Bone-targeting antibodies

Abstract

To provide antibodies modified with bone-targeting peptides, and to provide methods of using them to treat pathophysiological bone degeneration.SOLUTION: Provided are recombinant and chemically conjugated antibodies and fragments thereof modified with one or more poly-aspartate (poly-D) peptides (e.g., a D10 sequence) to improve localization of the antibodies or fragments to bone. Methods of making and using of these antibodies and fragments are also provided. In particular, antibodies that effectively target bone, such as anti-TGFβ antibodies, are disclosed.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 448,763, filed January 20, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on January 11, 2018, is named 022548_WO012_SL.txt and is 75,113 bytes.

[0003] FIELD OF THEINVENTION The present invention relates to antibodies modified with bone-targeting peptides and methods of their use to treat pathophysiological bone degeneration. [Background technology]

[0004] Proper bone development and maintenance is an important factor for normal health. In the average human, bone development occurs until about the age of 20 years, when bone mineral density is typically maximized. Thereafter, bone mineral density can decrease in the absence of proper diet and physical activity. However, normal bone maintenance requires homeostatic bone turnover, in which old bone is removed and replaced with new bone.

[0005] Yet, there are numerous diseases and conditions that can affect bone development and maintenance. For example, bone development is affected in diseases such as osteogenesis imperfecta, which impairs bone strength and makes children's bones weak and prone to fracture. Furthermore, with age, otherwise healthy individuals may experience a lack of homeostatic bone turnover, leading to osteoporosis, which reduces bone density over time, and ultimately to fragile bones and fractures.

[0006] Furthermore, there are certain diseases in which bone health is affected concomitantly with the primary disease and is involved in other concurrent sequelae, such as chronic kidney disease (CKD). CKD is a progressive disease in which renal function declines over time, often resulting in poor bone health and cardiovascular disease associated with altered bone turnover rates. Treatments that improve bone health have been shown to simultaneously reduce concomitant cardiovascular disease. Such reports suggest that normal bone turnover rates may affect, if not cause, other diseases. Thus, improved methodologies for regulating bone development and / or maintenance may have a wide range of direct or indirect effects on improving the health of individuals suffering from a number of different diseases and conditions.

[0007] TGFβ is a member of the transforming growth factor beta (TGFβ) superfamily and is important in bone formation during mammalian development (see Non-Patent Document 1). TGFβ appears to be important for bone homeostasis as well. Interestingly, TGFβ has been shown to be expressed at higher levels in patients with CKD, suggesting that this is a viable target for therapeutic intervention. Systemic treatment of the jck mouse model of CKD with anti-TGFβ antibodies demonstrated a reduction in the high bone turnover rate (Non-Patent Document 2). However, this study did not investigate the extent to which localization of anti-TGFβ antibodies in bone could improve therapeutic outcomes. Given that TGFβ is involved in numerous cellular processes, including DNA damage response, allergic immune responses, and wound epithelialization, to name just a few, a more targeted approach to control TGFβ activity is desirable to minimize potential undesirable side effects. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Chen et al., Int.J.Biol.Sci.8(2):272-88(2012) [Non-Patent Document 2] Liu et al., J. Bone Miner Res. 29(5):1141-57(2014) Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, more precise approaches to modulate TGFβ activity are needed to provide improved therapies for modulating bone development and / or maintenance. [Means for solving the problem]

[0010] Provided herein is an antibody, such as an anti-TGFβ antibody, that effectively targets bone. In a first aspect, the present disclosure provides an antibody, or an antigen-binding fragment thereof, that comprises a heavy chain, a light chain, and one or more polyaspartic acid (polyD) peptides. In a particular embodiment, the antibody or antigen-binding fragment comprises a heavy chain, a light chain, and one or more polyaspartic acid (polyD) peptides linked to the C-terminus of the heavy chain and / or the light chain.

[0011] In one embodiment, the antibody or antigen-binding fragment thereof exhibits at least a two-fold increase in localization to bone compared to an antibody having the same heavy and light chains but lacking one or more poly-D peptides.

[0012] In one embodiment, one or more poly-D peptides are linked to the antibody or antigen-binding fragment thereof by chemical conjugation. In another embodiment, one or more poly-D peptides are linked at the hinge region of the heavy chain. In a further embodiment, one or more poly-D peptides are linked to the N-terminus or C-terminus of the light chain. In yet a further embodiment, one or more poly-D peptides are linked to the antibody or antigen-binding fragment thereof by one or more spacers / linkers (e.g., polyethylene glycol (PEG) spacers and peptide linkers).

[0013] In one embodiment, one or more poly-D peptides are integral with the amino acid sequence of the heavy chain and / or one or more poly-D peptides are integral with the amino acid sequence of the light chain. Poly-D peptides that are "integral" with an amino acid sequence are included in the same polypeptide chain. For example, the integral poly-D peptides are translated from the same RNA strand as the heavy or light chain sequence, encoded in a recombinant DNA plasmid. In one embodiment, one or more poly-D peptides are integral with the N-terminus of the heavy chain and / or one or more poly-D peptides are integral with the C-terminus of the heavy chain. Two or more poly-D peptides are linked in tandem to the N-terminus or C-terminus of the heavy chain, separated by zero, one or more other amino acid residues (i.e., non-aspartic acid amino acids) or by a peptide linker. In a further embodiment, one or more poly-D peptides are integral with the N-terminus of the light chain and / or one or more poly-D peptides are integral with the C-terminus of the light chain. For example, two or more poly-D peptides are linked in tandem to the N-terminus or C-terminus of the light chain, separated by zero, one or more other amino acid residues (i.e., non-aspartic acid amino acids) or by a peptide linker. In one embodiment, the poly-D peptide is attached to the C-terminus of the heavy chain. In another embodiment, the poly-D peptide is attached to the C-terminus of the heavy chain. And a poly-D peptide is attached to the N-terminus of the heavy chain.

[0014] In one embodiment, the light chain does not comprise a poly D peptide, hi another embodiment, the heavy chain does not comprise a poly D peptide.

[0015] In one embodiment, one or more poly D peptides each independently contain 2 to 30 aspartic acid residues. For example, a poly D peptide may contain 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 aspartic acid residues. In another embodiment, one or more poly D peptides each independently contain 6, 7, 8, 9, 10, or 11 aspartic acid residues. In another embodiment, one or more poly D peptides each contain 10 aspartic acid residues; such peptides are referred to herein as "D10" (SEQ ID NO: 1). In some embodiments, the antibody or fragment may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 poly D peptides.

[0016] In another embodiment, the antibody isotype is IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE, or IgD. In another embodiment, the antibody isotype is IgG1 or IgG4. In another embodiment, the antibody or antigen-binding fragment specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3, such as one or more of human TGFβ1, TGFβ2, and TGFβ3.

[0017] In one embodiment, the antibody fragment is intended to have one or more polyaspartic acid (polyD) peptides. It is envisioned that the antibody fragment will exhibit at least a two-fold increase in bone localization compared to the same antibody fragment lacking one or more polyD peptides. The antibody fragment may be, for example, a Fab, F(ab')2, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, bispecific diabody, trispecific triabody, scFv-sc, minibody, IgNAR, V-NAR, hcIgG, or VhH, or any combination thereof. In one embodiment, the antibody fragment binds to one or more TGFβ1, TGFβ2, and TGFβ3, such as one or more human TGFβ1, TGFβ2, and TGFβ3. The antibody or antibody fragment herein may be fully human, humanized, or chimeric.

[0018] In a second aspect, the disclosure provides a method for producing a bone-targeting antibody or antigen-binding fragment thereof comprising the steps of providing an antibody heavy chain, providing an antibody light chain, providing one or more poly-D peptides attached to the heavy chain and / or one or more poly-D peptides attached to the light chain, and combining the heavy and light chains to produce the bone-targeting antibody or antigen-binding fragment thereof.

[0019] In one embodiment, the one or more poly D peptides attached to the heavy chain and / or the one or more poly D peptides attached to the light chain are joined by chemical conjugation, in another embodiment, the one or more poly D peptides attached to the heavy chain and / or the one or more poly D peptides attached to the light chain are joined recombinantly.

[0020] In a third aspect, the present disclosure provides an antigen-binding domain comprising an antigen-binding domain as set forth in any one of SEQ ID NOs: 2, 3, 4, and 5. The present invention provides an anti-TGFβ antibody that targets bone, comprising a heavy chain comprising an amino acid sequence (with or without the heavy chain C-terminal lysine) and a light chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 6, 7, 8, 11, and 12, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 6, then the heavy chain amino acid sequence is not SEQ ID NO: 2 (with or without the heavy chain C-terminal lysine).

[0021] In a fourth aspect, the disclosure provides an anti-TGFβ antibody that targets bone, comprising a heavy chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 13, 14, 16, and 17 (with or without the heavy chain C-terminal lysine), and a light chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 15, 18, 19, 20, 21, and 22, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 15, then the heavy chain amino acid sequence is not SEQ ID NO: 13 (with or without the heavy chain C-terminal lysine).

[0022] In a fifth aspect, the disclosure provides a human IgG4 antibody (e.g., mAb2 F6) comprising a heavy chain (with or without the heavy chain C-terminal lysine) comprising the amino acid sequence of SEQ ID NO: 14, and a light chain comprising the amino acid sequence of SEQ ID NO: 15. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the antibody specifically binds to TGFβ1.

[0023] In a sixth aspect, the disclosure provides a human IgG4 antibody (e.g., mAb2 F16) comprising a heavy chain (with or without the heavy chain C-terminal lysine) comprising the amino acid sequence of SEQ ID NO: 17, and a light chain comprising the amino acid sequence of SEQ ID NO: 15. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the antibody specifically binds to TGFβ1.

[0024] In a seventh aspect, the disclosure provides a human IgG4 antibody (e.g., mAb2 F11) comprising a heavy chain (with or without the heavy chain C-terminal lysine) comprising the amino acid sequence of SEQ ID NO: 16, and a light chain comprising the amino acid sequence of SEQ ID NO: 15. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the antibody specifically binds to TGFβ1.

[0025] In an eighth aspect, the disclosure provides a human IgG4 antibody (e.g., mAb2 F17) comprising a heavy chain (with or without the heavy chain C-terminal lysine) comprising the amino acid sequence of SEQ ID NO: 17, and a light chain comprising the amino acid sequence of SEQ ID NO: 18. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the antibody specifically binds to TGFβ1.

[0026] In a ninth aspect, the disclosure provides a human IgG4 antibody (e.g., mAb2 F12) comprising a heavy chain (with or without the heavy chain C-terminal lysine) comprising the amino acid sequence of SEQ ID NO: 16, and a light chain comprising the amino acid sequence of SEQ ID NO: 18. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the antibody specifically binds to TGFβ1.

[0027] In a tenth aspect, the disclosure provides a human IgG4 antibody (e.g., mAb2 F7) comprising a heavy chain (with or without the heavy chain C-terminal lysine) comprising the amino acid sequence of SEQ ID NO: 14, and a light chain comprising the amino acid sequence of SEQ ID NO: 18. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the antibody specifically binds to TGFβ1.

[0028] In an eleventh aspect, the present disclosure provides a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 (heavy chain C-terminus The present invention provides a human IgG4 antibody (e.g., mAb2F2) comprising a light chain comprising an amino acid sequence of SEQ ID NO: 18, the light chain comprising a nucleotide sequence (with or without the terminal lysine) and an amino acid sequence of SEQ ID NO: 18. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the antibody specifically binds to TGFβ1.

[0029] In a twelfth aspect, the disclosure provides an anti-TGFβ antibody that targets bone, comprising a heavy chain comprising an amino acid sequence encoded by any of the nucleic acid sequences set forth in SEQ ID NOs: 23, 24, 25, and 26 (with or without a codon for the heavy chain C-terminal lysine), and a light chain comprising an amino acid sequence encoded by any of the nucleic acid sequences set forth in SEQ ID NOs: 27, 28, 29, 30, 31, and 32, with the proviso that when the light chain amino acid sequence is encoded by the nucleic acid sequence set forth in SEQ ID NO: 27, the heavy chain amino acid sequence is not encoded by the nucleic acid sequence set forth in SEQ ID NO: 23 (with or without a codon for the heavy chain C-terminal lysine).

[0030] In a thirteenth aspect, the disclosure provides a human IgG4 antibody comprising a heavy chain (with or without a codon for the heavy chain C-terminal lysine) comprising the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 25, and a light chain comprising the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 27. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3.

[0031] In a fourteenth aspect, the disclosure provides a human IgG4 antibody comprising a heavy chain (with or without a codon for the heavy chain C-terminal lysine) comprising the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 26, and a light chain comprising the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 27. The antibody specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3.

[0032] In a fifteenth aspect, the disclosure provides a method of treating an individual for bone loss comprising administering to the individual an effective amount of an anti-TGFβ antibody or antigen-binding fragment thereof that targets bone, and detecting at least one of reduced TGFβ levels, reduced TGFβ activity, reduced bone loss, reduced rate of bone loss, increased bone density, increased bone strength, and reduced IL-11 levels.

[0033] In one embodiment, the individual is a human. In another embodiment, the anti-TGFβ antibody or antibody fragment specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3. In a further embodiment, the anti-TGFβ antibody comprises a heavy chain, a light chain, and one or more polyaspartic acid (polyD) peptides. The antibody exhibits at least a two-fold increase in bone localization compared to an antibody having the same heavy and light chains but lacking one or more polyD peptides. In one embodiment, the antibody isotype is any of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE, and IgD. In another embodiment, the antibody isotype is IgG1 or IgG4. In one embodiment, the individual has a bone disease, including chronic kidney disease and / or metastasis of cancer to bone. The bone disease may be osteogenesis imperfecta or osteoporosis. In one embodiment, an effective amount of an anti-TGFβ antibody or antibody fragment that targets bone is administered subcutaneously, intravenously, or intramuscularly.

[0034] In a sixteenth aspect, the disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of the invention and a pharma- ceutically acceptable carrier. For example, the antibody may comprise a heavy chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 2, 3, 4, and 5 (with or without the heavy chain C-terminal lysine), and a light chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 6, 7, 8, 11, and 12, with the proviso that when the light chain amino acid sequence is SEQ ID NO: 6, the heavy chain lysine is not a C-terminal lysine. In another embodiment, the antibody may comprise a heavy chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 13, 14, 16, and 17 (with or without the heavy chain C-terminal lysine), and a light chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 15, 18, 19, 29, 21, and 22, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 15, then the heavy chain amino acid sequence is not SEQ ID NO: 13 (with or without the heavy chain C-terminal lysine).

[0035] In a seventeenth aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain, the light chain, or both, of a bone-targeting anti-TGFβ antibody, wherein the heavy chain of the anti-TGFβ antibody comprises the amino acid sequence set forth in any of SEQ ID NOs: 13, 14, 16, and 17 (with or without the heavy chain C-terminal lysine) and the light chain of the anti-TGFβ antibody comprises the amino acid sequence set forth in any of SEQ ID NOs: 15, 18, 19, 20, 21, and 22, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 15, then the heavy chain amino acid sequence is not SEQ ID NO: 13 (with or without the heavy chain C-terminal lysine).

[0036] In an eighteenth aspect, the present disclosure provides an expression vector comprising a nucleic acid sequence encoding the heavy chain, the light chain, or both, of a bone-targeting anti-TGFβ antibody, wherein the heavy chain of the anti-TGFβ antibody comprises the amino acid sequence set forth in any of SEQ ID NOs: 13, 14, 16, and 17 (with or without the heavy chain C-terminal lysine) and the light chain of the anti-TGFβ antibody comprises the amino acid sequence set forth in any of SEQ ID NOs: 15, 18, 19, 20, 21, and 22, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 15, then the heavy chain amino acid sequence is not SEQ ID NO: 13 (with or without the heavy chain C-terminal lysine).

[0037] In a nineteenth aspect, the present disclosure provides a host cell comprising one or more expression vectors comprising a nucleic acid sequence encoding a bone-targeting anti-TGFβ antibody, wherein the heavy chain of the anti-TGFβ antibody comprises a heavy chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 13, 14, 16, and 17 (with or without the heavy chain C-terminal lysine) and a light chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 15, 18, 19, 29, 21, and 22, with the proviso that when the light chain amino acid sequence is SEQ ID NO: 15, the heavy chain amino acid sequence is not SEQ ID NO: 13 (with or without the heavy chain C-terminal lysine). In one embodiment, the host cell is a mammalian cell or a prokaryotic cell. In another embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell or an Escherichia coli (E. coli) cell.

[0038] In a twentieth aspect, the disclosure provides a method for producing a bone-targeting anti-TGFβ antibody or antigen-binding fragment thereof. The method comprises growing a host cell under conditions that allow for production of the antibody or antigen-binding fragment thereof. The host cell comprises (i) a nucleic acid sequence encoding a heavy chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 13, 14, 16, and 17 (with or without the heavy chain C-terminal lysine); and (ii) a nucleic acid sequence encoding a light chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 15, 18, 19, 29, 21, and 22, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 15, then the heavy chain amino acid sequence is not SEQ ID NO: 13 (with or without the heavy chain C-terminal lysine). In one embodiment, the method further comprises formulating the antibody or antigen-binding fragment thereof as a pharmaceutical composition comprising an acceptable carrier.

[0039] In a twenty-first aspect, the present disclosure provides a pharmaceutical composition comprising a bone-targeting anti-TGFβ antibody. The bone-targeting anti-TGFβ antibody comprises a heavy chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 2, 3, 4, and 5 (with or without the heavy chain C-terminal lysine), and a light chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 6, 7, 8, 11, and 12, with the proviso that when the light chain amino acid sequence is SEQ ID NO: 6, the heavy chain amino acid sequence is not SEQ ID NO: 2 (with or without the heavy chain C-terminal lysine), or The TGFβ antibody comprises a heavy chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 13, 14, 16, and 17 (with or without the heavy chain C-terminal lysine), and a light chain comprising an amino acid sequence set forth in any of SEQ ID NOs: 15, 18, 19, 29, 21, and 22, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 15, then the heavy chain amino acid sequence is not SEQ ID NO: 13 (with or without the heavy chain C-terminal lysine). In one embodiment, the pharmaceutical composition is formulated as a liquid pharmaceutical. In another embodiment, the pharmaceutical composition is formulated as a lyophilized pharmaceutical.

[0040] In a twenty-second aspect, the disclosure provides an anti-TGFβ antibody that targets bone. The heavy chain of the antibody comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 35. The light chain of the antibody comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 38. The antibody further comprises a D10 polypeptide at one or more of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain.

[0041] In a twenty-third aspect, the disclosure provides an anti-TGFβ antibody targeted to bone, wherein the heavy chain of the antibody comprises heavy chain complementarity determining regions (CDRs) 1-3 of SEQ ID NO:39 and light chain CDRs 1-3 of SEQ ID NO:40, and the antibody further comprises a D10 polypeptide at one or more of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain. In some embodiments, the antibody comprises a heavy chain variable domain (V) comprising the amino acid sequence of SEQ ID NO:39. H or HCVD), and a light chain variable domain (V L or LCVD).

[0042] In a twenty-fourth aspect, the disclosure is a bone-targeting anti-TGFβ antibody, wherein the heavy chain of the antibody comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 35; the light chain of the antibody comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 38; and the antibody further comprises an N-terminus of the heavy chain, a C-terminus of the heavy chain, an N-terminus of the light chain, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 39. or a polynucleotide sequence encoding a bone-targeting anti-TGFβ antibody, wherein the heavy chain of the antibody comprises heavy chain complementarity determining regions (CDRs) 1-3 of SEQ ID NO: 39 and light chain CDRs 1-3 of SEQ ID NO: 40, and the antibody further comprises a D10 polypeptide at one or more of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain.

[0043] In a twenty-fifth aspect, the present disclosure provides a bone-targeted antibody, such as a bone-targeted anti-TGFβ antibody or antigen-binding fragment of the invention, for use in the therapeutic methods described herein.

[0044] In a twenty-sixth aspect, the disclosure provides the use of a bone-targeted antibody, such as a bone-targeted anti-TGFβ antibody or antigen-binding fragment, of the invention for the manufacture of a medicament for the methods of treatment described herein.

[0045] Specific embodiments contemplated herein are further described below. These and other features and advantages of the present invention will be more fully understood from the following detailed description of the invention taken in conjunction with the accompanying claims. It should be noted that the claims are defined by the description therein, and not by the specific descriptions of the features and advantages set forth herein. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1 shows D10 peptide chemically conjugated to an anti-TGFβ (α-TGFβ) antibody. [Diagram 2] FIG. 1 shows a process for chemically conjugating a peptide bearing a linker (peptide linker) and a maleimide functional group to a reduced hinge region disulfide on a mouse IgG1 antibody. [Figure 3A] Figure 1 shows a reducing SDS-PAGE gel of the chemical conjugate of D10 peptide-linker with anti-TGFβ mouse IgG1, mAb1. The upper band represents the heavy chain and the lower band represents the light chain. [Figure 3B] FIG. 1 shows peptide-to-antibody ratio (PAR) values ​​versus peptide-maleimide:mAb ratio in the chemical conjugation reaction of Example 1, showing a linear increase in PAR with increasing numbers of peptides up to 8 mole:mol PAR. [Figure 4A] Figure 1 shows size exclusion chromatography of a 1:1 molar mixture of TGFβ1 and chemical conjugates of either Trastuzumab (Herceptin®) or anti-TGFβ antibody mAb1 with D10 peptide. SEC profile of chemical conjugates of D10 peptide with mAb1. [Figure 4B]Figure 1 shows size exclusion chromatography of a 1:1 molar mixture of TGFβ1 and chemical conjugates of either Trastuzumab (Herceptin®) or anti-TGFβ antibody mAb1 with D10 peptide. SEC profile of a 1:1 molar mixture of mAb1-D10 chemical conjugate and TGFβ1. [Figure 4C] Figure 1 shows size exclusion chromatography of 1:1 molar mixtures of TGFβ1 and chemical conjugates of either Trastuzumab (Herceptin®) or anti-TGFβ antibody mAb1 with D10 peptide. SEC profile of unmodified mAb1 is shown. [Figure 4D] Figure 1 shows size exclusion chromatography of a 1:1 molar mixture of TGFβ1 and chemical conjugates of either Trastuzumab (Herceptin®) or the anti-TGFβ antibody mAb1 with the D10 peptide.The SEC profile of a 1:1 molar mixture of mAb1 and TGFβ1 is shown. [Figure 4E] Figure 1 shows size exclusion chromatography of 1:1 molar mixtures of TGFβ1 and chemical conjugates of either Trastuzumab (HERCEPTIN®) or anti-TGFβ antibody mAb1 with D10 peptide. SEC profile of HERCEPTIN®. [Figure 4F] Figure 1 shows size exclusion chromatography of a 1:1 molar mixture of TGFβ1 and chemical conjugates of either Trastuzumab (Herceptin®) or anti-TGFβ antibody mAb1 with D10 peptide. SEC profile of chemical conjugates of D10 peptide with Herceptin®. [Figure 4G] Figure 1 shows size exclusion chromatography of a 1:1 molar mixture of TGFβ1 and chemical conjugates of either Trastuzumab (Herceptin®) or anti-TGFβ antibody mAb1 with D10 peptide. SEC profile of chemical conjugates of D10 and Herceptin® mixed with TGFβ1 in a 1:1 ratio. [Figure 5A]FIG. 1 shows A280 traces of hydroxyapatite chromatography of mAb1 and D10-mAb1 conjugate (x-axis=min, y-axis=absorbance (normalized)). [Figure 5B] FIG. 1 shows an SDS-PAGE gel of the flow-through (FT) and peak 4 fractions. [Figure 6A] Figure 1 shows hydroxyapatite chromatography of chemical conjugates with increasing numbers of peptides, with absorbance at 280 nm of the eluate for each conjugate (x-axis = minutes, y-axis = absorbance (normalized)). [Figure 6B] FIG. 1 shows the percentage of analyte bound (upper curve, circles, left scale) and retention time (lower curve, triangles, right scale) as a function of the number of conjugated peptides as determined by SDS-PAGE. [Figure 7] FIG. 1 shows an in vitro TGFβ neutralization assay performed with A549 cells using a control conjugate (PAR=0) and conjugates with an average of 4 or 9 peptides compared to unmodified mAb1. [Figure 8A] Figure 1 shows the time-dependent biodistribution of fluorophore-labeled mAb1 and 1 mg / kg (1 mpk) chemical conjugate containing approximately 4.5 peptides. The time at which the animals were imaged is indicated in each panel. For each picture, the mouse on the left received mAb1 and the mouse on the right received the chemical conjugate. The intensity of the images has been adjusted to reveal differences in distribution. [Figure 8B] Figure 8B shows the ratio of fluorescence seen in the region of interest corresponding to the distal femur and the heart in the image shown in Figure 8A. The circles correspond to the mAb1 antibody and the squares correspond to the D10 peptide conjugated to mAb1 (D10 mAb1). [Figure 9A]FIG. 1 shows a schematic representation of possible locations of D10 peptides on IgG subtype antibodies to generate a series of mAb1 fusion variant antibodies obtained by attachment of the D10 peptide to the heavy and / or light chain termini using recombinant methods. The addition site of the D10 peptide is indicated by a circle and the use of a peptide linker sequence is indicated by a wavy line (longer wavy lines represent longer linkers than shorter wavy lines). [Figure 9B] 9A shows a series of fusion variant antibodies (fusion variants) obtained by arranging peptides as shown in FIG. 9A. Recombinant fusion variants with various combinations of binding positions and number of peptides were generated. Small numbers under each figure indicate the identification number of each recombinant fusion variant referred to herein for clarity. As used herein, fusion variants are designated by either "fusion" or "F" followed by the desired variant number. For example, "fusion 1" and "F1" both refer to an antibody with the configuration of antibody "1" without the D10 peptide. Longer wavy lines represent longer linkers than shorter wavy lines. [Figure 10] FIG. 1 shows SDS-PAGE of the indicated purified recombinant mAb1 fusion variants under reducing (top gel) or non-reducing (bottom gel) conditions. [Figure 11] Figure 1. Thermal stability of recombinant mAb1 fusion variants as determined by differential scanning fluorimetry (DSF). The transition to a partially denatured form at each temperature is detected by an increase in dye fluorescence. The slope of the fluorescence increase with temperature (-d(RFU) / dT) was calculated and plotted against the temperature of the sample. The rate of denaturation is maximum at the minimum of the curve, which represents the midpoint of the thermal transition (Tm). For reference, the structure of each recombinant mAb1 fusion variant is shown diagrammatically. [Figure 12A] FIG. 9B shows neutralization of TGFβ in inducing IL-11 production by A549 cells in vitro by eight recombinant mAb1 fusion variants, as shown diagrammatically in FIG. 9B. [Figure 12B]FIG. 9B shows neutralization of TGFβ in inducing IL-11 production by A549 cells in vitro by eight recombinant mAb1 fusion variants, as shown diagrammatically in FIG. 9B. [Figure 13] FIG. 1 shows the affinity of recombinant mAb1 fusion variants and mAb1 chemical conjugates for hydroxyapatite as assessed by column chromatography on a ceramic hydroxyapatite column. [Figure 14] FIG. 1 shows the biodistribution of selected fluorophore-labeled recombinant mAb1 fusion variants and mAb1 chemical conjugates in CD-1 mice obtained by live imaging at various time points after administration. [Figure 15] Figure 1 shows the amount of fluorochrome-labeled antibodies, recombinant mAb1 fusion variants and mAb1 chemical conjugates localized to the spinal column after administration to CD-1 mice. Fluorescence was measured by an IVIS instrument over a 3 week period. The logarithm of maximum fluorescence within the ROI is shown. [Figure 16] 16 shows fluorescence images of excised vertebrae and femurs from mice administered recombinant mAb1 fusion variants mAb1 F6, mAb1 F16, and mAb1 F17, and mAb1 chemical conjugate ("Conj") at 10 and 21 days in the studies described in Example 13 and FIG. [Figure 17A] FIG. 13 shows fluorescence levels of mAb2 F1 and mAb2 F6 in 10 μL of serum, as well as in excised lumbar vertebrae, distal (trabecular) femur, kidney and heart after 24 and 96 hours as described in Example 15. [Figure 17B] FIG. 13 shows fluorescence levels of mAb2 F1 and mAb2 F6 in 10 μL of serum, as well as in excised lumbar vertebrae, distal (trabecular) femur, kidney and heart after 24 and 96 hours as described in Example 15. [Figure 18A]Figure 1 shows that bone targeting via mAb1-D10 (mAb1 F6) significantly impacts serum PK after a single dose. mAb1 F6 exhibits 13-14 fold lower serum exposure (AUC), faster serum clearance, and shorter serum half-life (t1 / 2) than mAb1 as measured by ELISA. Data are presented as mean ± SD; statistical significance (*p≦0.05, mAb1 F6 vs. mAb1) was observed as measured by analysis of variance (AVOVA), Dunnett's multiple comparison test. mAb1 is a murine inhibitory anti-TGFβ monoclonal antibody and mAb1 F6 is a recombinant murine inhibitory anti-TGFβ monoclonal antibody with aspartic acid polypeptide D10 attached to the C-terminus of the heavy chain of mAb1. Image / bone AUC is normalized to 1.0. The dose of each mAb1 F6 and mAb1 was 5 mg / kg. [Figure 18B] FIG. 1 shows that mAb1 F6 exhibits 22-fold higher exposure (AUC) in bone compared to mAb1 as measured by optical imaging. Data are presented as mean ± SD; statistical significance (*p≦0.05, mAb1 F6 vs. mAb1) was observed as measured by AVOVA, Dunnett's multiple comparison test. The dose of each mAb1 F6 and mAb1 was 1 mg / kg. [Figure 19] Figure 1: Peak-trough PK profiles of multiple doses. Bone targeting (with mAb1 F6) has a significant impact on peak-trough serum PK of multiple doses. mAb1 F6 shows lower serum concentrations than mAb1 at both 24 and 48 hours post-dose, as well as at the first dose and 23 hours post-dose. Fold differences were 3-4.5-fold lower for mAb1 F6 at 24 hours and 6-9-fold lower at 48 hours. Accumulation also appeared to be less for mAb1 F6 than for mAb1. Data are presented as mean ± SD; statistical significance (*p ≤ 0.05, mAb1 F6 vs. mAb1) was observed as determined by unpaired t-test. Serum concentrations were measured by mass spectrometry. [Figure 20]Figure 1 shows that bone-targeted (mAb1 F6) and mAb1 increase BV / TV (%) in a dose-dependent manner in G610C (OI) mice. Significant changes in BV / TV (%) were observed for both treatments at doses of 1 and 5 mg / kg compared to G610C mice treated with the control antibody 13C4 (mouse IgG1 antibody). G610C mice treated with 13C4 (13C4) showed a significant decrease in BV / TV compared to the WT background strain (WT 13C4). Data are presented as mean ± SD: statistical significance was observed (*p ≤ 0.05, mAb1 F6 vs. mAb1; #p ≤ 0.05, 13C4 vs. WT 13C4) as determined by one-way ANOVA. BV / TV (%) was measured by μCT imaging. [Figure 21] FIG. 1 shows that bone-targeted (mAb1 F6) and mAb1 increase maximum force to failure in a dose-dependent manner in G610C(OI) mice. Significant changes in maximum force to failure compared to 13C4-treated G610C mice were observed at 1 and 5 mg / kg for mAb1 F6, and only at 5 mg / kg for mAb1. G610C mice treated with antibody control (13C4) showed a significant decrease in maximum force to failure compared to the WT background strain. Data are presented as mean ± SD: statistical significance was observed (*p≦0.05, mAb1 F6 vs. mAb1; #p≦0.05, 13C4 vs. WT 13C4) as measured by one-way ANOVA. Maximum force to failure was measured by biomechanical compression testing. [Figure 22] Figure 1 shows the effect of mAb1 and mAb1 F6 on BV / TV in G610C mice. 5 mg / kg of antibody was administered at various frequencies (three times weekly, once weekly, once every 2 weeks, or once every 4 weeks) for 12 weeks. Antibody 13C4 was used as a control. Statistical significance was observed (*p≦0.05, mAb1 F6 vs. mAb1; #p≦0.05, 13C4 vs. WT 13C4) as measured by one-way ANOVA. BV / TV was measured by μCT imaging. [Figure 23] Figure 1 shows the effect of mAb1 and mAb1 F6 on maximum breaking force in G610C mice. 5 mg / kg of antibody was administered at various frequencies (three times per week, once per week, once every two weeks, or once every four weeks) for 12 weeks. Antibody 13C4 was used as a control. Statistical significance (*p≦0.05, mAb1 or mAb1 F6 vs. 13C4) was observed as measured by one-way ANOVA. Maximum breaking force was measured by biomechanical compression testing. [Figure 24] Figure 1 shows the effect of mAb1 and mAb1 F6 on BV / TV (%) and mean serum levels of antibodies in G610C mice. Antibodies were administered at 5 mg / kg once every 2 weeks or once weekly for 12 weeks. Antibody 13C4 was used as a control. Statistical significance was observed (*p≦0.05, mAb1 or mAb1 F6 vs. 13C4; #p≦0.05, 13C4 vs. WT 13C4) as measured by one-way ANOVA. BV / TV (%) was measured by μCT imaging. [Diagram 25] Figure 1 shows the effect of mAb1 and mAb1 F16 on BV / TV (%) in G610C mice. Antibodies were administered at 5 mg / kg three times weekly for 8 weeks. Antibody 13C4 was used as a control. Statistical significance was observed as measured by one-way ANOVA (*p≦0.05, mAb1 or mAb1 F16 vs. 13C4; #p≦0.05, 13C4 vs. WT 13C4). BV / TV (%) was measured by μCT imaging. [Figure 26] Figure 1 shows the effect of mAb1 and mAb1 F11 on BV / TV (%) in wild type mice. Antibodies were administered at 5 mg / kg three times weekly for 9 weeks. Statistical significance (*p≦0.05, comparing mAb1 or mAb1 F11 with 13C4) was observed as determined by one-way ANOVA. BV / TV (%) was measured by μCT imaging. [Figure 27]Figure 1 shows the total radiant efficiency in the lumbar spine of wild-type mice after receiving a single intraperitoneal injection of vehicle or fluorescently labeled mAb1 or mAb1 F6. Statistical significance (*p≦0.05, comparing mAb1 F6 with mAb1) was observed as determined by one-way ANOVA. [Figure 28] Figure 1 shows the total radiant efficiency in the hearts of wild-type mice after receiving a single intraperitoneal dose of vehicle or fluorescently labeled mAb1 or mAb1 F6. Statistical significance (*p≦0.05, comparing mAb1 with mAb1 F6) was observed as determined by one-way ANOVA. [Figure 29] Figure 1 shows the total radiative efficiency in the liver of wild-type mice after receiving a single intraperitoneal dose of vehicle or fluorescently labeled mAb1 or mAb1 F6. Statistical significance (*p≦0.05, comparing mAb1 with mAb1 F6) was observed as determined by one-way ANOVA. [Diagram 30] FIG. 1 shows the total radiative efficiency in the intestine of wild-type mice after receiving a single intraperitoneal dose of vehicle or fluorescently labeled mAb1 or mAb1 F6. [Diagram 31] Figure 1 shows the total radiant efficiency in the indicated tissues of wild-type mice 24 or 96 hours after receiving a single intraperitoneal dose of fluorescently labeled mAb2 or mAb2 D10. Statistical significance was observed as determined by t-test (*p≦0.05, comparing mAb1 with mAb1 F6). [Diagram 32] FIG. 1 shows the lumbar spine / serum total radiative efficiency ratios of wild-type mice 24 or 96 hours after receiving a single intraperitoneal dose of fluorescently labeled mAb2 or mAb2 D10. [Diagram 33] Figure 1 shows femur / serum total radiative efficiency ratios of wild-type mice 24 or 96 hours after receiving a single intraperitoneal dose of fluorescently labeled mAb2 or mAb2 D10. Statistical significance was observed (*p≦0.05, comparing mAb2 D10 vs. mAb2) as measured by t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The present invention provides antibodies and antigen-binding fragments thereof linked to one or more bone-targeting poly-D peptides such that the antibodies and fragments preferentially home to bone in patients in need thereof. The bone-targeting function of such antibodies or fragments allows the antibodies and fragments to specifically target bone tissue and reduce the patient's systemic exposure to the antibodies or fragments, thereby enhancing drug efficacy while minimizing undesirable adverse side effects.

[0048] As used herein, the term "poly-D peptide" refers to a peptide sequence having multiple aspartic acid or aspartate or "D" amino acids, such as about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or more aspartic acid amino acids (residues). In one embodiment, a poly-D peptide may contain about 2 to about 30, or about 3 to about 15, or about 4 to about 12, or about 5 to about 10, or about 6 to about 8, or about 7 to about 9, or about 8 to about 10, or about 9 to about 11, or about 12 to about 14 aspartic acid residues. In one embodiment, a poly-D peptide contains only aspartic acid residues. In another embodiment, a poly-D peptide may contain one or more other amino acids or similar compounds. As used herein, the term "D10" refers to a contiguous sequence of 10 aspartic acid amino acids, as seen in SEQ ID NO:1. In some embodiments, an antibody or antibody fragment of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 poly D peptides.

[0049] The poly-D peptide is linked to the antibody or antigen-binding fragment of interest via recombinant techniques or chemical conjugation. As used herein, the term "fusion variant" or "variant" refers to an assembled antibody construct (see FIG. 9B) that includes at least one of the heavy or light chains or antibody fragments or sub-portions that incorporates or is somehow linked to a poly-D peptide, such as the D10 sequence. For example, the poly-D peptide is linked to the antibody chain in the fusion variant by recombinant techniques (e.g., where the poly-D peptide sequence is integrated with the amino acid sequence of the heavy chain, light chain, or antibody fragment or sub-portion), chemical conjugation, or both.

[0050] As used herein, the term "chemical conjugate" refers to an assembled antibody comprising at least one heavy or light chain or antibody fragment or sub-portion to which one or more poly-D peptides are linked by chemical reaction, for example, to a cysteine ​​residue present in the amino acid sequence of the heavy chain, light chain, antibody fragment, or sub-portion. Exemplary cysteine ​​residues that may be used for conjugation are those in the heavy chain hinge region. Cysteine ​​residues or other residues suitable for conjugation may also be introduced into the antibody chain by mutagenesis. Spacers / linkers such as peptide linkers or chemical moieties (e.g., maleimide functional groups and polyethylene glycol (PEG)) may be used between the poly-D peptide and the antibody component in the conjugation. Methods for chemical conjugation of desired moieties to antibodies are well known in the art. Examples: See, for example, Behrens and Liu, mAbs 6:1, 46-53 (2014).

[0051] As used herein, the term "integrated" refers to the integration of a poly-D peptide with an antibody chain by recombinant techniques such that the poly-D peptide is transcribed from the same RNA transcript as the antibody chain and is present in the same polypeptide sequence as the antibody chain. In such cases, the poly-D peptide is linked to the antibody chain at either or both termini of the antibody chain, with or without a peptide linker or amino acid spacer, or is incorporated within the antibody chain without affecting proper folding of the antibody chain, assembly of the antibody molecule, or binding of the antibody to its antigen.

[0052] Exemplary formats of bone-targeting antibodies of the invention are shown in FIG. 9B (formats F2-F20). The bone-targeting peptide (represented by a circle) is attached or fused (e.g., integral) to one or both termini of the heavy and / or light chains of the antibody. In some embodiments, the bone-targeting peptide is not attached to the light chain via the N-terminus of the light chain. The attachment or fusion can be by a direct linkage (i.e., no spacer or linker) or via a spacer or linker (represented by a wavy line; e.g., a peptide linker). Specific examples of these formats are shown in Tables 1 and 7 below.

[0053] In the present invention, any suitable spacer or linker can be used to link the bone targeting peptide to the antibody of interest, for example, by recombinant technology or chemical conjugation.For example, a peptide linker having 1, 2, 3 or more repeats of G4S peptide (SEQ ID NO: 9) can be used.Other suitable peptide linkers can also be used.See, for example, Chen et al., Adv Drug Deliv Rev 65(10):1357-1369(2013).

[0054] Exemplary bone-targeted antibodies and antigen-binding fragments thereof The present invention discloses antibodies and antigen-binding fragments having one or more poly-D (poly-aspartic acid or poly-Asp) peptides (e.g., D10 sequences) attached thereto. These modified antibodies and fragments have improved localization to bone. In one particular embodiment, these antibodies are anti-TGFβ antibodies as described herein. Without wishing to be bound by theory, it is believed that effectively targeting anti-TGFβ antibodies to bone with one or more poly-D peptides may provide a new treatment for individuals with diseases characterized by pathophysiological bone degeneration associated with TGFβ.

[0055] However, although many of the embodiments and examples herein are expressed in the context of using α-TGFβ antibodies and D10 sequences, it is contemplated that other antibodies or proteins suitable for treating abnormal bone conditions or bone diseases may be modified with the bone-targeting moieties described herein. For example, therapeutic antibodies for treating bone loss, stimulating bone growth, or targeting abnormal cells (e.g., cancer cells) in bone are linked to one or more bone-targeting peptides described herein. Therapeutic antibodies may bind to proteins or peptides involved in bone formation or maintenance. Additionally, other bone-localizing or targeting peptides may be used.

[0056] As used herein, the terms "α-TGFβ antibody" and "anti-TGFβ antibody" are used interchangeably and refer to an antibody or antigen-binding fragment thereof specific for TGFβ1, TGFβ2, and / or TGFβ3. For example, at least one antigen-binding site (or paratope) of an α-TGFβ antibody or antigen-binding fragment thereof binds to an epitope found on human TGFβ1, TGFβ2, and / or TGFβ3.

[0057] In one embodiment, the contemplated α-TGFβ antibody-D10 construct comprises a chemical construct. The poly-D peptide is produced by jugation. For example, chemical conjugation is carried out by methods known in the art, such as those disclosed in U.S. Patent Nos. 7,763,712, 4,671,958, and 4,867,973, each of which is incorporated herein by reference. In another example, a peptide or other linker is used to attach the D10 peptide to the antibody (see FIG. 1 and FIG. 2). In a further embodiment, reduction of the thiol groups in the hinge region of the antibody (e.g., cysteine ​​residues in the hinge region) allows chemical conjugation of the poly-D peptide with a PEG spacer. Similarly, other cysteine ​​residues of the contemplated antibodies and antibody fragments, either naturally present in the antibodies and fragments or introduced by mutagenesis, are chemically conjugated with the poly-D peptide. One such possible assembly scheme of an α-TGFβ antibody chemically conjugated with a D10 peptide is shown in FIG. 2.

[0058] In another embodiment, the contemplated α-TGFβ antibody-D10 construct is made by recombinant expression in which the D10 sequence is added to the amino acid sequence of the heavy and / or light chain of the α-TGFβ antibody. For example, the nucleic acid sequence encoding the amino acid sequence of the heavy and / or light chain is modified to encode the D10 sequence expressed at either the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the heavy and / or light chain of the α-TGFβ antibody. Similarly, one or more D10 sequences are added at or near the hinge region of the amino acid sequence of the antibody heavy chain and / or within the amino acid sequence of the antibody light chain. Each nucleic acid sequence of the heavy and / or light chain with D10 may be incorporated into an expression vector and subsequently transfected into a host cell capable of expressing and translating the nucleic acid sequence into the corresponding amino acid sequence. Furthermore, the host cell can assemble the expressed amino acid sequence into a functional protein by combining each of the heavy and light chains with their complementary sequences to form the α-TGFβ antibody-D10 construct. Examples of contemplated recombinant α-TGFβ antibody-D10 fusion variants are shown in Figures 9A and 9B.

[0059] Although poly-D peptides are discussed herein, other similar peptides may be used to allow for the targeting of antibodies, other proteins, or peptides to bone.For example, the aspartic acid repeat sequence may have more or fewer residues than the D10 sequence, such as about 2, or about 4, or about 6, or about 8, or about 12, or about 14, or about 16, or about 18, or about 20, or about 30, or 6, 7, 8, 9, 10, or 11 residues.In addition, other natural amino acids with similar chemical properties, such as glutamate, or unnatural amino acids, and / or other chemically equivalent compounds, may be used in place of or in combination with aspartic acid.

[0060] In one embodiment, an antibody having one or more poly D peptides bound thereto is believed to exhibit at least about a 2-fold, or about a 3-fold, or about a 5-fold, or about a 10-fold, or about a 20-fold increase in localization to bone compared to the same antibody without the one or more poly D peptides.

[0061] In addition, although α-TGFβ antibodies are described herein, any antibody that binds to other proteins involved in bone formation or bone maintenance may be similarly modified to target the antibody to bone as desired. The antibodies or antigen-binding fragments contemplated herein may be from any species or may be hybrid antibodies combining heavy and light chains from different species and may be specific for any desired epitope. Furthermore, the antibodies that may be used herein are not limited by isotype and may be any of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE, or IgD. Antibody fragments may also be used. For example, the D10 sequence or other bone-targeting compounds are combined with Fab and / or Fc fragments or any other antibody fragments to achieve the desired results as described herein. Furthermore, the D10 sequence may be combined with Fab and / or Fc fragments or any other antibody fragments to achieve the desired results as described herein. is attached to scFv fragments and other similar fusion proteins. In another embodiment, the D10 sequence may be attached to an antibody with an S228P core-hinge mutation (numbered according to the EU numbering system; or alternatively S248P according to the Kabat system; see Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., United States Government Printing Office, 165-492 (1987); and Silva et al., Jour. Biol. Chem. 290:5462-5469 (2015)).

[0062] In further embodiments, the antibodies and / or other proteins contemplated herein are conjugated with additional molecules. For example, the antibodies or other proteins contemplated herein are conjugated with chemical labels that allow the antibodies / proteins to be tracked when injected or otherwise introduced into a subject. For example, radioactive labels, fluorescent compounds, etc. are attached to the antibodies / proteins to aid in their in vivo tracking. Furthermore, the antibodies and / or other proteins contemplated herein may also be conjugated with additional compounds that have therapeutic effects, such as small molecules, pharmaceuticals, anti-neoplastic agents, growth hormones, vitamins, etc., such that the antibodies and / or other proteins serve as vehicles for one or more such compounds.

[0063] In some embodiments, the bone-targeted anti-TGFβ antibody comprises a heavy chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 2, 3, 4, and 5, and a light chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 6, 7, 8, 11, and 12, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 6, then the heavy chain amino acid sequence is not SEQ ID NO: 2. An exemplary antibody is mAb1. F3, mAb1 F4, mAb1 F5, mAb1 F6, mAb1 F8, mAb1 F9, mAb1 F10, mAb1 F11, mAb1 F13, mAb1 F14, mAb1 F15, mAb1 F16, mAb1 F18, mAb1 F19, and mAb1 F20 (Table 1).

[0064] In other embodiments, the bone-targeted anti-TGFβ antibody comprises a heavy chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 13, 14, 16, and 17, and a light chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 15, 18, 19, 20, 21, and 22, with the proviso that if the light chain amino acid sequence is SEQ ID NO: 15, then the heavy chain amino acid sequence is not SEQ ID NO: 13. Exemplary antibodies are mAb2 F3, mAb2 F4, mAb2 F5, mAb2 F6, mAb2 F8, mAb2 F9, mAb2 F10, mAb2 F11, mAb2 F13, mAb2 F14, mAb2 F15, mAb2 F16, mAb2 F18, mAb2 F19, and mAb2 F20 (Table 7).

[0065] In some embodiments, antibodies of the invention, such as anti-TGFβ antibodies, do not have a C-terminal lysine on the heavy chain. The C-terminal lysine can be removed during production or by recombinant techniques (i.e., the coding sequence for the heavy chain does not contain a codon for a C-terminal lysine). Thus, also contemplated within the scope of the invention are antibodies comprising the heavy chain amino acid sequence of SEQ ID NO: 2 or 13 without the C-terminal lysine. A poly-D peptide is attached to the C-terminus of the heavy chain, with or without a C-terminal lysine.

[0066] Treatment method In one particular embodiment, a method of treating an individual, such as a human patient, for TGFβ-associated bone loss comprises administering to the individual an effective amount of an anti-TGFβ antibody that targets bone. The method may further comprise measuring or detecting a decrease in TGFβ levels or activity, a decrease in bone loss or rate of bone loss, an increase in bone density, and / or an increase in bone strength.

[0067] As used herein, an "effective amount" refers to an amount that, when administered to an individual in need thereof, For example, it refers to an amount of a therapeutic agent, such as an α-TGFβ antibody or antibody fragment, that improves the health of an individual by reducing the level or activity of TGFβ associated with bone, reducing bone loss or the rate of bone loss, increasing bone density, and / or increasing bone strength.

[0068] As used herein, the term "individual" refers to an animal. Examples of individuals include, but are not limited to, humans, livestock, household pets, and other animals. Further examples of individuals include animals with TGFβ-associated bone diseases.

[0069] In another embodiment, pharmaceutical antibody formulations or compositions, including aqueous liquid pharmaceutical formulations and lyophilized pharmaceutical formulations, are contemplated that contain one or more bone-targeted anti-TGFβ antibodies, such as chemical conjugates or recombinant fusion variants. Pharmaceutical compositions containing bone-targeted anti-TGFβ antibodies and / or antibody fragments may be formulated as described in U.S. Patent Application Publication No. US2014 / 0286933A9, which is incorporated herein by reference, or as otherwise known in the art.

[0070] In one particular embodiment, the method for treating bone disease comprises administering an effective amount of an anti-TGFβ antibody that targets bone to an individual with bone disease, such as bone disease associated with chronic kidney disease, cancer metastasis to bone, or abnormal metabolic state.In another particular embodiment, the method for treating osteogenesis imperfecta comprises administering an effective amount of an anti-TGFβ antibody that targets bone to an individual with osteogenesis imperfecta.In a further particular embodiment, the method for treating osteoporosis comprises administering an effective amount of an anti-TGFβ antibody that targets bone to an individual with osteoporosis.

[0071] In some embodiments, the patient is administered a bone-targeting antibody or antibody fragment of the invention. For example, the antibody or antibody fragment and the other therapeutic agent may be administered simultaneously or sequentially to the patient.

[0072] In some embodiments, the bone-targeting antibodies and other components used in the therapeutic methods of the invention are provided in a kit or article of manufacture.

[0073] How to generate antibodies The antibody or fragment of the present invention can be produced by methods well established in the art. The DNA sequences encoding the antibody heavy and light chains are inserted into an expression vector such that the genes are operably linked to the necessary expression control sequences, such as transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The antibody light chain coding sequence and the antibody heavy chain coding sequence may be inserted into separate vectors and may be operably linked to the same or different expression control sequences (e.g., promoters). In one embodiment, both coding sequences are inserted into the same expression vector and operably linked to the same expression control sequence (e.g., a common promoter), separate identical expression control sequences (e.g., promoters), or different expression control sequences (e.g., promoters). The antibody coding sequence can be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0074] In addition to the antibody chain genes, the recombinant expression vector may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Examples of regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus LTRs, and ribozymes. These include viral elements that direct high levels of protein expression in mammalian cells, such as those derived from viruses (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian promoters such as the native immunoglobulin and actin promoters.

[0075] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. For example, the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for use with methotrexate selection / amplification in dhfr- host cells), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0076] The expression vector encoding the antibody of the present invention is introduced into a host cell for expression. The host cell is cultured under conditions suitable for the expression of the antibody, and the antibody is then harvested and isolated. The host cell includes mammalian, plant, bacterial or yeast host cells. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and many other cell lines. Cell lines may be selected based on their expression levels. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells.

[0077] Furthermore, expression of the antibody may be enhanced using a number of known techniques, for example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions.

[0078] Tissue culture media for host cells may or may not contain animal derived components (ADCs) such as bovine serum albumin. In some embodiments, media without ADCs is preferred for human safety. Tissue culture may be performed using fed-batch, continuous perfusion, or any other method suitable for the host cells and the desired yield.

[0079] Pharmaceutical Compositions The antibody of the present invention can be prepared for suitable storage stability.For example, the antibody can be lyophilized or stored or reconstituted for use with pharma- ceutically acceptable excipients.For combination therapy, two or more therapeutic agents such as antibodies can be co-formulated, for example, mixed and provided in a single composition.

[0080] The term "excipient" or "carrier" is used herein to describe any ingredient other than the compound of the present invention. The choice of excipient will largely depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. "Pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, etc. , ethanol, and the like, as well as combinations thereof. In some cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, will be included in the composition. Further examples of pharma-ceutically acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibodies.

[0081] The pharmaceutical composition of the present invention is manufactured, packaged or sold in bulk as a single unit dose or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of active ingredient administered to a subject, or a convenient fraction of such dosage, such as half or a third of such dosage.

[0082] The pharmaceutical composition of the present invention is typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physical destruction of the target tissue and administration of the pharmaceutical composition through a break in the tissue, thus generally resulting in administration directly into the bloodstream, intramuscularly, or into an internal organ. Thus, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion; and kidney dialysis infusion techniques. Local perfusion is also intended. Preferred embodiments may include intravenous and subcutaneous routes.

[0083] A formulation of a pharmaceutical composition suitable for parenteral administration typically comprises the active ingredient combined with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations are prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations are prepared, packaged, or sold in unit dosage form, such as ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (e.g., pH 3-9), although for some applications they may be more suitably formulated as sterile nonaqueous solutions or as dry forms used with a suitable vehicle such as sterile pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired. Other parenteral dosage formulations that are useful include those that contain the active ingredient in microcrystalline form or in a liposomal formulation. Formulations for parenteral administration may be formulated for immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0084] In some embodiments, the antibodies or antigen-binding fragments of the invention are administered at 40, 20, or 15 mg / kg or less (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg / kg). In some further embodiments, the dose can be 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 mg / kg. The frequency of administration can be, for example, daily, every 2, 3, 4, or 5 days, weekly, biweekly, or 3 weeks, monthly, bimonthly, 3 months, 6 months, or 12 months, or as needed. The antibodies are administered intravenously (e.g., intravenously over a period of 0.5 to 8 hours). Administration may be by intravenous injection), subcutaneous, intramuscular, or any other route of administration appropriate to the condition and pharmaceutical formulation.

[0085] Exemplary Implementations Further specific embodiments of the present invention are described below.

[0086] 1. An antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, and one or more polyaspartic acid (poly D) peptides linked to (i) the C-terminus of the heavy chain, (ii) the C-terminus of the light chain, or (iii) both (i) and (ii).

[0087] 2. The antibody or antigen-binding fragment of embodiment 1, wherein the one or more poly-D peptides are linked to the antibody or antigen-binding fragment by chemical conjugation.

[0088] 3. The antibody or antigen-binding fragment of embodiment 2, wherein the one or more poly-D peptides are conjugated to the heavy chain at the hinge region.

[0089] 4. The antibody or antigen-binding fragment of embodiment 2 or 3, wherein the one or more poly-D peptides are conjugated to the antibody or antigen-binding fragment by a polyethylene glycol (PEG) spacer.

[0090] 5. The antibody or antigen-binding fragment of embodiment 1, comprising a poly-D peptide associated with the amino acid sequence of the heavy or light chain.

[0091] 6. The antibody or antigen-binding fragment of embodiment 5, comprising a poly-D peptide attached to the N-terminus of the heavy chain.

[0092] 7. The antibody or antigen-binding fragment of embodiment 5, comprising a poly D peptide attached to the C-terminus of the heavy chain.

[0093] 8. The antibody or antigen-binding fragment of embodiment 5, comprising a first polyD peptide associated with the N-terminus of the heavy chain and a second polyD peptide associated with the C-terminus of the heavy chain.

[0094] 9. The antibody or antigen-binding fragment of any one of embodiments 5 to 8, comprising a poly D peptide attached to the C-terminus of the light chain.

[0095] 10. The antibody or antigen-binding fragment of any one of embodiments 5 to 9, wherein the poly D peptide is fused to the heavy or light chain via a peptide linker.

[0096] 11. The antibody or antigen-binding fragment of embodiment 10, wherein the peptide linker comprises 1 to 3 repeats of the amino acid sequence GGGGS (SEQ ID NO: 9).

[0097] 12. The antibody or antigen-binding fragment of any one of the preceding embodiments, wherein the one or more poly D peptides each independently comprise 2 to 30 aspartic acid residues.

[0098] 13. The antibody or antigen-binding fragment of embodiment 12, wherein the one or more poly-D peptides each contain 10 aspartic acid residues (SEQ ID NO:1).

[0099] 14. The antibody or antigen-binding fragment of any one of the preceding embodiments, wherein the antibody is an IgG.

[0100] 15. The antibody or antigen-binding fragment of embodiment 15, wherein the antibody is IgG1 or IgG4.

[0101] 16. The antibody or antigen-binding fragment of any one of the preceding embodiments, wherein the antibody or antigen-binding fragment specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3.

[0102] 17. The antibody or antigen-binding fragment of embodiment 16, wherein the antibody comprises heavy chain complementarity determining regions (CDRs) 1-3 of SEQ ID NO: 13 and light chain CDRs 1-3 of SEQ ID NO: 15.

[0103] 18. The antibody comprises a heavy chain variable domain (V H ) amino acid sequence, and a light chain variable domain (V L 20. The antibody or antigen-binding fragment of embodiment 17, comprising the amino acid sequence:

[0104] 19. The antibody or antigen-binding fragment of embodiment 17 or 18, wherein the antibody comprises a human IgG4 constant region having a proline at position 228 (EU numbering).

[0105] 20. The antibody or antigen-binding fragment of embodiment 19, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 13, with or without the heavy chain C-terminal lysine, and the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 15.

[0106] 21. The antibody of embodiment 17, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 13, with or without the heavy chain C-terminal lysine, SEQ ID NO: 14, with or without the lysine immediately preceding the C-terminal D10 sequence, SEQ ID NO: 16, with or without the heavy chain C-terminal lysine, or SEQ ID NO: 17, with or without the lysine immediately preceding the C-terminal D10 sequence, and the light chain comprises the amino acid sequence of SEQ ID NO: 15, 19, 21, or 22.

[0107] 22. The antibody or antigen-binding fragment of embodiment 16, wherein the antibody is murine antibody 1D11 having the heavy and light chain amino acid sequences of SEQ ID NO: 2, with or without the C-terminal lysine, and SEQ ID NO: 6, respectively.

[0108] 23. An IgG4 antibody that binds to human TGFβ1, TGFβ2, and TGFβ3, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 14 (with or without the lysine immediately preceding the C-terminal D10 sequence) and the light chain comprises the amino acid sequence of SEQ ID NO: 15.

[0109] 24. An IgG4 antibody that binds to human TGFβ1, TGFβ2, and TGFβ3, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 17 (with or without the lysine immediately preceding the C-terminal D10 sequence) and the light chain comprises the amino acid sequence of SEQ ID NO: 15.

[0110] 25. The antibody or antigen-binding fragment of any one of the preceding embodiments, wherein the antibody or antigen-binding fragment shows at least a two-fold increase in localization to bone compared to an antibody having the same heavy and light chains but lacking the poly-D peptide.

[0111] 26. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of the preceding embodiments, and a pharma- ceutically acceptable excipient.

[0112] 27. A method for treating an individual having a bone condition that would benefit from inhibition of TGFβ, comprising administering to the individual an effective amount of an anti-TGFβ antibody or antigen-binding fragment of any one of claims 16-25.

[0113] 28. The method of embodiment 27, further comprising detecting at least one of: (1) a decrease in TGFβ levels; (2) a decrease in TGFβ activity; (3) a decrease in bone loss; (4) a decrease in the rate of bone loss; (5) an increase in bone density; (6) an increase in bone strength; and (7) a decrease in IL-11 levels.

[0114] 29. The antibody or antigen-binding fragment of any one of embodiments 16 to 25, for use in treating an individual having a bone condition that would benefit from inhibition of TGFβ.

[0115] 30. Use of an antibody or antigen-binding fragment according to any one of embodiments 16 to 25 for the manufacture of a medicament for treating an individual with a bone condition that would benefit from inhibition of TGFβ.

[0116] 31. The method according to embodiment 27, the antibody or antigen-binding fragment for use according to embodiment 29, or the use according to embodiment 30, wherein the individual is a human.

[0117] 32. The method, antibody or antigen-binding fragment for use, or use according to embodiment 31, wherein the human has osteogenesis imperfecta.

[0118] 33. The method, antibody or antigen-binding fragment for use, or use according to embodiment 31, wherein the human has bone loss or osteoporosis.

[0119] 34. The method, antibody or antigen-binding fragment for use, or use according to embodiment 31, wherein the human has chronic kidney disease.

[0120] 35. The method, antibody or antigen-binding fragment for use, or use according to embodiment 31, wherein the human is a cancer patient with bone metastases.

[0121] 36. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain, the light chain, or both, of the antibody or antigen-binding fragment according to any one of embodiments 1 to 25.

[0122] 37. An expression vector comprising the isolated nucleic acid molecule of embodiment 36.

[0123] 38. A host cell comprising an expression vector according to embodiment 37.

[0124] 39. The host cell according to embodiment 38, wherein the host cell is a mammalian cell.

[0125] 40. A method for producing an antibody or antigen-binding fragment according to any one of embodiments 1 to 25, comprising: providing a host cell comprising first and second nucleotide sequences encoding the heavy and light chains, respectively, of the antibody or antigen-binding fragment; growing the host cell under conditions that allow for the production of the antibody or antigen-binding fragment; Recovering the antibody or antigen-binding fragment. The manufacturing method comprising the steps of:

[0126] 41. The method of embodiment 40, wherein the first nucleotide sequence comprises SEQ ID NO: 23, 24, 25 or 26 (with or without the codon for the heavy chain C-terminal lysine) and the second nucleotide sequence comprises SEQ ID NO: 27, 29, 31 or 32.

[0127] 42. A method for producing a bone-targeting antibody or antigen-binding fragment, comprising: Providing an antibody or antigen-binding fragment thereof and one or more polyD peptides; and Attaching poly-D peptides to antibodies via covalent bonds by chemical conjugation The manufacturing method comprising the steps of:

[0128] 43. The method of any one of embodiments 40-42, further comprising formulating the antibody or antigen-binding fragment as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

[0129] 44. A method for producing a bone-targeting pharmaceutical composition, comprising: Providing an antibody or antigen-binding fragment according to any one of embodiments 1 to 25, and Mixing the antibody or antigen-binding fragment with a pharma- ceutically acceptable carrier. The manufacturing method comprising the steps of:

[0130] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0131] Working Example The following examples illustrate certain embodiments of the present invention and various applications thereof. They are provided for illustrative purposes only and should not be construed as limiting the invention.

[0132] TGFβ antibody The first anti-TGFβ antibody, referred to herein as mAb1, is a mouse IgG1 monoclonal antibody specific for human TGF-β1, TGF-β2, and TGF-β3 ("pan-specific") and is available from R&D Systems (clone #1D11, Minneapolis, MN). The mAb1 antibody served as the template in the examples.

[0133] The second anti-TGFβ antibody used in the examples, referred to herein as mAb2, is a human anti-TGFβ IgG4 antibody with the hinge mutation S228P (EU numbering). The mAb2 antibody is similar to the antibody disclosed in U.S. Pat. No. 9,090,685, which is incorporated herein by reference. The antibody mAb2 has a predicted molecular weight of 144KD when not glycosylated. The amino acid sequences of its heavy and light chains are SEQ ID NOs: 13 and 15, respectively. These two sequences are shown below. The variable domains are shown in italics and are designated herein as the heavy chain variable domain (HCVD, SEQ ID NO: 39) and the light chain variable domain (LCVD, SEQ ID NO: 40). The CDRs are indicated by boxes and are named heavy chain complementarity determining region 1 (HCDR1, SEQ ID NO:33); HCDR2 (SEQ ID NO:34); and HCDR3 (SEQ ID NO:35), and light chain complementarity determining region 1 (LCDR1, SEQ ID NO:36); LCDR2 (SEQ ID NO:37); and LCDR3 (SEQ ID NO:38). The glycosylation site in the heavy chain constant domain is in bold (N297).

[0134] [ka]

[0135] As described herein, other antibodies, antibody fragments, proteins, or peptides that bind to proteins involved in bone formation or maintenance may also be used. EXAMPLES

[0136] Preparation of chemical conjugates of D10 peptide and mAb1 α-TGFβ antibody The D10 peptide chemical conjugate of α-TGFβ antibody mAb1 was prepared as shown in Figure 2. mAb1 (2.0 mg) was exchanged into degassed borate buffer (25 mM sodium chloride, 1 mM DTPA, 20 mM sodium borate, pH 8.0) by three rounds of ultrafiltration using Amicon® Ultra 50 kDa MWCO centrifugal filters (EMD Millipore). The disulfides in the hinge region were then reduced with 12 mol:mol dithiothreitol (DTT) per mAb for 2 hours at 37°C. The product was desalted using Amicon® Ultra filters with 4 mL of degassed borate buffer. Aliquots (1 nmol, 150 μg) were reacted with increasing amounts (1-15 mol:mol) of D10-maleimide peptide (Ac-D10-C2-PEG12-C6-maleimide, where PEG12 consists of a defined length PEG containing 12 ethylene oxide groups) at 25°C. After 1.5 h, remaining unreacted thiol groups were blocked by the addition of 12 equivalents of N-ethylmaleimide, followed by incubation for 1.5 h. The products were desalted by ultrafiltration. Figure 3A shows SDS-PAGE of 0.5 μg of each product on a 4-12% NuPAGE gel stained with SimplyBlue™ (Thermo Scientific) and imaged with an Odyssey® near-infrared scanner (LiCor). Lane fluorescence profiles were integrated using AlphaView software (ProteinSimple Corp.) to determine peptide-to-antibody ratios (PARs). The small shift in mobility in each of the heavy or light chains is assumed to represent the addition of a single D10 peptide, which is consistent with a maximum of five distinguishable bands for the heavy chain and one for the light chain, consistent with the number of hinge region cysteines in each. The average number of peptides on the heavy and light chains was calculated separately from the sum of the product of the relative abundance of each minor band and its assigned peptide number. Since there are two of each chain in whole IgG, the PAR was calculated from the sum of twice their numbers from the heavy and light chains.The PAR values ​​versus peptide-maleimide:mAb ratios in the chemical conjugation reactions are shown in Figure 3B, which shows a linear increase in PAR with increasing numbers of conjugated peptides up to 8 mol:mol. EXAMPLES

[0137] Binding of mAb1-D10 chemical conjugate to TGF-β1 In this example, a panel of chemical conjugates of different PARs were prepared in the same manner as described in Example 1, except that the ratio of DTT was varied from 8 to 10 mol:mol and the maleimide-peptide:mAb was either 3 or 15 mol:mol. As a control, a human IgG1 (Herceptin®) chemical conjugate was prepared by reduction of its hinge disulfide with 3 mol:mol tris(2-carboxyethylphosphine) (TCEP) for 2 hours at 37° C. under argon, followed by reaction with 15 mol:mol maleimide-peptide overnight at 25° C., and purified by desalting using ultrafiltration.

[0138] To assess their ability to bind TGF-β1, the chemical conjugates were mixed with TGF-β1 at a 1:1 molar ratio followed by size-exclusion chromatography on a Superdex200 (GE Healthcare) in phosphate-buffered saline (PBS) at pH 7.2. The mAb1-D10 chemical conjugate alone produced a somewhat heterogeneous peak that eluted earlier than unmodified mAb1, as shown in Figure 4A, likely due to charge effects generated by the conjugated peptide. Addition of TGF-β1 to the conjugate produced a peak that eluted earlier than either mAb1 or the mAb1-D10 chemical conjugate, indicating the formation of a higher molecular weight complex (Figure 4B). Similarly, addition of TGF-β1 to the unmodified mAb1 antibody produced a shift to earlier retention times (Figures 4C, 4D). In contrast, chemical conjugation of D10 to Herceptin® caused a shift in the retention time of the antibody alone, similar to the mAb1 conjugate (Figures 4E, 4F), but addition of TGF-β1 (1 mole:mol) to the Herceptin®-D10 conjugate did not cause any change in its elution time or apparent MW (Figures 4F, 4G), indicating that binding to the conjugate did not occur as a result of conjugation. EXAMPLES

[0139] Peptide-dependent binding of mAb1-D10 chemical conjugates to hydroxyapatite In this example, chemical conjugates of D10 and mAb1 were generated by reducing the hinge region disulfides with 12 equivalents of DTT in 25 mM NaCl, 1 mM DTPA, 20 mM sodium borate, pH 8 for 2 hours at 37°C, followed by reaction with 2 mol:mol of 2,2'-dipyridyl disulfide (Sigma) to convert some of the free thiols to disulfides, as shown in Figure 2. This was followed by reaction with the D10-maleimide peptide as described in Example 1. The final product was purified by ultrafiltration. A portion (25 μg) was exchanged into 5 mM sodium phosphate, pH 7.4 on a spin column and chromatographed on a 100 μL column of ceramic hydroxyapatite (CHT) type II (BioRad) and eluted with a gradient of 5-500 mM sodium phosphate, pH 7.4 at a flow rate of 0.5 mL / min. Unmodified mAb1 was used as a control. A 280 The column profile is shown in Figure 5A. Approximately half of the conjugate bound and eluted near 0.2 M phosphate, although mAb1 showed only trace binding to the column. Fractions were concentrated and analyzed by SDS-PAGE (Figure 5B). The unbound (flow-through or "FT") fraction represented mostly unmodified mAb, while the main peak 4 contained a conjugate with an estimated PAR of 6 by SDS-PAGE. EXAMPLES

[0140] Effect of sequential peptide loading on binding to hydroxyapatite In this example, a series of D10 peptide chemical conjugates with various numbers of peptides as described in Example 1 were chromatographed on a CHT Type II column as described in Example 3. 280 The profile and plot of percent bound conjugate versus peak retention time are shown in Figure 6A. As can be seen in Figure 6B, The amount of conjugate bound increased up to a PAR of 3.8, plateaued, and began to decrease for more peptides. In contrast, the retention time of the conjugate on the resin (indicative of the strength of the interaction) increased with increasing peptide numbers up to 9. EXAMPLES

[0141] Effect of D10 peptide chemical conjugation to mAb1 on its ability to neutralize TGF-β1 in vitro In this example, a set of three mAb1-D10 conjugates was prepared as described in Example 1, as shown in FIG. 2. A control conjugate was prepared by omitting the D10-maleimide peptide during conjugation. The peptide loading (PAR) was determined to be 0, 5 or 9 for these three conjugates by SDS-PAGE analysis as shown in FIG. 3. The ability of these conjugates to inactivate TGF-β1 was then determined by co-incubation with TGF-β1 for 1 hour. Serial dilutions in growth medium were then added to human A549 cells expressing the human TGF-β1 receptor, followed by overnight incubation. The cellular response to active TGF-β1 was then determined by the release of IL-11 into the growth medium, which was detected by an ELISA assay specific for IL-11. The IL-11 response for the three conjugates and the mAb1 control is shown in FIG. 7. In all cases, half-maximal inhibition of IL-11 release (EC 50 ) occurred. Both conjugates (PAR=5 and PAR=9) and the mock conjugate lacking peptide (PAR=0) showed slightly better inhibition than mAb1. The number of conjugated peptides increased with the EC 50 had no effect on EXAMPLES

[0142] Effect of conjugated D10 peptide on the biodistribution of mAb1 administered to mice D10-mAb1 chemical conjugate was prepared as described in Example 1, as shown in Figure 2. mAb1 (19.6 mg) was exchanged into degassed borate buffer and reacted with 10.6 equivalents (1.06 μmol) of DTT for 1.5 hours at 37°C, followed by reaction with 6 equivalents of D10 peptide (Ac-D10-C2-PEG12-C6-maleimide) for 1.5 hours at 25°C under argon without buffer exchange. Free unconjugated thiols were blocked by addition of 12 equivalents of N-ethylmaleimide, followed by incubation at 25°C for 30 minutes. The final product was purified by ultrafiltration. SDS-PAGE was used to determine the peptide-to-antibody ratio (PAR of approximately 4.8). The light chain showed a minor band corresponding to the addition of a single peptide to a light chain cysteine ​​residue.

[0143] Conjugates and mAb1 antibody controls were labeled separately with AlexaFluor® 750 (Thermo Fisher) using conditions described by the manufacturer. Fluorescent test articles were administered to SKH-1 hairless mice, which were imaged with an IVIS instrument (Perkin Elmer) immediately after administration (0.3-1 h), and 4 h, as well as 1, 2, 3, 4, 7, 8, and 9 days later. Images of mice injected with 1 mg / kg labeled mAb1 and conjugate are shown in Figure 8A. Fluorescence of labeled mAb1 was distributed evenly throughout the body, and this pattern was maintained over 9 days, although with a decrease in intensity. In contrast, after 1 day, the mAb1-D10 conjugate was concentrated at or near the dorsal midline, limbs, and tail, consistent with localization to bone, but at barely detectable levels elsewhere in the body. Consistent with the images in FIG. 8A, the ratio of distal femur fluorescence compared to heart fluorescence measured in the images in FIG. 8A was significantly elevated by 24 hours (P<0.05) and remained significantly elevated up to 168 hours for the mAb1-D10 conjugate compared to the control (see FIG. 8B). The change was slight and was maintained over the nine days. EXAMPLES

[0144] Production of recombinant mouse anti-TGFβ-D10 antibody fusion variant A panel of recombinant anti-TGFβ-D10 peptide fusion variants shown in Figures 9A and 9B were produced as follows.

[0145] Heavy and light chain expression vector constructs A set of plasmids was generated for expressing mAb1 heavy and light chains with D10 peptides at various positions. In addition, two plasmids were generated for wild-type mAb1 heavy and light chains without D10 peptide. All codon-optimized sequences were synthetically generated (GeneArt), flanked by appropriate restriction enzyme sites designed in frame for cloning purposes. Three gene fragments encoding the mouse IgG1 constant region with or without the D10 peptide at the C-terminus, and the entire mAb1 wild-type light chain were cloned into the empty episomal mammalian expression vector pFF, an analogue of the pTT vector described by Durocher et al. (2002, Nucl. Acids Res. 30(2):E9), using ApaLI / HindIII restriction enzymes and subsequent ligation to generate "mIgG1_CH123_pFF", "mIgG1_CH123D10_pFF" and "mAb1_VLCL_pFF". Gene fragments encoding the variable regions with or without the N-terminal D10 peptide were cloned into the vector using ApaLI / EagI for the heavy chain and ApaLI / MfeI for the light chain. Gene fragments encoding the constant region of mouse Ig kappa light chain with C-terminal D10 and different lengths of G4S (SEQ ID NO: 9) spacer were cloned into "mAb1_VLCL_pFF" using MfeI / HindIII to replace the wild-type constant region. The correct DNA sequence expected for each construct was confirmed by DNA sequencing (ACGT, Inc.).

[0146] Construction and expression of fusion variants Fusion variants were made by selecting one of each of the heavy and light chain vectors for co-transfection to generate the fusion variants and no peptide control described in Table 1. The "PAR" (peptide to antibody ratio) reflects the total number of peptides expected to be added to the final expressed antibody in both its heavy and light chains. The fusion variant ID "mAb1 F1" represents the "wild type" ("wt") construct that is substantially identical to the mAb1 sequence without modification. "HC" represents the heavy chain of mAb1, "LC" represents the light chain of mAb1, "D10" represents the D10 peptide (SEQ ID NO:1), and "G4S" represents the spacer sequence consisting of gly-gly-gly-gly-ser (SEQ ID NO:9) that is incorporated into some constructs.

[0147] [Table 1]

[0148] To assess the ability of the desired recombinant fusion variants to be expressed, 16 variants from Table 1 were evaluated by co-transfection into Expi293F™ cells (Life Technologies) using conditions described by the manufacturer. After 4 days, expression was measured by SDS-PAGE of conditioned media. All variants were expressed at levels estimated to be 10-30 μg / mL. Slightly higher levels were observed after 5 and 7 days, but expression levels were variant-dependent. WT (mAb1 F1), HC-D10:LC-D10 (mAb1 F8), and HC-D10:LC-G4S-D10 (mAb1 F9) showed particularly high expression, whereas D10-HC:D10-LC (mAb1 F12) was poorly expressed.

[0149] Larger scale expression All 20 recombinant mAb1-D10 fusion variants were expressed in Expi293-F cells at a 30 mL scale. Conditioned medium (CM) was collected on day 6 and expression levels were assessed by non-reducing SDS-PAGE. Compared to the initial evaluation, slightly higher levels of expression (30-150 μg / ml) were observed, but the relative expression levels were consistent with smaller scale transfections. EXAMPLES

[0150] Characterization of recombinant mAb1-D10 fusion variants Expression levels and TGF-β1 binding Quantification of expression levels and ability of recombinant mAb1-D10 fusion variants to bind TGF-β1 was assessed using Octet® QK384. Octet® biosensors for mouse IgG were immersed in conditioned medium diluted 1:10 in sample diluent (PBS containing 0.01% BSA and 0.02% Tween 20, pH 7.4) or two-fold serial dilutions of purified mAb1 standard ranging from 1.25 to 100 μg / mL. Binding data were collected for 2 min while samples were shaken at 500 rpm. Titer was calculated using a four-parameter fit of the initial binding rates and comparison with those obtained from the standards. The biosensors were then washed with diluent for 1 min to remove the medium and re-establish a baseline, and then immersed in wells containing 40 nM TGF-β1 for 3 min at 1000 rpm to track binding. This was followed by a dissociation step by transferring the sensor to diluent for 3 min at 1000 rpm. As can be seen in Table 2, the concentrations of the fusion variants in the medium ranged from 11 to 178 μg / mL ("Octet concentration"), generally consistent with the trends observed by SDS-PAGE ("Expression level"). Furthermore, all of the fusion variants were able to bind TGF-β1, suggesting that the presence of the D10 peptide at any of the indicated positions did not severely affect antigen binding.

[0151] Mouse FcRn binding Mouse FcRn binding by recombinant mAb1-D10 fusion variants was evaluated using Octet QK384. All steps were performed at 1000 rpm. Conditioned medium diluted to achieve an antibody concentration of 10 μg / mL was contacted with anti-mouse IgG Fv biosensor for 5 minutes. The biosensor was then immersed in PBSP pH 6.0 (50 mM sodium phosphate, pH 6.0, 150 mM sodium chloride, 0.005% surfactant PO) to establish a baseline. The sensor was then transferred to a well containing soluble mouse FcRn diluted to 1 μM in PBSP pH 6.0 for 3 minutes, followed by dissociation in a well containing PBSP pH 6.0 for 3 minutes. As shown in Table 2, all variants bound to mouse FcRn.

[0152] Protein G binding Binding to Protein G by recombinant mAb1-D10 fusion variants was assessed using Octet QK384. Protein G biosensors were immersed in wells containing conditioned medium diluted to 10 μg / mL antibody in sample diluent and the signal was followed for 3 min at 1000 rpm. As shown in Table 2, all variants bound to Protein G.

[0153] [Table 2]

[0154] SDS-PAGE Purified WT construct and recombinant fusion variants mAb1 F2, mAb1 F6, mAb1 F7, mAb1 F11, mAb1 F12, mAb1 F16, and mAb1 F17 was analyzed on a 4-20% Tris-Glycine SDS-PAGE gel (Novex, Life Sciences) under reducing and non-reducing conditions and stained with Coomassie Blue. Visible light images collected by a ProteinSimple® Imager are shown in Figure 10. A slight reduction in mobility of the heavy and / or light chains was observed, consistent with the expected presence and number of D10 peptides for each. No impurities or covalent aggregates were detected.

[0155] Thermal stability by differential scanning fluorometry (DSF) The thermal stability of several recombinant mAb1-D10 fusion variants, representing all possible combinations of D10 peptides at the heavy and light chain termini, was determined by differential scanning fluorimetry using SYPRO® Orange (Thermo Scientific) as a reporter dye. The stability of the proteins at higher temperatures is predictive of their stability under typical storage conditions and therefore is useful for the manufacture and use of therapeutic agents. It is generally accepted that thermal stability can be used to evaluate their suitability for use. Thermal stability is assessed on a real-time PCR machine using dyes that show an increase in fluorescence upon binding to hydrophobic regions exposed by unfolding of the protein structure (Lo et al., 2004, Anal. Biochem. 332(1):153-9). Fluorescence of samples (10 μL at 0.5 mg per ml of protein) containing a 1:1000 dilution of SYPRO® Orange was followed at increasing temperatures on a CFX96 real-time PCR detection system. Data were analyzed using CFX Manager 3.0 (Bio-Rad Laboratories). Figure 11 shows the rate of change of fluorescence with temperature for several variants. Rate of change rather than absolute fluorescence is generally used to isolate dye binding to parts of the protein structure that are not affected by the structural transition. Negative deviations reflect an increase in the rate of fluorescence change, and the minimum represents the midpoint ("Tm") of the transition to the unfolded state. Several of the variants tested (mAb1 F6, mAb1 F11, mAb1 F16) showed Tm profiles indistinguishable from the unmodified antibody control ("WT" = mAb1 F1). The other two (mAb1 F2 and mAb1 F7) showed a significant decrease in Tm for the major transitions. Two variants (mAb1 F12 and mAb1 F17) showed the lowest Tm values. Notably, all four recombinant mAb1-D10 fusion variants containing the D10 peptide at the N-terminus of the light chain showed a significant decrease in Tm, suggesting that the placement of the peptide at this position on the mAb1 antibody destabilizes its structure. Conversely, the placement of the D10 peptide at either end of the heavy chain did not correlate with any change in Tm. The Tm values ​​of the predominant transitions for several fusion variants observed as shown in Figure 11 are summarized in Table 3.

[0156] [Table 3] EXAMPLES

[0157] Potency of recombinant mAb1-D10 fusion variants in neutralizing TGF-β1 in vitro The potency of recombinant mAb1-D10 fusion variants in neutralizing TGF-β1 activity was determined by in vitro inhibition of IL-11 secretion by A549 tumor cells in response to TGF-β1 added to the culture medium. The procedure was performed as described in Example 3. Several representative fusion variants from Example 7 were selected based on their affinity for protein G (as an indication of their ease of purification). The TGF-β1 inhibition profiles for eight variants (mAb1 F1, mAb1 F2, mAb1 F6, mAb1 F7, mAb1 F11, mAb1 F12, mAb1 F16, and mAb1 F17) are shown in Figures 12A and 12B. All fusion proteins had ECs similar to those of the mAb1 control. 50 showed. EXAMPLES

[0158] Binding of TGF-β1 by recombinant fusion variants The recombinant mAb1-D10 fusion variants of Example 7 were also quantitatively assessed for TGF-β1 binding using surface plasmon resonance (SPR) detected by Biacore®. The equilibrium constant K DTo determine the affinity of purified variants, samples containing the variants were passed over a sensor chip with amine-coupled TGF-β1. A target immobilization level of TGF-β1 below 100 response units (RU) was chosen to minimize potential avidity effects arising from neighboring TGF-β1 molecules on the chip. The level of immobilization was determined by the change in response units after NHS / EDC activation of the chip surface and before quenching with ethanolamine. Fusion variants, WT fusion variant F1, and mAb1 control were diluted to 30, 10, 3, 1, and 0.37 nM in HBS-EP buffer and passed over the TGF-β1 chip at 30 μL / min for 3 min, followed by dissociation in the same buffer for 5 min. Between tests, the chip surface was regenerated and bound antibodies were removed by passing two 30 s injections of 40 mM HCl over the chip at a flow rate of 75 μL / min. As shown in Table 4, the equilibrium constants (K D ) was within 2.4-fold of the mAb1 control. Unexpectedly, the maximum signal (RU) induced by the fusion variants decreased with increasing number of peptides, with variant F17 (D10-HC-D10 / D10-LC, PAR=6) showing the lowest signal. This is in contrast to the potency assay in A549 cells described in Example 9, which showed that all fusion variants tested were comparable or more potent than either mAb1 or the WT construct in neutralizing TGF-β1. A possible explanation for this decrease could be reflected from electrostatic repulsion between the chip matrix (carboxymethyldextran) and the negatively charged D10 peptide.

[0159] [Table 4] EXAMPLES

[0160] Binding of recombinant mAb1-D10 fusion variants to hydroxyapatite The recombinant mAb1-D10 fusion variants from Example 7 were also tested for binding to a ceramic hydroxyapatite (CHT) column as described in Example 3 to assess their potential to bind to bone mineral structures. Recombinant mAb1-D10 fusion variants (25 μg each in 5 mM sodium phosphate pH 7.4) were applied to a 100 μL column of hydroxyapatite (CHT type II, BioRad) and incubated with 0.005–0.5 M N phosphate. a + The proteins in the mobile phase eluted from the column were analyzed using a gradient of A 280 To ensure consistency of column performance, a standard (usually mAb1-D10 chemical conjugate) was tested at the beginning and end of each set of experiments. Figure 13 shows the A of each experiment. 280 The profiles are shown in Table 5 below. Retention times (RT) and bound fractions are shown. All but one of the recombinant fusion variants bound nearly quantitatively, but showed differences in affinity, as reflected by their retention times. The rank order of retention times did not correlate well with the PAR, suggesting that the position of the peptide significantly affected binding. Notably, the strongest binding (highest RT) was observed for variant F7 (PAR4), while variant F17 (PAR6) showed slightly weaker binding. One variant (F6, PAR2) showed weaker binding than most of the PAR4 variants, but was significantly better than the other PAR2 variants (F2 and F11). One variant (F2) with a peptide only at the N-terminus of the light chain showed only weak binding (23%), as reflected by its RT and bound fraction. The mAb1-D10 chemical conjugate ("CC"; PAR of about 4) produced two peaks, with 84% eluting at an earlier RT than all PAR4 fusion variants.

[0161] [Table 5] EXAMPLES

[0162] Biodistribution of recombinant fusion variants in mice The biodistribution of a selected subset of recombinant mAb1-D10 fusion variants (F6, F16, and F17) and mAb1-D10 chemical conjugates, generated as described in Example 6, was determined after tail vein injection in CD-1 mice. Recombinant variants were selected based on several factors, including expression and purification yield, TGF-β1 binding affinity, cellular potency, and binding to hydroxyapatite. These variants included examples containing 2, 4, or 6 D10 peptides to assess whether targeting recapitulates binding to hydroxyapatite or whether it is a function of the number of peptides. Proteins were expressed in HEK293 cells and purified on protein A. The three recombinant mAb1 fusion variants were analyzed in detail in vitro and in vivo. In these, the D10 peptide was recombinantly added either only to the C-terminus of the heavy chain (mAb1 F6), to both the N- and C-termini of the heavy chain (mAb1 F16), or to the N- and C-termini of the heavy chain and the C-terminus of the light chain (mAb1 F17). The mAb1 F6, F16 and F17 recombinant variants have peptide-to-antibody ratios (PAR) of 2, 4 and 6, respectively. The mAb1-D10 peptide chemical conjugate (PAR of about 4.8), which showed bone targeting in the study in Example 6, was selected as a positive control.

[0163] Recombinant fusion variants and chemical conjugates were labeled by reaction with Dylight® 800-4×PEG NHS ester (Thermo Scientific) at a dye:protein molar ratio of approximately 5:1 in 50 mM sodium borate pH 8.65. The degree of labeling (DOL) was maintained within 20% (approximately 1.2 mol:mol) by adjusting the dye:protein ratio. The labeled protein was then administered at 1 mg / kg to CD-1 mice via tail vein injection. Anesthetized animals were subsequently imaged with an IVIS small animal near-infrared imager (Perkin Elmer) at 24, 48, 168, and 504 hours (3 weeks) after administration. To confirm delivery to bone, femurs and vertebrae were collected from each group of animals at 240 and 504 hours.

[0164] As shown in the dorsal view of Figure 14, all mAb1-D10 fusion variants and chemical conjugates were enriched in the dorsal midline near the spine and remained there for 3 weeks (504 hours), although significant differences were observed between the signal intensities of the test articles. A region of interest (ROI) including the portion of the spine between the shoulder and pelvis was used for quantification, as shown in Figure 14. The maximum radiative efficiency within the ROI normalized to DOL was calculated and plotted as shown in Figure 15. The recombinant fusion variant F6, which has a D10 peptide at each of its heavy chain C-terminus, showed the highest level in the spine of all constructs over the entire course of the study (3 weeks). After 48 hours, the ranking of signal intensity within the ROI was variant F6>variant F16>variant F17≈chemical conjugate, and this order was maintained throughout the course of the study (Figure 15). The area under the curve (AUC) of maximum radiant efficiency within the ROI over time minus background autofluorescence (from vehicle-only control animals) calculated using Phoenix® WinNonlin® software (Pharsight) is shown in Table 6. There was an 8-22-fold increase in bone exposure (AUC) compared to the mAb1 control, with moderate differences (up to 2.5-fold) between the D10-containing constructs. Variant F6 showed the highest increase compared to mAb1 (21.8-fold, p<0.05). Tissue half-life calculated using WinNonlin® similarly showed a greater than 10-fold increase in half-life in bone for variant F6 compared to mAb1 (p<0.05).

[0165] Vertebrae and femurs from representative animals of each cohort were isolated at 240 and 504 hours, separated from surrounding tissue, and imaged (Figure 16). The relative fluorescence intensities of these samples were consistent with the dorsal image signals observed in live animals, indicating that they reflect the presence of recombinant fusion variants and chemical conjugates in bone.

[0166] [Table 6] EXAMPLES

[0167] Production of recombinant human anti-TGFβ-D10 antibody fusion variants An expression vector for producing mAb2 with D10 sequence at the heavy chain C-terminus (human anti-TGFβ IgG4 antibody with hinge mutation S228P) (i.e., mAb2 HC-D10 / mAb2 wt LC (SEQ ID NO: 14 / SEQ ID NO: 15), which has a configuration corresponding to that in variant F6 (see FIG. 9B), hereafter referred to as mAb2 F6; see Table 7) was generated similarly as described in Example 7. Expi293F cells were transfected with miniprep DNA, conditioned medium (60 mL) was harvested after 6 days, and the protein product was purified on a HiTrap Protein A (GE Healthcare).

[0168] [Table 7] EXAMPLES

[0169] Biodistribution of human anti-TGFβ-D10 antibody fusion protein in mice Recombinant mAb2 variant F6 (mAb2 F6) and mAb2 control antibodies were labeled with AlexaFluor® 647 (Thermo Scientific) and administered intraperitoneally to C57BL / 6 mice at a dose of 1 mg / kg. After 24 and 96 hours, some mice were sacrificed and the spine and femur were excised and imaged with the IVIS instrument. Serum samples (10 μL) obtained at the time of sacrifice were imaged in parallel. The average total radioefficiencies for the distal femur (trabecular) ROI and lumbar spine are shown in Figures 17A and 17B. Table 8 summarizes the relative intensities. These results show a large increase in the amount of antibody in the lumbar spine and femur at 96 hours as a result of recombinant addition of D10 to mAb2, with a significant decrease in serum.

[0170] [Table 8] EXAMPLES

[0171] Pharmacokinetics of a single-dose murine anti-TGFβ-D10 antibody fusion protein in serum and bone in mice In this example, the pharmacokinetics of a mouse anti-TGFβ-D10 antibody fusion protein was measured in mice.

[0172] A single dose of mAb1 or recombinant mAb1 F6 (see Table 2) was administered intraperitoneally to G610C mice (osteogenesis imperfecta animal model; n=12 per time point) and blood samples were collected 4 hours or 2, 7, 15, 22, and 43 days after administration. Optimized ELISAs were utilized to detect and quantitate serum concentrations of relevant antibodies.

[0173] For bone imaging, a single dose of fluorophore-labeled mAb1, recombinant mAb1 F6, or various other D10 surrogates was administered intravenously to nude CD-1 mice (n=3 per time point) and in vivo optical imaging was performed 4 hours or 1, 2, 4, 7, 10, and 21 days after administration. Fluorescent images of the mouse spine were generated, which allowed for relative test article comparison between mAb1 and mAb1 F6 in bone (not shown).

[0174] The pharmacokinetic profiles in serum and bone are seen in Figures 18A and 18B, respectively, and give the pharmacokinetic parameters in Table 9 below.

[0175] The results revealed a fundamental contrast in pharmacokinetics between mAb1 and mAb1 F6 in serum and bone after a single dose. mAb1 F6 showed 13-fold lower AUC(exposure) in serum and 22-fold higher exposure in bone compared to mAb1. In addition, mAb1 F6 has a 14-fold shorter t than mAb1 in serum 1 / 2 Finally, in bone, mAb1 F6 showed an 11-fold longer t than mAb1. 1 / 2and correspondingly showed 17-fold slower clearance. These attributes may be advantageous for the human form of mAb1-D10 in clinical areas where peripheral (serum) inhibition of TGFβ may be undesirable from a safety standpoint, while higher exposure in bone may enhance efficacy.

[0176] [Table 9] EXAMPLES

[0177] Peak-trough serum pharmacokinetics of multiple doses of murine anti-TGFβ-D10 antibody fusion protein in mice In this example, a multiple-dose peak-trough pharmacokinetic study was performed in an animal model of osteogenesis imperfecta.

[0178] mAb1 and mAb1 F6 (see Table 2) were administered intraperitoneally to G610C mice (n=10) at concentrations of 0.3 mg / kg and 1 mg / kg three times per week for 8 weeks (total of 24 doses) and blood samples were taken after dose 1 and dose 23 (at the beginning and end of the study), 24 hours and 48 hours. Results are shown only for the 1 mg / kg dose (see Figure 19). Optimized mass spectrometry assays were utilized to detect and quantitate serum concentrations of relevant antibodies.

[0179] The results are displayed in Table 10 below. The results revealed a fundamental contrast in pharmacokinetics between mAb1 and mAb1 F6 in serum after both doses 1 and 23. Significantly lower serum concentrations were observed for mAb1 F6 compared to mAb1 at both 24 and 48 hours post-dose for both doses 1 and 23. In addition, the slope between 24 and 48 hours post-dose for mAb1 F6 was steeper compared to mAb1 for both doses 1 and 23, suggesting that mAb1 F6 leaves serum (systemic circulation) at a faster rate than mAb1, likely due to its higher affinity for bone (hydroxyapatite). Finally, while both mAb1 F6 and mAb1 appear to accumulate in serum from doses 1 to 23, mAb1 F6 appears to accumulate at a reduced concentration compared to mAb1 (mAb1 F6: 2.5-3.5-fold accumulation, mAb1: 4-5.5-fold accumulation from doses 1 to 23). These attributes may be advantageous for a human form of mAb1 F6 in clinical areas where peripheral (serum) inhibition of TGFβ may be undesirable from a safety perspective.

[0180] [Table 10] EXAMPLES

[0181] Multiple-dose efficacy study in the lumbar spine with mAb1 and mAb1 F6 In this example, a multiple-dose efficacy study was performed in an animal model of osteogenesis imperfecta to determine the efficacy of bone-targeted (mAb1 F6) versus non-targeted mAb1 on bone density and strength.

[0182] mAb1 and mAb1 F6 were administered intraperitoneally to G610C mice at 0.3, 1 and 5 mg / kg, three times weekly for 8 weeks. After the final administration, mice were necropsied and the 6th hip bone was imaged by μCT to determine bone volume relative to total body volume (BV / TV) and subjected to biomechanical testing to ascertain maximum breaking force (bone strength).

[0183] Results are shown in Figures 20 and 21. Significant changes in BV / TV (%) were observed for both treatments at doses of 1 and 5 mg / kg compared to G610C mice treated with 13C4. Significant changes in maximum breaking force were observed for mAb1 F1 at 1 and 5 mg / kg, and for mAb1 at 1 mg / kg only, compared to G610C mice treated with 13C4. G610C mice treated with antibody control (13C4) showed a significant decrease in both BV / TV and maximum breaking force compared to the WT background strain. These results demonstrate that both treatments induce similar dose-related changes in BV / TV and maximum breaking force in G610C mice with this regimen of three times weekly administration. There was indeed a trend towards improved efficacy of mAb1 F6 on bone strength, as half of the 5 mg / kg cohort of mice exhibited substantially higher maximum breaking force values ​​(>40 Newtons) than mice treated with mAb1 at 1 mg / kg or 5 mg / kg. EXAMPLES

[0184] Dosing frequency study in the lumbar spine using mAb1 and mAb1 F6 In this example, a dose frequency study was performed in an animal model of osteogenesis imperfecta to determine the appropriate dose frequency for mAb1 F6 and to achieve its optimal efficacy of the bone-targeted antibody.

[0185] mAb1 and mAb1 F6 were administered intraperitoneally at 5 mg / kg to G610C mice at various frequencies (three times weekly, once weekly, once every two weeks, or once every four weeks) for 12 weeks. Pharmacokinetic (PK) serum samples were taken at the beginning and end of the study to ascertain peak and trough levels for both mAb1 and mAb1 F6. After the final dose, mice were necropsied and the 6th hip bone was imaged by μCT to determine bone volume relative to total volume (BV / TV) and subjected to biomechanical testing to ascertain maximum breaking force (bone strength).

[0186] The results are shown in Figures 22, 23 and 24. For mAb1, significant changes in BV / TV (%) were observed at 3 times per week, 1 time per week, 1 time per 2 weeks and 1 time per 4 weeks compared to G610C mice treated with 13C4. For mAb1 F6, significant changes in BV / TV (%) were observed at 3 times per week and 1 time per week compared to G610C mice treated with 13C4. mAb1 treatment showed significantly higher BV / TV compared to mAb1 F6 treatment at dosing frequencies of 1 time per 2 weeks and 1 time per 4 weeks. For mAb1, significant changes in maximum breaking force compared to G610C mice treated with 13C4 were observed at 3 times per week, 1 time per week and 1 time per 2 weeks. For mAb1 F6, significant changes in maximum breaking force compared to G610C mice treated with 13C4 were observed at 3 times per week and 1 time per week. G610C mice treated with control antibody (13C4) showed a significant decrease in BV / TV and a trend towards lower maximum breaking force compared to the WT background strain.

[0187] These results demonstrate that both mAb1 and mAb1 F6 can induce similar maximal effects in BV / TV and maximum breaking force in G610C mice. mAb1 appears to have an advantage in durability of efficacy compared to mAb1 F6, maintaining significant efficacy when administered once every 4 weeks for BV / TV and once every 2 weeks for maximum breaking force. However, the average PK serum samples in equally effective dosing regimens (mAb1, once every 2 weeks; mAb1 F6, once weekly) resulted in approximately 38 μg / mL and 8 μg / mL for mAb1 and mAb1 F6, respectively. This suggests that serum exposure may be less with mAb1 F6, which may provide a safety advantage to OI patients. EXAMPLES

[0188] Administration frequency study in the lumbar spine using mAb1 F16 In this example, a dose frequency study was performed in an animal model of osteogenesis imperfecta to determine the appropriate dose frequency for mAb1 F16 and achieve its optimal effect on bone density.

[0189] mAb1 and mAb1 F16 were administered intraperitoneally to G610C mice at 5 mg / kg, three times weekly for 8 weeks. After the final administration, mice were necropsied and the 6th hip bone was imaged by μCT to determine bone volume relative to total body volume (BV / TV).

[0190] The results are shown in Figure 25. Significant changes in BV / TV (%) were observed for both mAb1 and mAb F16 at 5 mg / kg compared to G610C mice treated with 13C4. G610C mice treated with control antibody (13C4) showed a significant decrease in BV / TV compared to the WT background strain. These results demonstrate that both mAb1 and mAb F16 induce similar dose-related changes in BV / TV in G610C mice under this dosing regimen. EXAMPLES

[0191] Dosage frequency study in bone using mAb1 F11 In this example, a dosing frequency study was performed in wild type mice to determine the appropriate dosing frequency for mAb1 F11 and achieve its optimal effect on bone density.

[0192] mAb1 and mAb1 F11 were administered intraperitoneally to wild-type mice at 5 mg / kg, three times weekly for 11 weeks. In vivo μCT was taken at several time points during the in-life portion of the study. Data are presented for bone volume relative to total volume (BV / TV%) only at 9 weeks post-treatment.

[0193] The results are shown in Figure 26. Significant changes in BV / TV (%) were observed for both treatments at a dose of 5 mg / kg compared to wild-type mice treated with 13C4. These results demonstrate that both mAb1 and mAb1 F11 induce similar dose-related changes in BV / TV in wild-type mice at this dosing regimen. EXAMPLES

[0194] Biodistribution of mouse and human bone-targeted anti-TGF-β antibodies mAb1 F6 and mAb2 F6 in mice In this example, a study was performed to compare the biodistribution of fluorescently labeled mAb1, mAb1 F6, mAb2, and mAb2 D10 (D10 conjugated to the heavy chain C-terminus of mAb2; mAb2 F6) in wild-type mice. Mice were administered a single intraperitoneal dose of each test article and vehicle, and were euthanized at various time points for tissue collection. Among other tissues harvested (data not shown), lumbar spine, heart, liver, and intestine were collected at 1, 4, 10, 20, 43, and 98 days after administration of mAb1 and mAb1 F6. Tissues were also sampled at 24 and 96 hours after administration of mAb2 and mAb2 D10.

[0195] The results are shown in Figures 27-33. Figures 27-30 show the total radiative efficiency (TRE) in tissues from mice administered mAb1, mAb1 F6, or vehicle at days 1-98 post-treatment. The lumbar spine shows a robust and persistent presence of mAb1 F6 compared to mAb1, with a significantly higher total radiative efficiency (TRE) at each time point. In the heart and liver, mAb1 F6 shows a much lower TRE compared to mAb1. Finally, in the intestine, no significant differences were observed between vehicle and any of the test articles.

[0196] These results suggest that mAb1F6 is more potent in other tissues (e.g., heart and liver). The results demonstrate that TGF-β is characterized by high bone affinity, which in turn results in lower exposure to TGF-β. The results also demonstrate the safety benefits of targeting the site of TGF-β inhibition to the bone, while limiting systemic TGF-β inhibition and reducing adverse side effects. In the intestine, the absence of any TRE compared to the vehicle reveals that the fluorophore maintained its label on the respective antibody. Previous data have shown that if the fluorophore did not maintain its label on the antibody, it would be detected in the intestine.

[0197] Figures 31-33 show TRE in tissues from mice administered either mAb2 or mAb2 D10. Figure 31 demonstrates the higher bone affinity of mAb2 D10 compared to mAb2. Significantly, higher TRE is observed in the femur for mAb2 D10 compared to mAb2. The same overall trend was observed in the lumbar spine. The lumbar spine / serum and femur / serum ratios in mice administered mAb2 also strongly support the bone targeting ability of mAb2 D10 compared to the ratios in mice administered mAb2 (Figures 32 and 33).

[0198] Although the present invention has been described in detail and with reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the present invention as defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as being particularly advantageous, it is contemplated that the present invention is not necessarily limited to these specific aspects of the present invention. In some embodiments, the values ​​disclosed herein may alternatively vary in amounts of ±10, 20, or 30% from the disclosed values ​​and still remain within the scope of the contemplated invention.

[0199] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in practicing or testing the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, shall control. Although a number of documents are cited herein, this citation is not an admission that any of these documents form part of the common general knowledge in the art. Furthermore, unless otherwise required by context, singular terms shall include plurals and plural terms shall include singular. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Throughout this specification and the embodiments, the terms "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of other integers or groups of integers. Additionally, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an antibody" means one or more antibodies.

[0200] For purposes of describing and defining the present invention, the term "substantially" is utilized herein to express the degree of inherent uncertainty that results from a quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to express the extent to which a quantitative representation may vary from the stated standard without resulting in a change in the basic functionality of the subject matter at issue. It is used in the following cases.

[0201] As used herein, the term "about" refers to ±10% of a given amount, except that when referring to an indivisible object, such as an amino acid or other object where the amount in question would lose its identity, "about" refers to ±1 of the indivisible object. For example, about 2% water refers to 1.8%-2.2% water, while about 6 amino acid residues refers to 5-7 amino acid residues.

[0202] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination with each other or exclusively with each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z."

[0203] The sequences referred to herein are provided in Table 11 below and in the Sequence Listing.

[0204] [Table 11]

[0205] [Table 12]

[0206] [Table 13]

[0207]

Table 14

[0208]

Table 15

[0209]

Table 16

[0210]

Table 17

[0211]

Table 18

Claims

1. An antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, and one or more polyaspartic acid (poly D) peptides linked to the C-terminus of the heavy and / or light chain.

2. 2. The antibody or antigen-binding fragment of claim 1, wherein the one or more poly-D peptides are linked to the antibody or antigen-binding fragment by chemical conjugation.

3. 3. The antibody or antigen-binding fragment of claim 2, wherein the one or more poly-D peptides are conjugated to the heavy chain at the hinge region.

4. 4. The antibody or antigen-binding fragment of claim 2 or 3, wherein the one or more poly-D peptides are conjugated to the antibody or antigen-binding fragment by a polyethylene glycol (PEG) spacer.

5. The antibody or antigen-binding fragment of claim 1 , comprising a poly-D peptide associated with the amino acid sequence of the heavy or light chain.

6. The antibody or antigen-binding fragment of claim 5 , comprising a poly-D peptide attached to the N-terminus of the heavy chain.

7. The antibody or antigen-binding fragment of claim 5 , comprising a poly D peptide attached to the C-terminus of the heavy chain.

8. 6. The antibody or antigen-binding fragment of claim 5, comprising a first poly-D peptide associated with the N-terminus of the heavy chain and a second poly-D peptide associated with the C-terminus of the heavy chain.

9. The antibody or antigen-binding fragment of any one of claims 5 to 8, comprising a poly D peptide attached to the C-terminus of the light chain.

10. The antibody or antigen-binding fragment of any one of claims 5 to 9, wherein the poly-D peptide is fused to the heavy or light chain via a peptide linker.

11. The antibody or antigen-binding fragment of claim 10, wherein the peptide linker comprises 1 to 3 repeats of the amino acid sequence GGGGS (SEQ ID NO: 9).

12. The antibody or antigen-binding fragment of any one of claims 1 to 11, wherein the one or more poly-D peptides each independently comprise from 2 to 30 aspartic acid residues.

13. The antibody or antigen-binding fragment of claim 12, wherein the one or more poly-D peptides each contain 10 aspartic acid residues (SEQ ID NO: 1).

14. The antibody or antigen-binding fragment of any one of claims 1 to 13, wherein the antibody is an IgG.

15. The antibody is IgG １ or IgG ４ 16. The antibody or antigen-binding fragment of claim 15,

16. The antibody or antigen-binding fragment of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment specifically binds to one or more of TGFβ1, TGFβ2, and TGFβ3.

17. 17. The antibody or antigen-binding fragment of claim 16, wherein the antibody comprises heavy chain complementarity determining regions (CDRs) 1-3 of SEQ ID NO:13 and light chain CDRs 1-3 of SEQ ID NO:

15.

18. The antibody comprises a heavy chain variable domain (V Ｈ ) amino acid sequence of the light chain variable domain (V Ｌ 17. The antibody or antigen-binding fragment of claim 16, comprising the amino acid sequence:

19. The antibody is a human IgG with a proline at position 228 (EU numbering). ４ 19. The antibody or antigen-binding fragment of claim 17 or 18, comprising a constant region.

20. 17. The antibody or antigen-binding fragment of claim 16, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 13 and the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:

15.

21. The antibody of claim 17, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 13, 14, 16 or 17, and the light chain comprises the amino acid sequence of SEQ ID NO: 15, 19, 21 or 22.

22. 17. The antibody or antigen-binding fragment of claim 16, wherein the antibody comprises the heavy and light chain amino acid sequences of SEQ ID NOs: 2 and 6, respectively.

23. An IgG antibody that binds to human TGFβ1, TGFβ2, and TGFβ3, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO:14 and the light chain comprises the amino acid sequence of SEQ ID NO:

15. ４ antibody.

24. An IgG antibody that binds to human TGFβ1, TGFβ2, and TGFβ3, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO:17 and the light chain comprises the amino acid sequence of SEQ ID NO:

15. ４ antibody.

25. 25. The antibody or antigen-binding fragment of any one of claims 1 to 24, wherein the antibody or antigen-binding fragment exhibits at least a two-fold increase in localization to bone compared to an antibody having the same heavy and light chains but lacking the poly-D peptide.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 25 and a pharma- ceutically acceptable excipient.

27. 26. A method of treating an individual having a bone condition that would benefit from inhibition of TGFβ, comprising administering to the individual an effective amount of an anti-TGFβ antibody or antigen-binding fragment of any one of claims 16 to 25.

28. The antibody or antigen-binding fragment of any one of claims 16 to 25 for use in treating an individual having a bone condition that would benefit from the inhibition of TGFβ.

29. 26. Use of the antibody or antigen-binding fragment of any one of claims 16 to 25 for the manufacture of a medicament for treating an individual with a bone condition that would benefit from the inhibition of TGFβ.

30. The method of claim 27, the antibody or antigen-binding fragment for use of claim 28, or the use of claim 29, wherein the treatment results in at least one of: (1) a decrease in TGFβ levels; (2) a decrease in TGFβ activity; (3) a decrease in bone loss; (4) a decrease in the rate of bone loss; (5) an increase in bone density; (6) an increase in bone strength; and (7) a decrease in IL-11 levels in the individual.

31. The method according to claim 27, and the antibody for use according to claim 28, wherein the individual is a human. or an antigen-binding fragment thereof, or the use according to claim 29.

32. 32. The method, antibody or antigen-binding fragment for use, or use of claim 31 , wherein the human has osteogenesis imperfecta.

33. 32. The method, antibody or antigen-binding fragment for use, or use of claim 31 , wherein the human has bone loss or osteoporosis.

34. 32. The method, antibody or antigen-binding fragment for use, or use of claim 31 , wherein the human has chronic kidney disease.

35. 32. The method, antibody or antigen-binding fragment for use, or use of claim 31 , wherein the human is a cancer patient with bone metastases.

36. 26. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain, the light chain, or both, of the antibody or antigen-binding fragment of any one of claims 1 to 25.

37. 37. An expression vector comprising the isolated nucleic acid molecule of claim 36.

38. A host cell comprising the expression vector of claim 37.

39. The host cell of claim 38 , wherein the host cell is a mammalian cell.

40. A method for producing the antibody or antigen-binding fragment according to any one of claims 1 to 25, comprising the steps of: providing a host cell comprising first and second nucleotide sequences encoding the heavy and light chains, respectively, of the antibody or antigen-binding fragment; growing the host cell under conditions that allow for the production of the antibody or antigen-binding fragment; Recovering the antibody or antigen-binding fragment. The manufacturing method comprising the steps of:

41. 41. The method of claim 40, wherein the first nucleotide sequence comprises SEQ ID NO:23, 24, 25 or 26 and the second nucleotide sequence comprises SEQ ID NO:27, 29, 31 or 32.

42. A method for producing the bone-targeting antibody or antigen-binding fragment according to any one of claims 1 to 25, comprising: Providing an antibody or antigen-binding fragment thereof and one or more polyD peptides; and Attaching poly-D peptides to antibodies via covalent bonds by chemical conjugation The manufacturing method comprising the steps of:

43. 43. The method of any one of claims 40-42, further comprising formulating the antibody or antigen-binding fragment as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

44. A method for producing a bone-targeted pharmaceutical composition, comprising the steps of: Providing an antibody or antigen-binding fragment according to any one of claims 1 to 25, and Mixing the antibody or antigen-binding fragment with a pharma- ceutically acceptable carrier. The manufacturing method comprising the steps of:

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