cells of B. abortus strain 1119-3 in phenol-treated buffer. The concentration in each 60 mL vial is approximately 10 ⁹ particles/mL. A stock of 5 × 10 ⁹ was washed and prepared as follows. First, 60 mL vials were removed from the refrigerator and mixed thoroughly. Then, 500 mL of VA was transferred to 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 obtain a stock of ^5x109 particles/ml, which was aliquoted and frozen at -80°C.

Animal keeping conditions.

Animals were housed in Micro-Isolator hard-bottomed communal cages during the acclimatization and study periods. Animals were maintained on a standard light cycle of 12 hours dark, 12 hours light (lights on at 6:30 a.m., lights off at 6:30 p.m.) at a room temperature of 2123°C and humidity of 30-70%. During the acclimatization and study periods, animals were allowed access to

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Experimental conditions.

Determination of antibody activity in a BMP reporter gene assay.

In a typical assay, ^0.6 x 104 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 following day, antibodies diluted in PBS were added, followed by BMP6 to a final concentration of 10 ng/ml. The volume was adjusted to 50 µl with serum-free EMEM to give a final serum concentration of 1%. BMP2/4/7 activation was performed in parallel as interim assays. The BrighGlo assay (Promega) was performed 24 h after antibody addition according to the manufacturer's instructions using an Envision plate reader (PerkinElmer). Data were calculated as the percent inhibition for each antibody compared to full reporter activation by the control antibody.

Single dose pharmacokinetic study in rats.

The primary PK study in rats was not designed to determine classical PK parameters with definitive statistical significance, but rather to estimate the serum half-life of the test antibody. Three animals were administered a single intravenous dose of the antibody.

To study the PK/PD dose-response relationship in mice, animals were equally divided into two separate cohorts. Each cohort included mice that received both the vehicle and the compound. One cohort was analyzed on days 2 and 4, while the second cohort was examined on days 6 and 8 after antibody injection. The reason for separating the cohorts was to reduce the need for periodic blood sampling, so that the impact on serum iron levels was kept to a minimum. The animal groups are shown in Table 10.

Table 10

Study design, animal distribution and doses of test drug

Experiment Group Quantity Dose (mg/kg, IV) Frequency FC/FD mouse HlgG testing 5 0.5 One-time 0.05 mg/kg ANTIBODY 6 5 0.05 One-time 0.1 mg/kg ANTIBODY 6 5 0.1 One-time 0.5 mg/kg ANTIBODY 6 5 0.5 One-time Mouse BA anemia False (without VA) 6 0 0 VA, EPO+ control hlgG 6 2 One-time BA, EPO+ ANTIBODY 5 6 2 One-time BA, EPO+ ANTIBODY 6 7 2 One-time BA, EPO+ ANTIBODY 7 6 2 One-time

Induction of anemia during inflammation in mice and therapeutic treatment. ^5x108 VA particles for injection were prepared as follows (example for 10 mice). The initial concentration should be ^2.5x109 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%, a total of 2.2 mL of ^2.5x109 particles/mL is required. Stock 1.1 mL VA + 1.1 mL PBS. Administration of VA 1-8 days before ESP treatment has been shown to result in a weakening of the HGB response after 6-7 days.

C57BL/6 mice were injected with VA (3 x 108 particles/mouse), and serum IL6 levels were measured after 5 hours using ELISA (KMC0061, Life Technologies) to determine the inflammatory response. Animals with IL6 concentrations below the 95% confidence interval of the mean for all VA-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-positive results when including animals that did not have sufficient inflammation with an attenuated response to ESP. Following the exclusion process, mice were injected with antibodies intravenously as indicated on day 6 and received EPO (subcutaneous injection of 100 g/kg darbepoetin alfa, Amgen) at a dose of 100 mg/kg on day 7 relative to VA treatment. The response to ESP and antibody therapy was measured after 6 days.

Pharmacokinetic, pharmacodynamic and endpoint studies.

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

For the study of VA-induced anemia and antibody treatment, the final blood draw was performed in BD Microtainer EDTA-coated tubes at the end of cardiac puncture. Whole blood was used for the complete blood count (CT-) on the 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 differences between groups (p < 0.05 was considered significant) in hematological parameters. Data are presented as mean ± SEM.

Results.

Biological activity of antagonist antibodies to BMP6 in cellular assays of BMP-dependent transcription.

All three antagonist antibodies to BMP6-5, 6, and 7 completely inhibited the bioactivity of recombinant human BMP6-induced BMP reporter (BRE-luc) activity in the human hepatoma cell line Hep3B (IC ₅₀ = 0.4 nM compared with 0.3 nM rhBMP6) and, thus, were active at an Ag/mAb molar ratio of 1:1 or better. The antibodies demonstrated good selectivity for related proteins of the BMP family, including BMPs 2, 5, and 7, with a 500-fold or greater window. See Fig. 1.

Acute pharmacokinetic and pharmacodynamic profiles of BMP6 antagonist antibodies in rats.

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

Serum antibody concentrations were measured over time following a single antibody injection. Samples were collected at 1 and 6 h, 1, 2, 4, 8, 16, and 28 days after administration of the dose (10 mg/kg, intravenously).

Table 11

Main parameters in the primary single-dose PK study in rats

Parameters ANTIBODY 5 ANTIBODY 6 ANTIBODY 7 T _1/2 (days) 9.1 7.8 9.2 C _tach (mcg/ml) 140.7 189.0 146.2 Average retention time (days) 8.6 7.0 6, 9

Total antibody 7 concentrations (free and BMP6-bound) were also measured in rat and cynomolgus monkey sera after single intravenous injections of antibody 7 (10, 3, 1, 0.3, 0.1, and 0.03 mg/kg in rats; 3 mg/kg in monkeys) at the indicated time points using ELISA with an LLOQ of 46 ng/mL (dashed line) in rats and an LLOQ of 0.2 μg/mL (dashed line) in monkeys. The results are shown in Figs. 9 (rat) and 12 (monkey).

Dose-dependent response of serum iron parameters after treatment with anti-BMP6 antibodies in mice and cynomolgus monkeys.

To further determine the dose-dependent response of iron metabolism to anti-BMP6 antibody treatment, naive C57BL/6 mice were administered 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 scaffold, 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 concentrations 2 days after treatment. However, only at the 0.5 mg/kg dose was there a strong long-term effect on iron metabolism, observed for up to 8 days after injection. See Fig. 3. These results suggest that dose-dependent saturable neutralization of the target can be readily achieved with potent antagonist anti-BMP6 antibodies.

See Fig. 3, dose-dependent effect of antibody 6 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: The left panel represents the quantitative analysis of serum hepcidin concentration after a single injection of antibody 6 or control human IgG, while the right panel represents the serum iron concentration.

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

The results are shown in Fig. 11.

These results indicate that the anti-BMP6 antibodies of the present invention are capable of inducing a dose-dependent increase in serum iron. The effect was sustained and lasted for at least two weeks after antibody administration.

The effect of anti-BMP6 antibody on serum iron parameters was also studied in cynomolgus macaques. Male cynomolgus macaques received a single intravenous injection of antibody 7 at a dose of 3 mg/kg. On the indicated days after injection, serum samples were collected and analyzed 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 the x line. An increase in serum iron and suppression of serum hepcidin were observed 24 hours after antibody administration, with the effect maintained (relative to pre-dose levels) until the end of the 28-day study. These results indicate that the anti-BMP6 antibodies of the invention potently induce hepcidin expression and reduce circulating iron concentrations in non-human primates.

Effect of BMP6 antibodies on red blood cell indices in inflammation-induced ES-resistant anemia in mice.

Experiments were conducted to evaluate the therapeutic utility of anti-BMP6 antibodies in a mouse model of anemia during inflammation. See Fig. 4. Mice treated with Brucella abortus antigen (BA) developed anemia within 6 days. Anemic animals were treated with anti-BMP6 antibody plus recombinant erythropoietin (EPO) starting one day later, and the effect of antibody therapy on anemia development was monitored at day 13 relative to BA. HGB and HCT values decreased from the start of treatment to day 13, with resistance observed with EPO alone. Combination treatment with BMP6 antibody and EPO effectively restored the response to EPO and significantly increased HGB and HCT levels. This effect 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 enzyme due to a deficiency of functional iron in the erythropoiesis compartment.

See Fig. 4, Therapeutic treatment with anti-BMP6 antibody in the ES-resistant anemia of inflammation mouse model. Top: Experimental scheme of the VA-induced ES-resistant anemia of inflammation model. Bottom: Erythropoiesis indices 13 days after VA treatment. HGB: hemoglobin; HCT: hematocrit; RETA: reticulocyte count; RET-HE: reticulocyte equivalent hemoglobin.

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

Example 3. Clinical plan for testing antibodies to BMP6 in humans.

Clinical trial 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, erythropoietin (EPO) and typically require intermittent administration of exogenous EPO and intravenous (IV) iron infusions to maintain their iron levels.

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

In summary, the goal of therapy using isolated antibodies or antigen-binding fragments thereof that bind human BMP6 as described herein is to mobilize sequestered iron, which may then reduce EPO and iron supplementation requirements and increase Hgb levels, which together are expected to improve patient treatment efficacy. This is a first-in-human, single-dose 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 chronic hemodialysis patients. The objective of this study is to evaluate whether therapy using isolated antibodies or antigen-binding fragments thereof that bind human BMP6 will warrant further clinical development in anemia associated with chronic kidney disease.

Research plan.

Study design.

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

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

Part 1.

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

During Part 1, a screening visit will be held to determine patient eligibility for study inclusion (Fig. 6). Eligible patients will be admitted to the study center and re-evaluated for eligibility during the first visit prior to study initiation. All pre-study safety assessment results must be obtained and reviewed prior to dosing.

Figure 6 provides an overview of the design of Part 1 of the study. Patients are asked to report to the study center on Day 1, immediately following their routine dialysis visit. They will then receive an infusion of an antibody or its 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 (e.g., Friday or Saturday), and will occur after the dialysis session on that day. However, if this is not possible, dosing may occur on a dialysis day not preceding the two days before the next dialysis. After dosing, the first two patients in Part 1 will be hospitalized for at least 48 hours for safety and PK/PD assessment. Patients will return to the study center 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 on approximately day 85. Study visits, including all laboratory tests except post-dialysis PK assessments, must occur before the patient's scheduled dialysis visit.

Part 1 will be initiated 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 after a single dose of the isolated antibody or antigen-binding fragment that binds human BMP6, and the transferrin saturation level (TSAT), as discussed in the statistical evaluation section 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 at 1 week post-dose) and an increase in Hgb. If iron is mobilized but Hgb does not increase by at least 0.5 g/dL by 29 days post-dose, clinical data will be analyzed to assess potential confounding factors (e.g., blood loss due to excessive venotomy outside the study). The applicable Investigators and the Sponsor's 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) the results of the preclinical toxicology study. Subsequent cohorts will not receive doses until the Investigators and the Sponsor indicate that the study may

- 75 039579 but it is safe to continue.

Figure 7 shows the dose adjustment algorithm for Part 1. Blood work, including Hgb measurements, will be performed before dialysis. The starting dose will be 0.01 mg/kg. In Part 1, patients are assigned to one of up to 6 unmasked dose cohorts, with up to 6 patients in each. The minimum FAD of the isolated antibody or its antigen-binding fragment that binds human BMP6, as defined above, will be the lowest dose group selected for Part 2. The dose for each subsequent cohort can be increased or decreased, as shown in Figure 7. 7. If the lowest possible dose (0.001 mg/kg) results in a median increase in Hgb>0.5 g/dL, this will be the minimum FAD, and the lower dose selected for Part 2 and the next highest dose evaluated 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 the minimum FAD. If additional dose cohorts are needed in Part 1, these cohorts will be added as described herein.

Each Part 1 cohort will include 6 patients. The first 2 patients in the first Part 1 cohort will receive a dose at least 7 days apart. The timing of subsequent doses for patients in the Part 1 cohort will depend on availability, taking into account the respective center's schedule and resources. All patients in Part 1 will be followed for 12 weeks after dosing.

Part 2.

The objectives of Part 2 are to (a) evaluate safety, PK, PD, and tolerability and (b) determine efficacy based on changes in Hgb in response to a single dose of an antibody that binds human BMP6 compared with 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 with a 1:1:1 randomization into three groups. If in Part 1 the trough FAD is also the highest dose evaluated (0.1 mg/kg), then Part 2 will contain only two groups: trough FAD and placebo. In this case, 40 patients will be randomized into two groups with a 1:1 randomization ratio. The sample size in Part 2 may be adjusted based on the variability in the change from the pre-study Hgb level in Part 1.

Figure 8 shows the study flow chart for Part 2. During Part 2, a screening visit will be held to determine patient eligibility for study inclusion. Eligible patients will be re-evaluated according to the inclusion criteria during the first visit prior to study initiation. All pre-study safety assessment results must be obtained and reviewed prior to dosing.

Patients are asked to report to the study center on Day 1, immediately following their routine dialysis visit. Patients will then receive an infusion of Ab or placebo, as determined at randomization. If possible, dosing should preferably occur on the day of dialysis two days before the next dialysis (e.g., Friday or Saturday) and will occur after the dialysis session on that day. However, if this is not possible, dosing may occur on a dialysis day not preceding the two days before the next dialysis. Patients will return to the study center on Days 4 and 6, and then weekly for follow-up assessments. During subsequent visits, patients undergo standard safety assessments, and PK data will also be collected for 85 days. Study visits may occur after the scheduled dialysis visit to ensure compliance with the patient's dialysis schedule. All patients enrolled in Part 2 will be followed for 12 weeks after the Ab (or placebo) dose.

Selection of the dose of EPO (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's assessment and will be verified against standard treatment recommendations (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 investigator's discretion, in addition to center-based recommendations for controlling Hgb levels above target levels.

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

Patients receiving loading doses of IV iron (100 mg/week) will be excluded from the study. Patients receiving weekly maintenance doses of IV iron (<100 mg/week) may be included in this study. The weekly maintenance dose of IV iron will be maintained at the beginning of the first week of AT dosing.

Iron indices will be monitored during the first week after the AT dose is administered, with emergency iron therapy and IV iron maintenance therapy following standard of care recommendations according to the hemodialysis unit operating protocol. Center protocols will be reviewed as part of the center's assessment and will be reviewed for compliance with standard of care recommendations (KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease 76 039579

Anemia Work Group 2012).

Justification of the research design.

Rationale for a two-stage research design.

The rationale for having two parts in a single patient population is the need to determine the minimum FAD that is safe and effective, while minimizing the number of patients and cohorts exposed to potentially subtherapeutic doses. Part 2 evaluates the efficacy of the minimum FAD and one dose level above the minimum FAD (as defined in Part 1), compared to the placebo group.

Part 1 is designed to evaluate single-dose safety, tolerability, PK/PD, and trough FAD of Ab in an open-label study. The trough FAD will be determined based on each median change in Hgb in the dose cohort over 29 days after Ab dosing. The rationale for defining FAD criteria is that clinically significant responses to EPO may require up to 4 weeks after changing the EPO dose. If Ab mobilizes iron in the target population, this may allow the current EPO dose in patients to exert a more pronounced erythropoietic effect. The 29-day Hgb cutoffs listed in the FAD criteria are based on clinically significant and safe increases in Hgb in response to the EPO dose (~0.5 g/dL over 29 days). The rationale for seeking the trough FAD rather than the maximal effect is that an excessive Hgb response poses a safety risk in this patient population, as reflected by the Hgb target limits in current standard-of-care recommendations (Kidney Disease Improving Global Outcomes (KDIGO) Anemia Working Group 2012). The objectives of the safety and tolerability assessments in Part 1 are to (a) identify safety signals and (b) inform the decision to make a dose adjustment, ensuring that the doses selected for Part 2 (trough FAD + 1 higher dose) are appropriate for further evaluation of safety and efficacy compared with placebo. While Part 1 is overpowered to obtain an objective safety assessment, the placebo arm and larger samples in Part 2 will provide an objective safety assessment at the trough FAD and one higher dose. In addition to safety, tolerability, and PK/PD, Part 2 is designed to evaluate efficacy compared with placebo in a double-blind study. Efficacy assessment 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 provides a quantitative measure 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-in-human (FIH) study will be conducted in patients on chronic hemodialysis (HD) rather than in healthy volunteers (HV). Evaluation of the safety, tolerability, and PK/PD response to anti-human BMP6 antibodies in HV will likely not be translatable to patients on chronic HD for several reasons:

Unlike HV, patients with chronic HD 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 most appropriately 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 efficiently excreted in the urine, resulting in low circulating levels. In contrast, hepcidin is less efficiently and transiently filtered during dialysis, resulting in higher circulating levels in patients with chronic HD (Zaritsky et al. 2010). Furthermore, normal kidneys dynamically regulate endogenous EPO levels, and changes in Hgb in response to Ab may not be apparent. Therefore, the safety and tolerability associated with hepcidin modulation via the BMP6 pathway and the action of Ab on Hgb are most appropriately assessed in patients with chronic HD.

Justification of the target population of patients.

This study was designed to evaluate antibodies against human BMP6 in the setting of EPO-hyporesponsive iron deficiency anemia. Established clinical guidelines (KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease Anemia Work Group 2012) define hyporesponsiveness to EPO as the need for two EPO dose increments, each 50% higher than the stable dose, to maintain a stable Hgb concentration. The proposed inclusion criteria were developed to select stable patients with chronic HD with anemia and clinical indicators of iron limitation: elevated ferritin and low TSAT (TSAT = serum iron/total iron-binding capacity; TSAT closely correlates with serum iron). In addition, when adjusting EPO and IV iron doses, a strict standard of care targeting Hgb, TSAT, and ferritin will be followed. This regimen reduces the risk of exceeding the required Hgb targets, since changes in iron and hematological parameters will continue to be managed according to the standard of care. In addition, patients receiving loading doses of IV iron within 1 week prior to study entry will be excluded. Patients receiving IV maintenance iron may be included (if all other inclusion criteria are met). The rationale for including these patients is that the current standard of care in the United States dictates that Hgb and TSAT should be maintained within narrow limits, and therefore, completely discontinuing maintenance iron therapy to meet the inclusion criteria for the lower limit of TSAT would result in the inclusion of patients with a TSAT risk below 25% who require a course of IV iron in loading doses according to the standard of care. However, eligible patients on IV maintenance iron will maintain their weekly IV iron dose at the beginning of the AT dosing week and will resume IV maintenance iron therapy only when determined by the center's protocol according to the standard of care, based on monitored iron indices.

Justification of dosage/administration regimen, duration of treatment.

Rationale for the initial dose.

The maximum recommended starting dose (MRND) was calculated based on the no-observed-adverse-effect level (NOAEL) in 13-week (14-dose) GLP toxicology studies conducted in rats and cynomolgus monkeys. Animals received weekly intravenous bolus doses of 0.1, 1, 10, and 100 mg/kg. The 1 and 100 mg/kg dose groups (only) were subsequently observed for 16 weeks in rats or 24 weeks in cynomolgus monkeys after administration of the final dose of the isolated antibody or its antigen-binding fragment 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 accumulated iron and the requirement for erythropoiesis. PK parameters for non-clinical species were derived from toxicokinetic (TK) data collected during toxicology studies for IND registration. Corresponding PK parameters in patients were then estimated using allometric scaling, and these parameters were used to predict the concentration of free isolated antibody or its antigen-binding fragment that binds human BMP6 as a function of time in patients for a given dose. When comparing TK data from toxicology studies with model-based PK of Ab in patients, it was suggested that a dose 10 times lower than the NOAEL/HED would be predicted to produce (at least) a 10-fold increase based on Ab concentration.

The peak serum iron levels observed in response to Ab in animal studies may underestimate the predicted iron response to Ab in humans, as HD patients treated with intravenous iron likely have larger tissue iron stores than healthy animals. However, unlike healthy, nonanemic animals, HD patients are expected to utilize released serum iron for erythropoiesis; therefore, animal models may overestimate the duration of the increase in iron levels. The liver pathology observed in the 13-week studies was not observed in the 4-week studies, suggesting that toxicity was due to cumulative serum iron exposure rather than a response to an acute iron release.

To account for expected differences in accumulated iron between non-clinical species and patients, the MRND was also predicted using an analysis modeling serum iron concentrations. The cumulative serum iron exposure that resulted in toxic effects was represented as the area under the iron curve (Fe AUC). In this method, the Fe AUC calculated for the NOAEL dose (0.1 mg/kg) was considered sufficiently safe. The Fe AUC at the predicted MRND in patients was then calculated and compared with the Fe AUC in non-clinical species at the NOAEL. Because the model-based serum iron exposure at the MRND in patients was >10-fold lower than at the NOAEL in non-clinical species, a reduction in the NOAEL/HED with a safety factor of 10 was considered sufficient to estimate the MRND. The predicted MRND was therefore 0.01 mg/kg.

Justification for dose adjustment.

For this study, the maximum test dose (xmax) will be the HED corresponding to the NOAEL in each of the two toxicology studies for IND status: 0.1 mg/kg. The minimum feasible test dose (xmin) is the lowest technically feasible dose based on compatibility studies: 0.001 mg/kg. The MRND (xO) will be assessed according to safety, TSAT, and Hgb criteria (Fig. 7). If dose xO results in a median change in Hgb <0.5 g/dL relative to predose, the selection of x+ (Fig. 7) will be guided by linear extrapolation on the natural logarithmic scale (expecting a sigmoidal dose-response relationship) between xO and xmax. Preliminary doses for this dose escalation are provided above. These preliminary doses may be adjusted based on data review at the 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

- 78,039,579 doses, and a decision will be made whether to refine the protocol by including 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 by including additional cohorts.

If xO instead results in a median change in Hgb >0.5 g/dL relative to the pre-dose level, 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 the minimum 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 in the interval (xmin, xO) on a natural logarithmic scale until the minimum FAD is determined or until 6 doses (cohorts) have been assessed. Preliminary doses in the interval (xmin, xO) are provided above. Such preliminary doses may be adjusted based on data review during an 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 relative to pre-dose levels.

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

Justification for the choice of the comparison drug.

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

Purpose and timing of interim analyses/scheme adaptations.

In Part 1, following completion of the postdose assessment at week 4 in each cohort of 6 patients, an informal interim analysis will be conducted to inform dose adjustment decisions for the next cohort. Safety and PD markers will be reviewed by all members of the dose adjustment team, including the applicable Investigators and representative(s) from the Sponsor. New cohorts will be included only if safety and tolerability are confirmed and if PD conditions are met, as described in Fig. 7. There will be up to 5 informal interim analyses in Part 1. A formal interim analysis is planned after all patients in the final Part 1 cohort have completed the post-dose assessment at week 4 to evaluate the clinical effects of study doses and potentially initiate additional preclinical studies, and may report body temperature, blood pressure, pulse rate, ECG results, blood chemistry, hematological iron indices, EPO resistance index, and adverse events reported through day 29 in the final Part 1 cohort in subsequent clinical trials.

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

Risk and benefit.

The anticipated benefits for patients participating in this study may include a reduction in the need for EPO and IV iron 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 inclusion criteria and careful clinical monitoring of all patients (and hospitalization of the first two patients in Part 1) during the first 48 hours after Ab administration.

Potential risks associated with iron mobilization include: (a) redistribution of iron to tissues and organs such as the spleen, liver, heart, pancreas, and pituitary gland, and (b) slightly increased susceptibility to bacterial infection, particularly in patients with indwelling vascular catheters. Some of the inclusion criteria reduce the risk of complications. Increased liver function tests may be noted due to iron redistribution. Liver function will be monitored in parallel with iron levels and hematological parameters. Exceeding standard-of-care Hgb targets may lead to polycythemia. Hgb monitoring, EPO therapy, and iron therapy may be considered.

HD patients treated with IV iron may have larger tissue iron stores than healthy animals; therefore, the maximum serum iron levels observed in patients treated with an isolated antibody or antigen-binding fragment thereof that binds human BMP6 may exceed those observed in animal studies. However, unlike healthy animals, HD patients are expected to utilize released serum iron for erythropoiesis; therefore, animal models may overestimate the duration of the increase in iron levels. The model-predicted Ab exposure (e.g., Cmax, AUC) at MRND is expected to be 10-fold less than that observed at the NOAEL in preclinical studies. This exposure is not expected to result in serum iron exposure levels (AUC) associated with elevated liver transaminases and single-cell necrosis in the liver observed in preclinical studies. Dose escalation of AT to the NOAEL will occur after the described safety assessments. Clinical experience with patients with chronic iron overload, as well as patients receiving parenteral iron, is unlikely to predict the effects that may result from acute increases in intracellular iron caused by AT. Therefore, the potential risk of AT-induced acute iron toxicity is likely low. Acute iron toxicity can affect the heart, liver, and/or pancreas. Clinical manifestations of acute iron toxicity may include cardiac conduction abnormalities, elevated liver transaminases, and impaired glucose tolerance/hyperglycemia. Severe acute iron toxicity may also include metabolic acidosis, electrolyte disturbances, and neurological manifestations. If acute iron toxicity develops, patients may undergo 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 collected from each patient in the study over 115 days. Additional samples may be collected to monitor any safety data. This is not considered hazardous for this population.

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

Chronic hemodialysis is associated with significant fetal and maternal morbidity and mortality. In a retrospective cohort study comparing women on chronic hemodialysis (267 births) with women who had undergone kidney transplantation (264 births), women on hemodialysis demonstrated a higher incidence of placental abruption, blood transfusion, small fetus, intrauterine fetal death, and maternal death (Saliem et al. 2015). Therefore, women of childbearing potential should use highly effective contraception to prevent pregnancy during administration of the isolated antibody or its 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 clinical symptomatic functional iron deficiency anemia, defined as anemia, in the presence of clinically sufficient iron stores, as determined by ferritin and transferrin saturation levels. Part 1 includes a plan to evaluate up to 36 patients, initially in 6 cohorts (6 patients/cohort). If after 6 cohorts no effects on TSAT and Hgb are observed and there are no safety concerns (as determined by the applicable Investigators and representative(s) from the Sponsor), up to 2 additional cohorts of 6 patients may be included (for a total of 48 patients in Part 1). Part 2 consists of a maximum of 3 groups (2 dose levels selected for further evaluation from Part 1 and a placebo group), with up to approximately 20 patients per group (for a total of 60 patients in Part 2). Thus, a total of approximately 96 patients (up to a maximum of 108) are planned to be included, of whom 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 meeting the inclusion criteria.

Patient selection must be completed with verification of all inclusion criteria during screening and the first visit prior to treatment initiation. A corresponding record (e.g., a control form) of verification of inclusion criteria must be maintained with the original documentation at the study center. Deviation from any inclusion criterion will result in exclusion of patients from 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 before any assessment is performed. If consent cannot be expressed in writing, it must be formally recorded.

- 80 039579 verified and witnessed, ideally through 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. Conducting appropriate hemodialysis at least twice weekly in end-stage renal disease; appropriate is defined as Kt/V>1.2 at the last monthly assessment before screening.

5. Receiving chronic erythropoietin (EPO) therapy according to the dialysis center's anemia treatment protocol. The EPO dose was not increased by 50% or more within 14 days prior to the study. EPO therapy should include only short-acting agents (not darbepoietin) and administered intravenously (not subcutaneously).

6. Hgb>8.5, including Hgb>8.5 and <11.5 g/dL, and not increased by >0.5 g/dL relative to pre-test level in the previous 14 days.

7. Ferritin 200-2000 ng/ml (inclusive) for at least 28 days prior to the start of the study (may include screening).

8. TSAT<30%, at least once, within 90 days before the start of the study, and TSAT<30% at the start of the study.

Exclusion criteria (both parts).

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

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

2. Case of hypersensitivity to the study drug or to 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 episodes of AV fistula thrombosis within 6 months prior to screening.

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

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

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

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

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 indwelling 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 the start of the study.

13. Receipt of a loading dose (100 mg/week) of intravenous iron preparation for 1 week before the start of the study.

14. History of drug or alcohol dependence within 12 months prior to administration of the dose or confirmation of such dependence based on laboratory tests performed during screening.

15. Positive hepatitis B surface antigen test result.

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

17. Women of childbearing potential may be enrolled in this study if they use highly effective contraception for at least 125 days after administration of a dose of 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; periodic abstinence (e.g., calendar, ovulatory, symptothermal, postovulatory methods) and withdrawal are not acceptable methods of contraception), b. male/female sterilization, c. use of oral, injectable, or implantable hormonal contraceptive methods, or

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

Treatment.

Treatment in study.

The investigational therapy in this study is an antibody or antigen-binding fragment that binds human BMP6, such as a fully human IgG1 anti-BMP6 antibody. The antibody is provided as a liquid solution. The stock concentration will be diluted at the center according to the administered dose. The infusion time will be maintained at a relatively constant level across 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 matching placebo (vehicle control). The active substance, the anti-human BMP6 antibody, and placebo will be supplied as liquid in vials. The excipients in the active substance and placebo are identical.

Treatment groups.

In Part 1, patients are allocated to one of up to 6 dose cohorts of 6 patients each. Part 1 is open-label treatment. The starting dose, maximum dose, and rationale for dose adjustments are described above. Preliminary doses for Part 1 are provided in Table 12 (Hgb <0.5 g/dL at MRND) and Table 13 (Hgb >0.5 g/dL at MRND).

Table 12

Pre-dose levels for Part 1

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

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

Table 13

Pre-dose levels for Part 1

For Hgb greater than or equal to 0.5 g/dL at the MRND dose level Preliminary dose Increase of the previous relative to 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 fuck 5 0.0040 mg/kg fuck 6 0.0063 mg/kg fuck

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

The treatments in the study were defined as follows.

A: Single dose of placebo.

B: single dose of anti-human BMP6 Ab at the minimum FAD as defined in Part 1.

C: A single dose of anti-human BMP6 Ab at one dose level above the minimum FAD as defined in Part 1.

Concomitant Therapy All prescription medications, over-the-counter medications, and significant non-pharmacological therapies (including physical therapy and blood transfusions) administered or taken during the period defined in the inclusion criteria, prior to study entry, and during the study should be recorded in the Concomitant Therapies/Significant Non-Pharmacological Therapies sections of the CRF. Therapy records should include details regarding brand name, single dose and unit, frequency and route of administration, dates of initiation and discontinuation, and the reason for therapy.

Efficacy/pharmacodynamics.

Efficacy assessments are defined below. Samples for efficacy assessments will be collected at various time points. Hematology 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 sample collection schedule is deemed suboptimal for the iron-PK relationship, the sample collection schedule may be modified in subsequent cohorts in Part 1.

Set of iron indices.

Anti-human BMP6 antibodies are 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 reticulocyte hemoglobin (CHr). These parameters will be measured in serum using validated assays.

The safety assessments are defined below.

Objective examination.

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

Significant findings present prior to the initiation of the drug study should be included in the screening of relevant information on past/current illnesses in the eCRF. Significant findings obtained after the initiation of the drug study that meet the definition of an adverse event should be recorded during the adverse event screening in the eCRF.

Vital signs.

Body temperature.

Blood pressure (BP).

Pulse, height and weight.

Height.

Body weight.

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

Laboratory research.

Clinically significant deviations in laboratory test results will be assessed against the criteria defining an adverse event and recorded as such if the criteria are met. Repeated assessments are mandatory until the result(s) return to normal or the change is no longer clinically significant.

Hematology.

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

Clinical chemistry.

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

Electrocardiogram (ECG).

PR interval, QRS prolongation, heart rate, RR, QT, QTc. The Fredericia (QTcF) correction formula should be used for clinical decision making.

Pregnancy and fertility assessments.

Pregnancy testing is necessary for all affected women regardless of reported reproductive/menopausal status.

This study will involve serum pregnancy tests. If the result is positive, the patient must discontinue participation.

If performed at screening and study initiation, 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 considered during each intercohort informal interim analysis as part of the dose adjustment assessment during Part 1.

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

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

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

The concentration of free anti-human BMP6 antibodies will be determined using an ELISA assay. The expected lower limit of quantification (LLOQ) is 10 pg/mL.

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

Concentrations of free versus human At.

The BMP6 will be expressed in µg/mL. All concentrations below the LLOQ or missing data will be noted as such in the concentration data lists. Concentrations below the LLOQ will be treated as zero in summary statistics only for concentration data. They will not be included in the calculation of PK parameters.

PK analysis samples remaining after determination of free anti-human BMP6 Ab may be used for research evaluations or other bioanalytical purposes (e.g. cross-validation between different centers, stability assessment).

The following pharmacokinetic parameters will be determined (when possible) using a 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-time data. The linear trapezoidal rule will be used to calculate AUC. The terminal elimination half-life of the antibody or antigen-binding fragment that binds human BMP6 (t1/2) will also be estimated from the data, if possible.

Other assessments.

Immunogenicity.

The ELISA assay will be used to detect antibodies against human BMP6. IgG samples remaining after immunogenicity analysis may be used for research evaluation or other bioanalytical purposes (e.g., cross-validation between different centers).

Research assessments.

Biomarkers objectively measure and evaluate indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions (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. A 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 achieving target Hgb levels.

Based on the above-described biological process, research biomarker assessments include, but are not limited to, hepcidin (measured by LC-MS).

Further research evaluations may explore the potential roles of bone absorption markers and also consider inflammation as a factor contributing to the mechanism of action.

The research objectives are as follows: to evaluate the relationships between hepcidin levels and several key indices, such as iron indices and ERI; to investigate the dynamics between primary and secondary endpoints and exploratory biomarkers in parallel; to evaluate pharmacogenetics; to evaluate immunogenicity.

Sample(s) will be collected at different time(s). Additional information on sample collection, numbering, processing, and transportation 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 adverse effects.

Furthermore, recent advances in genotyping technologies have made whole-genome methods feasible. Whole-genome methods can also be applied to the limited scope of current research, as described above.

Soluble biomarkers.

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

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

Other biomarkers.

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

- 84,039,579 immunogenicity assays (IAAs) will be collected at various time points. The immunogenicity of anti-human BMP6 antibodies will be assessed by measuring antibodies that recognize the anti-human BMP6 antibody.

Sources used

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, Abenhaim HA (2015) Pregnancy outcomes among renal transplant recipients and patients with end-stage renal disease on dialysis. J Perinat Med (Electronic publication ahead of print) http://www.ncbi.nlm.nih.gov/pubmed/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, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this specification pertains.

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

The invention formula is non-limiting and is presented below.

Although specific aspects and claims have been described in detail herein, this has been done by way of example, for illustrative purposes only, and is not intended to limit the scope of the appended claims or the scope of the claimed subject matter of any corresponding future application. In particular, it is contemplated by the inventors of the present invention that various substitutions, changes, and modifications may be made to the present description without departing from the scope and spirit of the description as defined by the claims. The selection of a starting nucleic acid, a desired clone, or a library type is believed to be routine for one of ordinary skill in the art, having knowledge of the aspects described herein. Other aspects, advantages, and modifications are considered to be within the scope of the following claims. Those skilled in the art will know, or will be able to ascertain using no more than routine experimentation, many equivalents to specific aspects of the invention described herein. Such equivalents are intended to be included in the following claims. Clarification of the claimed scope in later filed corresponding applications may be due to limitations of patent laws in various countries and should not be interpreted as a waiver 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. -