WO2025081137A1 - Bone targeting therapeutics - Google Patents

Abstract

The present disclosure relates to bone targeting therapeutic conjugates and uses thereof. In particular, the disclosure relates to bone targeting therapeutic conjugates comprising a therapeutic antibody for treatment of a bone disease and a bone targeting moiety.

Description

BONE TARGETING THERAPEUTICS

PRIORITY STATEMENT

This application claims priority to U.S. Provisional Application No. 63/589,880, filed October 12, 2023, the entire contents of which are incorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AR069620 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The text of the computer readable sequence listing filed herewith, titled “UM_42458_601_SequenceListing.xml”, created October 11, 2024, having a file size of 82,744 bytes, is hereby incorporated by reference in its entirety.

FIELD

The present disclosure relates to bone targeting therapeutic conjugates and uses thereof. In particular, the disclosure relates to bone targeting therapeutic conjugates comprising a therapeutic antibody for treatment of a bone disease and a bone targeting moiety.

BACKGROUND

Affinity targeting of proteins to the bone surface has been reported for over two decades. However, only one bone-targeted biotherapeutic is now approved for human use. Moreover, little is known about the key features necessary for optimal protein delivery - either from the standpoint of the cargo protein (e.g., size, circulation time) or the affinity ligand (affinity, avidity, site of attachment). Accordingly, what is needed are bone-targeted therapeutic agents that retain functional activity when anchored to the bone surface.

SUMMARY In some aspects, provided herein are bone targeting therapeutic conjugates (referred to herein as “conjugates”). In some embodiments, bone targeting therapeutic conjugates comprise a therapeutic antibody for treatment of a bone disease, and a bone targeting moiety. In some embodiments, the therapeutic antibody for treatment of a bone disease is an anti-sclerostin antibody, an anti-RANKL antibody, or an anti-TGFp antibody.

Any suitable bone targeting moiety may be used. In some embodiments the bone targeting moiety is a bisphosphonate. For example, in some embodiments the bone targeting moiety is a bisphosphonate such as alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate, or zoledronate. In some embodiments the bone targeting moiety is a bone targeting peptide. In some embodiments, the bone targeting peptide is an acidic oligopeptide. For example, in some embodiments the bone targeting peptide is an acidic oligopeptide comprising 6-25 combined aspartic acid and/or glutamic acid residues. In some embodiments the bone targeting peptide is an acidic oligopeptide comprising 6-20 combined aspartic acid and/or glutamic acid residues. In some embodiments the bone targeting peptide is an acidic oligopeptide comprising 6-15 combined aspartic acid and/or glutamic acid residues. In some embodiments, the bone targeting peptide is an acidic oligopeptide comprising 10-20 aspartic acid residues. In some embodiments, the bone targeting peptide is deca-aspartate (Dio (SEQ ID NO: 1). In some embodiments, the bone targeting peptide is icosa-asparate (e.g. comprises 20 aspartate residues) (D20) (SEQ ID NO: 8). In some embodiments, the therapeutic antibody is for the treatment of osteoporosis.

In some embodiments, the at least one bone targeting moiety is conjugated to a heavy chain of the therapeutic antibody for treatment of a bone disease. In some embodiments, the at least one bone targeting moiety is directly conjugated to the heavy chain. In some embodiments, the at least one bone targeting moiety is conjugated to the heavy chain by a linker. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-4 repeating GGGGS (SEQ ID NO: 9) units.

In some embodiments, a first bone targeting moiety is conjugated to a first heavy chain of the therapeutic antibody for treatment of a bone disease and a second bone targeting moiety is conjugated to a second heavy chain of the therapeutic antibody for treatment of a bone disease. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each a bisphosphonate. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each an acidic oligopeptide. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each an acidic oligopeptide comprising 10-20 aspartic acid residues. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each deca-aspartate (Dio (SEQ ID NO: 1). In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each icosa-asparate (e.g. comprises 20 aspartate residues) (D20) (SEQ ID NO: 8).

In some embodiments, the first bone targeting moiety is directly conjugated to the first heavy chain and the second bone targeting moiety is directly conjugated to the second heavy chain of the therapeutic antibody for treatment of the bone disease. In some embodiments, the first bone targeting moiety is conjugated to the first heavy chain by a linker. In some embodiments, the second bone targeting moiety is conjugated to the second heavy chain by a linker. In some embodiments, the first bone targeting moiety is conjugated to the first heavy chain by a linker and the second targeting moiety is conjugated to the second heavy chain by a linker. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-4 repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker for the first bone targeting moiety and the linker for the second bone targeting moiety are different. In some embodiments, the linker for the first bone targeting moiety and the linker for the second bone targeting moiety are the same.

In some aspects, provided herein are methods of treating a bone disease in a subject. In some embodiments, methods of treating bone disease comprise providing a bone targeting therapeutic conjugate described herein (e.g. a bone targeting therapeutic conjugate comprising a therapeutic antibody for treatment of a bone disease and a bone targeting moiety) to a subject having or suspected of having a bone disease. In some embodiments, the bone disease is osteoporosis. In some embodiments, the therapeutically effective dose of the conjugate is less than a therapeutically effective dose of the therapeutic antibody for treatment of the bone disease. In some embodiments, the subject is a human. In some embodiments, the therapeutically effective dose of the conjugate is less than 3 mg/kg when the conjugate is provided to the subject monthly. In some embodiments, the conjugate is provided to the subject by injection.

DESCRIPTION OF THE DRAWINGS

FIGs. 1 A-1D show bisphosphonate targeting of fluorescent proteins. FIG. 1 A shows mCherry-Dio, Dio-mCherry structure and mCherry protein modification via sortase/sortag labeling enables site-specific modification with a single BP at N or C terminal of the protein. Sequences shown in FIG. 1A are LPETGG (SEQ ID NO: 3), HHHHHH (SEQ ID NO: 4), LPET (SEQ ID NO: 2), GSKGSLPETGG (SEQ ID NO: 5), and DDDDDDDDDD (SEQ ID NO: 1). FIG. IB, FIG. IC, FIG. ID, and FIG. IE are fluorescence microscopy images. FIG. IB shows that site-specific single-site protein modification with a single ALD at the C-terminus demonstrate robust binding to both hydroxyapatite and bovine bone chips and superior targeting to hydroxyapatite disks and bovine bone chip substrates compared to non-specific-NHS- modification and untargeted control. FIG. IC shows that site-specific modification and bisphosphonate conjugation enable efficient targeting of mCherry, GFP, and mCardinal proteins using the same method. FIG. ID shows that both C-terminal and N-terminal site-specific modification of mCherry and GFP result in effective targeting in vitro, while FIG. IE shows that only N-terminal Dio-mCherry showed bone-surface targeting .

FIGs. 2A-2D show a comparison of two bone localizing motifs in vivo. Mice were injected intravenously with 2 mg/kg mCherry- ALD, Dio-mCherry, or unmodified mCherry mixed with tracer amount of ¹²⁵I-labeled proteins. At 4 hours, organs were perfused transcardially. FIG. 2A is a graph showing that mCherry-ALD, DIO-mCherry and unmodified mCherry showed similar blood clearance and in vivo. FIG. 2B is a graph showing that all proteins showed rapid kidney clearance. FIG. 2C is a graph showing that both bisphosphonate and Dio affinity ligands resulted in selective accumulation on the bone in %ID/g on the femur and vertebrae. FIG. 2D are fluorescence images showing that both mCherry-ALD and D10- mCherry efficiently localize to the femur trabeculae four hours following administration in vivo. n = 5 for mCherry-ALD group, n = 3 for DIO-mCherry and unmodified mCherry group. Data presented as Mean ± SEM. ns = non-significant, * p < 0.05, ** p < 0.01. FIGs. 3A-3D show in vivo bone localization efficacy of BP-targeted mCherry. Mice were injected intravenously with 2 mg/kg or 5 mg/kg of ¹²⁵I-labclcd mChcrry-ALD. At 4 hours, organs were perfused transcardially. No significant difference were seen on blood pharmacokinetics (FIG. 3A), biodistribution to bone (FIG. 3B) or other organs (FIG. 3C). mCherry-ALD was injected at 5 mg/kg to healthy mice for time-course study. Fluorescence images are shown in FIG. 3D. mCherry-ALD gradually accumulate over the first few hours (peak at approximately 4 hrs post-injection). By 24 hours post-injection, the fluorescent signal has decreased, and there is an appearance of isolated green fluorescence (white arrowheads), suggesting potential inactivation and catabolism of bone- anchored mCherry. n = 5 for 2mg/kg mCherry-ALD group, n = 3 for 5mg/kg group. Data presented as Mean ± SEM. ns = nonsignificant.

FIGs. 4A-4G show bivalent binding and half-life extension greatly enhance bone accumulation in vivo. To investigate the role of binding avidity and circulation time, a bivalent mCherry-Fc fusion was synthesized, with the affinity ligand fused to the C-terminus of each heavy chain (FIG. 4A). This fusion is referred to as mCherry-Fc-Dio. FIG. 4B are fluorescence microscopy images showing that Dio-mCherry and mCherry-Fc-Dio target to the bone similarly compared to mCherry-Fc and unmodified mCherry . FIG. 4C is a graph showing blood PK analysis demonstrating marked prolongation of circulation time with mCherry-Fc and mCherry- Fc-DlO vs. mCherry-ALD and mCherry-Dio. The relatively small (but significant) difference between mCherry-Fc-Dio and mCherry-Fc presumably reflects target-mediated drug disposition (TMDD). FIG. 4D is a graph showing that mCherry-Fc-Dio shows an order of magnitude increase in %lD/g on bone at 4 hours compared to mCherry-ALD and mCherry-Dio, and FIG. 4E is a graph showing a further 2-3 fold increase in both femur and vertebrae at 24 hrs. FIG. 4F is a graph showing that a much lower dose accumulates in the kidney and spleen. FIG. 4G are fluorescence images demonstrating a continuous bone accumulation of mCherry-Fc-Dio observed at 24h compared to 4h. mCherry-Fc shows selective accumulation in bone as well. However, fluorescence imaging (scale = 200 um) suggests that this may result from marrow uptake, rather than accumulation at the bone surface. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n > 3 for each condition and time point. n=5 for 4h study, n=4 for 24h study. Data presented as Mean ± SEM. ns = non-significant. * p < 0.05, *** p < 0.001, **** p < 0.0001. FIGs. 5A-5E show characterization of anti-(mouse) Sclerostin-Dio. FIG. 5 A is an illustration depicting that anti-Sclcrostin (anti-Scl, also referred to as anti-SOST)) blocks sclerostin signaling on osteoblasts and osteoclasts, increasing bone mass. Anchoring of anti- SOST to the bone surface should increase local scavenging, but could theoretically decrease efficacy if systemic neutralization or release and diffusion away from the bone surface are required for in vivo function. FIG. 5B is a graph showing binding affinity to immobilized recombinant murine sclerostin as measured using ELISA. The two proteins demonstrate nearly identical affinities - anti-SOST (i.e. anti-Scl) (KD = 3.4nM) and anti-SOST-Dio (i.e. anti-Scl- Dio) (Ko=4.2nM). Female mice were injected intravenously with 5 mg/kg ¹²⁵I-labeled anti-Scl, and anti-sclerostin-Dio. At 24, 72 and 168 hours, organs were perfused transcardially. FIG. 5C shows clearance of anti-SOST and anti-SOST-Dio antibodies All proteins maintained efficient circulation, clearing the blood in 7 days. FIG. 5D shows antibody accumulation in the femur, and FIG. 4E shows antibody accumulation in the vertebrae. Selective accumulation of anti-Scl- Dio was seen in the femur and vertebrae at 1 day. Bone accumulation of anti-Scl-Dio increased from 3 days to 7 days post administration, whereas anti-Scl decreased to undetectable levels, n = 2 for binding affinity assay, n = 4 for in vivo animal study. Data presented as Mean ± SEM. ns = non-significant. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

FIG. 6 is a schematic showing an exemplary method of ovariectomy-induced Osteoporosis. Healthy adult female mice were subjected to a bilateral ovariectomy to develop an osteoporosis phenotype and treated with 5 mg/kg drug or vehicle control, weekly, by intravenous injection. After four weeks, mice were sacrificed, and tissues harvested for histomorphometric analysis by microcomputed tomography.

FIG. 7A-7I show results from microcomputed tomography analysis demonstrating the therapeutic efficacy of Anti-Scl-DlO in femur trabecular bone. FIG. 7A shows three-dimensional renderings of the region of interest. FIG. 7B, 7C, 7D, 7E, 7F, 7G, and 71 show quantitative analysis of various parameters. FIG. 7B shows bone volume to total volume fraction. FIG. 7C shows connective density. FIG. 7D shows structural model index. FIG. 7E shows trabecular number. FIG. 7F shows trabecular thickness. FIG. 7G shows trabecular spacing. FIG. 7H shows bone mineral density. FIG. 71 shows total mineral density. Abbreviations for quantitative analysis: BV/TV = bone volume to total volume fraction; Conn Dens = connectivity density; SMI = structural model index; Tb.N = trabecular number; Tb.Th = trabecular thickness; Tb.Sp = trabecular spacing; BMD = bone mineral density; TMD = total mineral density. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 8 mice (n = 16 femurs) for each condition and time point.

FIGS. 8A-8D show results from microcomputed tomography analysis demonstrating the therapeutic efficacy of Anti-Scl-DlO in femur cortical bone. FIG. 8A shows three-dimensional renderings of the region of interest. FIG. 8B shows bone marrow to total volume fraction. FIG. 8C shows bone mineral density. FIG. 8D shows total mineral density. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 8 mice (n = 16 femurs) for each condition and time point.

FIGs. 9A-9I show results from microcomputed tomography analysis demonstrating the therapeutic efficacy of Anti-Scl-DlO in L4 vertebral trabecular bone. FIG. 9A shows three- dimensional renderings of the region of interest. FIG. 9B, FIG. 9C, FIG. 9d, FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9H, and FIG. 91 show quantitative analysis of various parameters. FIG. 9B shows bone volume to total volume fraction. FIG. 9C shows connective density. FIG. 9D shows structural model index, FIG. 9E shows trabecular number. FIG. 9F shows trabecular thickness. FIG. 9G shows trabecular spacing. FIG. 9H shows bone marrow density. FIG. 91 shows total mineral density. Abbreviations for quantitative analysis (B): BV/TV = bone volume to total volume fraction; Conn Dens = connectivity density; SMI = structural model index; Tb.N = trabecular number; Tb.Th = trabecular thickness; Tb.Sp = trabecular spacing; BMD = bone mineral density; TMD = total mineral density. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 8 for each condition and time point.

FIG. 10 is a schematic showing site-specific modification of mCherry with bisphosphonate affinity ligands. mCherry was produced with either an N- or C-terminal ‘sortag,’ enabling attachment of short, FITC-lysine, azidolysine-containing peptides via the bacterial transpeptidase, sortase (peptide sequences shown with modified lysine as [K]). Azide-modified fluorescent protein (e.g., mCherry) was then reacted via copper-free click chemistry with an excess of bisphosphonate-DBCO (BP-PEG4-DBCO to yield a final product, containing a single BP at either terminus. Sequences shown in the figure are GGGKGGSK (SEQ ID NO: 6) and GSKGLPETGG (SEQ ID NO: 7). FIGs. 1 1 A-1 IB show characterization of anti-sclerostin and bivalent anti-sclerostin-Dio. A. HPLC trace of anti-sclcrostin and anti-sclerostin-Dio. B. SDS-PAGE gel of anti-sclcrostin and anti-sclerostin-Dio. Proteins were run under Reducing(R) or non-reducing (NR) conditions on SDS-PAGE gel.

FIGs. 12A-12D show blood and bone PK and vital organ biodistribution of IgG-Dio in adult male and female mice. Animals were administered a single 5mg/kg dose of ¹²⁵I-labeled IgG-Dio. FIG. 12A shows blood PK; most error bars are too small to see. FIG. 12B shows bone PK on femur and FIG. 12C shows bone PK on vertebrae. FIG. 12D shows uptake by vital organs at 24 hours post-injection. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 4 for male mice and 3 for female mice at each time point, data presented as Mean ± SEM.

FIGS. 13A-13D show the role of avidity in bone surface targeting of non-functional IgG. FIG. 13A is a schematic depicting the structure of IgG, monovalent IgG-Dio (Dio affinity ligand only on knobs heavy chain) and bivalent IgG-Dio. FIG. 13B shows normalized HPLC traces showing relative elution time of the three proteins on SEC, FIG. 13C shows blood PK following a single 5mg/kg dose of each protein. FIG. 13D shows biodistribution to femur and vertebrae at 24 hours post-injection. Adult male mice were used in this experiment. * p < 0.05, ** p < 0.01, **** p < 0.0001. n = 4 for each group. Data presented as Mean ± SEM. Biodistribution to the femur and vertebra was significantly increased in bivalent IgG-DlO compared to monovalent IgG-DlO.

FIG. 14A-14C show that valence and targeting ligands affect biodistribution to the femur and vertebrae in various IgG conjugates. FIG. 14A shows blood pharmacokinetics. FIG. 14B shows biodistribution in urine, lung, liver, kidney, heat, spleen, brain, vertebrae, and femur. FIG. 14C shows biodistribution in vertebrae and femur. The bivalent IgG-DlO-DlO and bivalent IgG- D20-D20 conjugates showed increased biodistribution to the vertebrae and femur compared to monovalent IgGlO and compared to bivalent IgG-ElO-ElO.

FIG. 15A-15D show hydroxyapatite binding affinity curves determined based upon radioimmunoassay with 1251 labeling for various conjugates. FIG. 15A shows results for bivalent IgG-DlO-DlO, FIG. 15B shows results for monovalent IgG-DlO, FIG. 15C shows results for bivalent IgG E10-E10, and FIG. 15D shows results for bivalent IgG-D20-D20. The results demonstrate that bivalent IgG-DlO-DlO and IgG-D20-D20 have similar affinities to hydroxyapatite in vitro, each of which arc greater than the affinity of monovalent IgG-DlO. IgG- D10-D10 has a 5-fold more significant affinity to the bone than monovalent IgG-DlO, similar to bivalent IgG-ElO-ElO, supporting the use of bivalent IgG-DlO-DlO.

FIG. 16A shows confinnation of successful production and purification of anti-RANKL and bivalent bone targeted anti-RANKL (anti-RANKL-DlO-DlO) by western blot and FIG. 16B shows confirmation of expected properties by HPLC.

FIG. 17A shows biodistribution in various organs following administration of IgG, bivalent IgG, anti-RANKL, and bivalent anti-RANKL-DlO antibodies to adult mice. FIG. 17B shows distribution in vertebrae and femur. FIG. 17C shows blood pharmacokinetics. FIG. 17D is a graph showing binding affinity to immobilized recombinant murine RANKL as measured using ELISA.

FIG. 18A shows successful production and isolation of anti-TGFp a bone targeted, bivalent conjugate thereof (anti-TGFP-D10-D10). FIG. 18B shows confirmation of expected properties by HPLC.

FIG. 19A shows biodistribution in various organs following administration of IgG, bivalent IgG-DlO-DlO, anti-TGFp, and bivalent bone targeted anti-TGFp-D10-D10 to adult mice. FIG. 19B shows biodistribution in the vertebrae and femur. The figure legend for FIG. 19B applies to FIG. 19A. FIG. 19C shows blood pharmacokinetics. As shown, the bivalent bone targeted anti-TGFp demonstrates increased biodistribution in the vertebrae and femur compared to non-targeted anti-TGFp. No significant differences in blood pharmacokinetics or distribution in other organs was seen.

FIG. 20A and FIG. 20B are western blots demonstrating successful production and purification of various IgG conjugates with and without different linkers. FIG. 20C shows HPLC traces of the various conjugates.

DEFINITIONS

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods arc described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the ait to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide amphiphile” is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth.

For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

As used herein, the modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to ±10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9 - 1.1. Other meanings of “about” may be apparent from the context, such as rounding off; for example, “about 1” may also mean from 0.5 to 1.4.

As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method stcp(s), etc. and excludes any unrccitcd fcaturc(s), clcmcnt(s), method stcp(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and/or “consisting essentially of’ embodiments, which may alternatively be claimed or described using such language.

As used herein, the term “antibody” and “antibodies” are used herein in the broadest sense and are inclusive of antibodies and fragments thereof (i.e. antibody fragments). The term “antibody” refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi- specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, antibody fragments, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001/058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, TgM, TgD, IgA, and IgY), class (for example, IgGl , IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

“Antibody fragment” as used herein refers to a portion of an intact antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9y. 1126-1129 (2005)) (e.g., comprises the antigen-binding site or variable region). The term “antibody” is inclusive of antibody fragments. Any antigen-binding fragment of the antibody described herein is within the scope of the present disclosure. The antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’)i fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N- terminal variable (VH) region and three C-terminal constant (CHI, CH2, and Cm) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda ( ), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which arc located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the 0 sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

“CDR” is used herein to refer to the “complementarity determining region” within an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted “CDR1,” “CDR2,” and “CDR3,” for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region that binds the antigen. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be referred to as a “molecular recognition unit.” Crystallographic analyses of antigen-antibody complexes have demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as “Kabat CDRs”. Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as “LI,” “L2,” and “L3,” or “Hl,” “H2,” and “H3,” where the “L” and the “H” designate the light chain and the heavy chain regions, respectively. These regions may be referred to as “Chothia CDRs,” which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, FASEB J., 9: 133-139 (1995), and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain aspects use Kabat- or Chothia-defined CDRs.

As used herein, when an antibody or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (K_a) of at least 10⁷ M'¹ (e.g., >10⁷ M’¹, >10⁸ M’¹, >10⁹ M’¹, >1O¹⁰ M’¹, >10ⁿ M’ ^l, >10¹² M'¹, >10¹³ M'¹, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (sec, e.g., Clackson ct al. Nature, 352: 624-628 (1991)); and Marks ct al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

“Humanized” forms of non-human (e.g., rodent) antibodies are antibodies that have been modified to increase their similarity to variants produced naturally in humans. Humanized antibodies may be chimeric antibodies that contain a minimal sequence derived from the non- human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

The term “bispecific” antibody as used herein denotes an antibody that has at least two binding sites each of which bind to different epitopes of the same antigen or a different antigen. The term “multi specific” antibody as used herein denotes an antibody that has binding specificities for at least two different sites.

As used herein, the terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof).

As used herein, the terms “prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention.

The terms “subject” and “patient” are used interchangeably herein and refer to any animal. In some embodiments, the subject is a mammal, including, but not limited to, mammals of the order Roden tia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some aspects, the mammal is a human. In some aspects, the human is an adult aged 18 years or older. In some aspects, the human has or is suspected of having osteoporosis.

Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION

The choice of bone-targeting affinity ligand and the effect of avidity and protein circulation time were investigated herein, to determine the key determinants of bone surface targeting of protein therapeutics. Alendronate (ALD) and deca-aspartate (Dio. DDDDDDDDDD (SEQ ID NO: 1)) were site-specifically attached to fluorescent proteins, enabling direct comparison of their ability to target intact proteins to the bone surface, while simultaneously allowing evaluation of the functional activity of surface anchored proteins. The combination of bone affinity and enhanced circulation time was investigated by fusing mCherry to the Fc- fragment of human IgGi and two Dio (SEQ ID NO: 1) affinity ligands, resulting in a marked increase in bone accumulation and retention. Finally, bone surface targeting was found to increase the efficacy of a therapeutic anti-sclerostin antibody in a murine model of osteoporosis, with significant increases in bone mass and quality as compared to untargeted antibody and relevant controls. The results presented herein demonstrate that optimal bone surface targeting enhances the skeletal accumulation and retention of functional proteins, and that this strategy enhances the efficacy of therapeutic antibodies, thereby allowing for potentially lower dosing regimens and extended dosing intervals without compromising therapeutic effects. Accordingly, the bone targeted therapeutics developed herein provide for improved treatments for osteoporosis and other bone -related diseases.

In some aspects, provided herein are bone targeting therapeutic conjugates. In some embodiments, provided herein is a bone targeting therapeutic conjugate (a “conjugate”) comprising a therapeutic antibody for treatment of a bone disease and at least one bone targeting moiety.

In some embodiments, the bone targeting therapeutic conjugates provided herein display improved pharmacokinetic properties compared to the therapeutic agent in the absence of the bone targeting moiety, resulting in efficacy at a lower dose and/or less frequent dosing interval compared to the therapeutic agent alone. For example, the bone targeting therapeutic conjugates provided herein may possess increased circulation time following administration, increasing avidity, and/or increased affinity for the target. In some embodiments, one or more of these desirable properties of the conjugates provided herein convey a lower therapeutically effective dose to the conjugate compared to the therapeutically effective dose of the therapeutic agent (e.g. the therapeutic antibody absent the bone targeting moiety). Accordingly, the bone targeting therapeutic conjugates provided herein are particularly advantageous compared to therapeutic antibodies with undesirable side effects and/or risks associated with high doses or prolonged use, as they can be used at a lower therapeutically effective dose and/or administered with less frequency to the subject.

In some embodiments, the therapeutic antibody for treatment of a bone disease is an anti- sclerostin antibody. In some embodiments, the therapeutic antibody for treatment of a bone disease is a humanized anti-TGFp antibody. In some embodiments, the therapeutic antibody for treatment of a bone disease is an anti-receptor activator of nuclear factor kappa beta ligand (RANKL) antibody (i.e. anti-RANKL) antibody. In some embodiments, the therapeutic antibody for treatment of a bone disease is a humanized antibody (e.g. a humanized anti- sclerostin, a humanized anti-TGFp, or a humanized anti-RANKL antibody). The term “antibody” is used in the broadest sense and is inclusive of antibodies and fragments thereof.

In some embodiments, the therapeutic antibody for treatment of a bone disease is an anti- sclerostin antibody. In some embodiments, the anti-sclerostin antibody comprises a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) with SEQ ID NO: 49 and a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) with SEQ ID NO: 51. In some embodiments, the anti-sclerostin antibody comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49 and a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 51. In some embodiments, the anti-sclerostin antibody comprises a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49 and a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 51. In some embodiments, the anti- sclerostin antibody comprises a heavy chain comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 49 and a light chain comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 51 . In some embodiments, the anti- sclerostin antibody is humanized. In some embodiments, the anti- sclerostin antibody is an IgG antibody.

In some embodiments, the therapeutic antibody for treatment of a bone disease is an anti- RANKL antibody. In some embodiments, the anti-RANLK antibody comprises a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) with SEQ ID NO: 42 and a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) with SEQ ID NO: 44. In some embodiments, the anti-RANKL antibody comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42 and a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44. In some embodiments, the anti-RANKL antibody comprises a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 42 and a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 44. In some embodiments, the anti-RANLK antibody comprises a heavy chain comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 42 and a light chain comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 44. In some embodiments, the anti-RANKL antibody is humanized. In some embodiments, the anti-RANKL antibody is an IgG antibody.

In some embodiments, the therapeutic antibody for treatment of a bone disease is an anti- TGF0 antibody. In some embodiments, the anti-TGFp antibody comprises a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) with SEQ ID NO: 36 and a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) with SEQ ID NO: 38. In some embodiments, the anti-TGFp antibody comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 36 and a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 38. In some embodiments, the anti-TGFp antibody comprises a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 36 and a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 38. In some embodiments, the anti-TGFp antibody comprises a heavy chain comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 36 and a light chain comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 38. In some embodiments, the anti-TGFp antibody is humanized. In some embodiments, the anti-TGFp antibody is an IgG antibody.

In some embodiments, the conjugate comprises at least one bone targeting moiety. In some embodiments, the at least one bone targeting moiety is a bisphosphonate. In some embodiments, the at least one bone targeting moiety is a bone targeting peptide. For example, in some embodiments the targeting peptide is an acidic oligopeptide comprising six or more acidic residues (e.g. aspartic acid, glutamic acid). In some embodiments, the targeting peptide comprises 6-25 combined aspartic acid and/or glutamic acid residues. For example, in some embodiments the targeting peptide comprises 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6- 17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, or 7-8 combined aspartic acid and/or glutamic acid residues. In some embodiments, the target peptide comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, or 25 combined aspartic acid and/or glutamic acid residues. In some embodiments the targeting peptide is an acidic oligopeptide comprising 6-20 combined aspartic acid and/or glutamic acid residues. In some embodiments the targeting peptide is an acidic oligopeptide comprising 6-15 combined aspartic acid and/or glutamic acid residues. In some embodiments, the targeting peptide is an acidic oligopeptide comprising 10-20 aspartic acid residues. In some embodiments, the targeting peptide comprises 10 aspartic acid residues, also referred to as deca-aspartate (Dio (SEQ ID NO: 1). In some embodiments, the targeting peptide comprises 20 aspartic acid residues, also referred to as icosa-aspartate (D20 (SEQ ID NO: 8).

The term “bisphosphonate” refers to a class of compounds characterized by two phosphonate groups, and are also referred to as disphosphonates. The structure of bisphosphonates is shown below:

The R groups determine the chemical properties of a bisphosphonate and distinguishes individual types of bisphosphonates from one another. Exemplar)' bisphosphonates include, for example, alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate, zoledronate, and salts and derivatives thereof. In some embodiments, the bisphosphonate is alendronate (ALD), also referred to as alendronic acid or alendronate sodium. Alendronate has the structure:

The term “conjugate” indicates that the therapeutic antibody for the treatment of a bone disease and the at least one bone targeting moiety are conjugated together. In some embodiments, the at least one bone targeting moiety is conjugated to a portion of the therapeutic antibody for the treatment of a bone disease. For example, in some embodiments the at least one bone targeting moiety is conjugated to a heavy chain of the therapeutic antibody for the treatment of a bone disease. In some embodiments, the at least one bone targeting moiety is directly conjugated to the heavy chain of the therapeutic antibody. In some embodiments, the at least one bone targeting moiety is conjugated to the heavy chain by a linker. In some embodiments, the linker is a glycine-rich linker. A “glycine-rich” linker indicates that at least 50% of the residues in the linker are glycine residues. In some embodiments, the linker is a glycine-rich linker comprising 5-20 residues, wherein at least 50% of the residues are glycine residues. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-10 repeating GGGGS (SEQ ID NO: 9) units. For example, in some embodiments the linker comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3 repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, a first bone targeting moiety is conjugated to a first heavy chain of the therapeutic antibody for the treatment of bone disease and a second bone targeting moiety is conjugated to a second heavy chain of the therapeutic antibody for the treatment of a bone disease. Such an conjugate is referred to herein as “bivalent”. In some embodiments, a single bone targeting moiety is conjugated to a single heavy chain of the therapeutic antibody for the treatment of a bone disease. Such a conjugate is referred to herein as “monovalent”.

In some embodiments, the conjugate is bivalent. In some embodiments, the conjugate comprises a first bone targeting moiety conjugated to a first heavy chain of the therapeutic antibody for the treatment of bone disease and a second bone targeting moiety conjugated to a second heavy chain of the therapeutic antibody for the treatment of a bone disease. In some embodiments, the first bone targeting moiety is directly conjugated to the first heavy chain of the therapeutic antibody. In some embodiments, the second bone targeting moiety is directly conjugated to the second heavy chain of the therapeutic antibody for the treatment of a bone disease. In some embodiments, the first bone targeting moiety is directly conjugated to the first heavy chain and the second bone targeting moiety is directly conjugated to the second heavy chain.

In some embodiments, the first bone targeting moiety is conjugated to the first heavy chain of the therapeutic antibody for treatment of a bone disease by a linker. In some embodiments, the second bone targeting moiety is conjugated to the second heavy chain of the therapeutic antibody for treatment of a bone disease by a linker. In some embodiments, the linker for the first bone targeting moiety and the linker for the second bone targeting moiety are different. In some embodiments, the linker for the first bone targeting moiety and the linker for the second bone targeting moiety are the same. In some embodiments, the linker is a glycine- rich linker. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-10 repeating GGGGS (SEQ ID NO: 9) units. For example, in some embodiments the linker comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3 repeating GGGGS (SEQ ID NO: 9) units.

In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each a bisphosphonate. For example, in some embodiments the first bone targeting moiety and the second bone targeting moiety are each a bisphosphonate such as alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate, or zoledronate. In some embodiments, the first bone targeting moiety and the second bone targeting moiety arc each the same bisphosphonate. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each a different bisphosphonate. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each alendronate.

In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each an acidic oligopeptide. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each an acidic oligopeptide comprising 6-25 combined aspartic acid and/or glutamic acid residues. In some embodiments, the first bone targeting moiety and the second bond targeting moiety are each an acidic oligopeptide comprising 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, or 25 combined aspartic acid and/or glutamic acid residues. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each an acidic oligopeptide comprising 10-20 aspartic acid residues. For example, in some embodiments the first bone targeting moiety and the second bone targeting moiety are each deca-asparate (Dio (SEQ ID NO: 1). For example, in some embodiments the first bone targeting moiety and the second bone targeting moiety are each D20 (SEQ ID NO: 8). In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each the same acidic oligopeptide. In some embodiments, the first bone targeting moiety and the second bone targeting moiety are each a different acidic oligopeptide.

In some embodiments, provided herein is a conjugate comprising an anti-sclerostin antibody (e.g. an anti-sclerostin antibody having a heavy chain having at least 80% sequence identity to SEQ ID NO: 49 and a light chain having at least 80% identity to SEQ ID NO: 51) conjugated to the bone targeting moiety Dio (SEQ ID NO: 1). In some embodiments, the conjugate is monovalent. In some embodiments, the conjugate is bivalent (anti-Scl-DlO-DlO). In some embodiments, the first D10 moiety is directly conjugated to a first heavy chain of the anti-sclerostin antibody and the second D10 moiety is directly conjugated to a second heavy chain of the anti-sclerostin antibody. In some embodiments, each D10 moiety is conjugated to their respective heavy chain of the anti-sclerostin antibody by a linker. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is a glycine-rich linker comprising 5-20 residues. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1 -4 repeating GGGGS (SEQ ID NO: 9) units.

In some embodiments, provided herein is a conjugate comprising an anti-sclerostin antibody (e.g. an anti-sclerostin antibody having a heavy chain having at least 80% sequence identity to SEQ ID NO: 49 and a light chain having at least 80% identity to SEQ ID NO: 51) conjugated to the bone targeting moiety D20 (SEQ ID NO: 8). In some embodiments, the conjugate is monovalent. In some embodiments, the conjugate is bivalent (anti-Scl-D20-D20). In some embodiments, the first D20 moiety is directly conjugated to a first heavy chain of the anti-sclerostin antibody and the second D20 moiety is directly conjugated to a second heavy chain of the anti-sclerostin antibody. In some embodiments, each D20 moiety is conjugated to their respective heavy chain of the anti-sclerostin antibody by a linker. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is a glycine-rich linker comprising 5-20 residues. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-4 repeating GGGGS (SEQ ID NO: 9) units.

In some embodiments, provided herein is a conjugate comprising an anti-RANKL antibody (e.g. an anti-RANKL antibody comprising a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 42 and a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 44) conjugated to the bone targeting moiety Dio (SEQ ID NO: 1). In some embodiments, the conjugate is monovalent. In some embodiments, the conjugate is bivalent (anti-RANKL-DlO- D10). In some embodiments, the first D10 moiety is directly conjugated to a first heavy chain of the anti-RANKL antibody and the second D10 moiety is directly conjugated to a second heavy chain of the anti-RANKL antibody. In some embodiments, each D10 moiety is conjugated to their respective heavy chain of the anti-RANKL antibody by a linker. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is a glycine- rich linker comprising 5-20 residues. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-4 repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, provided herein is a conjugate comprising an anti-RANKL antibody (c.g. an anti-RANKL antibody comprising a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 42 and a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 44) conjugated to the bone targeting moiety D20 (SEQ ID NO: 1). In some embodiments, the conjugate is monovalent. In some embodiments, the conjugate is bivalent (anti-RANKL-D20- D20). In some embodiments, the first D20 moiety is directly conjugated to a first heavy chain of the anti-RANKL antibody and the second D20 moiety is directly conjugated to a second heavy chain of the anti-RANKL antibody. In some embodiments, each D20 moiety is conjugated to their respective heavy chain of the anti-RANKL antibody by a linker. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is a glycine- rich linker comprising 5-20 residues. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-4 repeating GGGGS (SEQ ID NO: 9) units.

In some embodiments, provided herein is a conjugate comprising an anti-TGFp antibody (e.g. an anti- TGFp antibody comprising a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 36 and a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 38) conjugated to the bone targeting moiety Dio (SEQ ID NO: 1). In some embodiments, the conjugate is monovalent. In some embodiments, the conjugate is bivalent anti-TGFP-D10-D10). In some embodiments, the first D10 moiety is directly conjugated to a first heavy chain of the anti-TGFp antibody and the second D10 moiety is directly conjugated to a second heavy chain of the anti- TGFp antibody. In some embodiments, each D10 moiety is conjugated to their respective heavy chain of the anti-TGFp antibody by a linker. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is a glycine-rich linker comprising 5-20 residues. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-4 repeating GGGGS (SEQ ID NO: 9) units.

In some embodiments, provided herein is a conjugate comprising an anti-TGFp antibody (e.g. an anti- TGFP antibody comprising a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 36 and a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 38) conjugated to the bone targeting moiety D20 (SEQ ID NO: 1). In some embodiments, the conjugate is monovalent. In some embodiments, the conjugate is bivalent (anti-TGFP-D20-D20). In some embodiments, the first D20 moiety is directly conjugated to a first heavy chain of the anti-TGFp antibody and the second D20 moiety is directly conjugated to a second heavy chain of the anti- TGFP antibody. In some embodiments, each D20 moiety is conjugated to their respective heavy chain of the anti-TGFp antibody by a linker. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is a glycine-rich linker comprising 5-20 residues. In some embodiments, the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units. In some embodiments, the linker comprises 1-4 repeating GGGGS (SEQ ID NO: 9) units.

In some aspects, provided herein are compositions comprising a conjugate described herein. In some embodiments, the bone targeting therapeutic conjugates and compositions provided herein are used in methods of treating a bone disease in a subject.

In some aspects, provided herein are methods of treating a bone disease in a subject, comprising providing to the subject a bone targeting therapeutic conjugate provided herein. As described above, the targeting therapeutic conjugate comprises a therapeutic antibody for treatment of the bone disease and a bone targeting moiety.

The bone disease is not limited to any particular bone disease to be treated. In some embodiments, the bone disease is characterized by a loss of bone mineral density, bone mass, bone structure, and/or bone strength. In some embodiments, the bone disease is osteoporosis. In some embodiments, the bone disease is osteopenia. In some embodiments, the bone disease is Paget’s disease. In some embodiments, the bone disease is cancer that has metastasized to the bone of the subject. In some embodiments, the bone disease is caused at least in part by pathological expression or activity of one or more factors that are targeted by the therapeutic antibody used in the conjugate herein.

In some embodiments, a therapeutically effective dose of the conjugate is less than a therapeutically effective dose of the therapeutic agent for treatment of the bone disease (e.g. when administered to the subject not as a part of the conjugate described herein). The term “therapeutically effective dose” or “therapeutically effective amount” indicate the dose/amount of an agent required to see a desired clinical effect. For bone disease, for example, a clinically effective amount may improve bone density, bone mass, bone strength, promote bone formation, and/or reduce the risk of fractures in a subject.

The “therapeutically effective dose” is inclusive of both the dose delivered per dosing session, and the frequency of dosing sessions given to the subject over time. Accordingly, the conjugate having a therapeutically effective dose less than the therapeutically effective dose of the therapeutic agent for treatment of the bone disease can indicate that the dose delivered per dosing session is smaller and/or that the dosing occurs less frequently than the dose of the therapeutic agent itself, without being part of the conjugate described herein. The results presented herein demonstrate that the conjugates require smaller doses and less frequent dosing interval to achieve therapeutic efficacy. For example, original trials of anti-sclerostin antibody in mice demonstrated efficacy at a dose of 25 mg/kg dosed intravenously every two days (J. Bone Miner. Res. 24, 578-588 (2009)). Herein, the conjugate anti-Scl-DlO (comprising anti-sclerostin antibody and the targeting moiety DIO (SEQ ID NO: 1)) is shown to be efficacious at 5 mg/kg, dosed weekly. Accordingly, the anti-Scl-DlO conjugate requires both a smaller (5 mg/kg, compared to 25 mg/kg) and a less frequent dosing interval (weekly, as opposed to every two days) to achieve therapeutic efficacy for treatment of bone disease. This lower therapeutically effective dose is beneficial due to minimized risk of undesirable side effects and/or toxicity to the subject.

In some embodiments, the therapeutically effective dose of the conjugate is less than 3 mg/kg when provided to the subject monthly. In some embodiments, the conjugate comprises anti-sclerostin antibody and the targeting moiety DIO (SEQ ID NO: 1 ), and the therapeutically effective dose of the conjugate is less than 3mg/kg when provided to the subject monthly. This is less than the therapeutically effective dose of romosozumab (Evinity®), a clinically approved anti-sclerostin antibody for treatment of osteoporosis.

EXAMPLES

Example 1

Site-Specific attachment of bone-targeting affinity ligands to fluorescent proteins To enable rigorous comparison of bone-targeting agents, methods for attachment of either bisphosphonate or dcca-aspartatc (Dio, SEQ ID NO: 1) affinity ligands to equivalent sites on fluorescent model proteins were developed herein. While Dio (SEQ ID NO: 1) was genetically fused to the N- or C-terminus, bisphosphonates were site-specifically conjugated following the scheme shown in Figure 1A. First, mCherry was synthesized with N- or C-terminal sortags (Curr Protoc Protein Sei. 2009 Apr: Chapter 15:15.3.1-15.3.9) and the bacterial enzyme sortase was used to site-specifically attach a short FITC-labeled, azidolysine-containing peptide to either terminus. Azide-modified mCherry was then reacted via copper- free click chemistry with bisphosphonate-DBCO (BP-PEG4-DBCO), resulting in a final product containing a single BP moiety covalently attached to either terminus.

Binding of C-terminal modified mCherry-BP to hydroxyapatite and bovine bone chips was evaluated in vitro. As a positive control, mCherry which had been decorated with surface BP in a non-site-specific manner was used. As shown in Figure IB, both C-terminal and non-site- spccific conjugation of BP resulted in binding of functional mCherry to hydroxyapatite and bone chips, whereas no fluorescent signal was detectable in the absence of BP modification. This approach was repeated with two other fluorescent proteins, eGFP and mCardinal, and nearly identical results were observed (Figure 1C). Likewise, bone targeting and surface function were consistent whether the BP affinity ligand was attached to the N- or C-terminus of the fluorescent proteins (Figure ID). Finally, in vitro binding of equimolar concentrations of BP-conjugated and Dio-fused mCherry was compared. While N-terminal Dio-mCherry demonstrated binding and surface-bound fluorescence roughly equivalent to that of BP-mCherry, no bone-surface targeting was observed when the Dio peptide was fused to the C-terminus of mCherry (Figure ID).

Comparison of bone-surface targeting of BP- and DlO-mCherry in vivo

A quantitative comparison of bone-surface accumulation of BP- and Dio-modified mCherry following intravenous injection was next performed in vivo, with untargeted mCherry as a control. To ensure that the three proteins were equivalent except for the affinity ligand, both Dio-mCherry and untargeted mCherry were synthesized with a sortag and modified with FITC- labeled, azide containing peptide, analogous to BP-mCherry. Each protein was radioiodinated and administered at an equimolar 2mg/kg dose. Animals were transcardially perfused to clear residual blood from the organs. As shown in Fig 2A, the three proteins cleared the blood quickly via renal filtration, with < 5% of the injected dose (ID) in the blood and most of the radioactive signal in the kidney and/or urine at 4 hours post-injection (Fig 2B). In spite of this rapid clearance, both BP- and Dio-modified mCherry demonstrated selective accumulation in skeletal tissues, with higher concentrations (0.75 ± 0.14, vs. 0.60 ± 0.06 vs. 0.31 ± 0.06 %ID/g on femur, 0.61 ± 0.13, 0.49 ± 0.06, 0.26 ± 0.07 %ID/g on vertebrae, BP-mCherry, Dw-mCherry, unmodified mCherry, respectively) observed in both femur and vertebrae compared to untargeted mCherry (Fig 2C). To confirm that this quantitative increase reflected anchoring of protein to the bone surface, calcified tissue was sectioned and confocal imaging was performed. As shown in Figure 2D, both BP- and Dio-modified mCherry were visualized on the surface of trabecular bone, confirming both localization and functional activity in vivo. Co-localization of red fluorescent protein and FITC-labeled peptide allowed discrimination of surface-anchored BP- and Dio-mCherry from red fluorescent signal observed in the bone marrow in all experimental groups (presumed to be autofluorescence).

Dose- and time-dependence of bone surface targeting in vivo

The effect of dose on accumulation of bone targeted protein in skeletal tissues was next evaluated. Given the relatively fast blood clearance and large surface area of mineralized bone in the body, it was hypothesized that the pharmacokinetics (PK) would be largely independent of dose (e.g., bone accumulation would not saturate). Indeed, injection of 2mg/kg and 5mg/kg doses of BP-mCherry resulted in nearly identical blood, bone, and organ distribution, when expressed as a percent of the injected dose (Figure 3A-C). Fluorescence imaging of bone tissue from animals injected with 5mg/kg BP-mCherry was next performed (Figure 3D). Consistent with radiotracing results, higher dose produced brighter fluorescent signal, facilitating study of the time dependence of bone surface targeting. As shown, the amount of fluorescent protein anchored to the bone surface gradually increased over the first few hours, reaching an apparent peak at approximately 4 hours post-injection. By 24 hours, fluorescent signal was visibly diminished - either due to clearance of intact protein, loss of activity, or catabolism. Interestingly, some isolated green fluorescent signal at the 24 hour time point was observed, suggesting that surface-anchored mCherry had been inactivated or catabolized, leaving only the surface-anchored FITC label (Figure 3D). Prolonging circulation time markedly increases bone surface accumulation

The relatively short circulation time of bone-targeted mCherry , together with evidence of gradual accumulation on the bone surface, suggested that prolongation of blood PK might significantly enhance bone surface targeting. To further investigate, mCherry was fused to the Fc-fragment of human IgGi. Deca-aspartate affinity ligands were fused to the C-terminus of each chain (Figure 4A). Dio was chosen over bisphosphonate in this case due to greater ease of synthesis and purification, as well as the similarity to the design of asfotase alfa. Dw-mCherry (without Fc) and mCherry-Fc (i.e., without Dio affinity ligands) were used as controls.

As shown in Figure 4B, Dio-mCherry and mCherry-Fc-Dio demonstrated similar function in vitro, with roughly equal binding to bovine bone chips at equimolar concentrations. In contrast, addition of the Fc fragment greatly prolonged circulation time, with mCherry-Fc and mCherry-Fc-DlO demonstrating 77- and 108-fold increases in plasma exposure, comparing to unmodified mCherry (AUC 732 ± 50 vs. 1024 ±45 vs. 9.5 ± 0.9, mCherry-Fc-Dio, mCherry-Fc, and unmodified mCherry, respectively) (Figure 4C). The increased plasma half-life revealed a relatively small, but significant, difference in blood PK between bone-targeted and untargeted mCherry-Fc, presumably reflecting target-mediated drug disposition (TMDD). Likewise, mCherry-Fc-DlO showed a several-fold increase in bone-specific accumulation, with 6.73 ± 1.89 and 5.05 ± 0.87 %ID/g at the femur and vertebrae, respectively, compared to 2.12 ± 0.29 and 1.23 ± 0.25 %ID/g for mCherry-Fc at 4 hours post-injection. By 24 hours, mCherry-Fc-Dio bone accumulation had further increased to 13.59 ± 1.6 and 11.42 ± 1.27 %ID/g on femur and vertebrae, with a bone:blood ratio of 213.5 ± 60.4 and 179.1 ± 47.1 (femur and vertebrae respectively) (Figure 4D-E). Other than bone, the organ biodistribution of mCherry-Fc-DlO and mCherry-Fc were similar, suggested that skeletal tissues were the primary site of accumulation of the targeted protein (Figure 4F). Interestingly, mCherry-Fc itself showed some selective accumulation bone - namely, an increase in bone uptake from 4 to 24 hours despite decreasing blood concentration (Figure 4E). Fluorescence imaging suggested that the majority of this uptake occurred in the bone marrow (where numerous cell types express Fc receptors), as opposed to on the trabecular surface, where only mCherry-Fc-DlO was observed (Figure 4G).

Sex is a variable for bone targeting and accumulation Sex as a variable for bone targeting was also investigated. The bone targeted but nonfunctional antibody IgG-Dio was used to evaluate whether sex -based differences in bone targeting or pharmacokinetics would be observed. Adult male and adult female mice were administered a single 5mg/kg dose of ¹²⁵I-labeled IgG-Dio The blood PK, bone PK, and organ biodistribution was evaluated and compared. Results are shown in FIG. 12. While blood PK and distribution to vital organs were closely matched across the two sexes, there were significant differences in the timing and magnitude of bone accumulation, especially in vertebrae.

Role of avidity in bone targeting

The role of avidity in bone surface targeting of the non-functional IgG was further investigated. The multichain IgG structure was used to directly evaluate the impact of binding avidity on bone surface targeting. Non-functional IgG (no affinity ligands) was compared to monovalent IgG-DlO (i.e., affinity ligand on just one of the two heavy chains), bivalent IgG-DlO (i.e., affinity ligands on both heavy chains), bivalent IgG-ElO, and bivalent IgG-D20.

To produce the monovalent IgG-Dio, ‘knob-into-hole’ mutations were first introduced in the Fc region of mouse IgGi. The sequence encoding the Dio affinity ligand was then added to the C-terminus of the knob Fc fragment. Transfection was then performed with the three plasmids in a 1:1:1 ratio (i.e., light chain: knob heavy chain-Dio:hole heavy chain). Protein production and purification were identical to that described for non-functional IgG and bivalent IgG-Dio. All proteins were stored at -80 °C in aliquots to avoid repeated freeze-thaw cycles.

Results are shown in FIG. 13. The bivalent molecule demonstrated significantly higher bone accumulation at 24 hours post-injection, indicating avidity is a key determinant of bone surface targeting of IgGs.

To further evaluate the role of avidity in bone targeting in vivo, adult male mice were dosed with a single dose of 1251-labelled antibody at 5 mg/kg. Non-functional IgG (no affinity ligands) was compared to monovalent IgG-DlO (i.e., affinity ligand on just one of the two heavy chains) and bivalent IgG-DlO (i.e., affinity ligands on both heavy chains), bivalent IgG-ElO, and bivalent IgG-D20. For the bivalent IgG-D20 conjugate, a GGGGS (SEQ ID NO: 9) linker was used to conjugate the D20 (SEQ ID NO: 8) bone targeting moiety to each chain of the IgG. Blood pharmacokinetics was assessed over 24 hours, and biodistribution was evaluated at 24 hours. Results are shown in FIG. 14. Valence and targeting ligands significantly affected biodistribution to the femur and vertebrae, as shown, but did not substantially alter biodistribution to other organs. Similar to in vitro results, bivalent IgG-DlO-DlO and IgG-D20- D20 localized to the bones with similar affinity, superior to monovalent IgG-DlO or bivalent IgG-ElO-ElO, all of which localized to the bone greater than IgG.

Monovalent and bivalent IgG were further evaluated for their affinity to hydroxyapatite in vitro. Antibodies were labeled with 1251 and affinity to hydroxyapatite was evaluated in vitro by radioimmunoassay. Results are shown in FIG. 15. Bivalent IgG-DlO-D 10 and IgG-D20-D20 have similar affinities to hydroxyapatite in vitro based on radioimmunoassay with 1251 labeling. IgG-DlO-DlO has a 5-fold more significant affinity to the bone than monovalent IgG-DlO, similar to bivalent IgG-ElO-ElO, supporting the preferential use of bivalent IgG-DlO-DlO.

Bone surface targeting of therapeutic antibodies

It was next evaluated whether affinity targeting to the bone surface could enhance the functional activity of a clinically-relevant protein therapeutic. A murine analogue of the FDA- approved monoclonal antibody, Romosozumab, was initially selected. The antibody binds and inhibits murine sclerostin, a small protein expressed predominantly by osteocytes which potently inhibits bone formation and stimulates bone resorption. Whether bone targeting enhanced antibody concentration and scavenging of sclerostin at the bone surface was evaluated. The primary site of action and the extent to which systemic neutralization of sclerostin might be responsible for its therapeutic activity was further evaluated. Similarly, whether anchoring the antibody to the bone surface might restrict diffusion within the bone extracellular space, thereby limiting sclerostin binding and decreasing therapeutic activity, was investigated (Figure 5A).

To evaluate these unknowns, targeted (anti-Scl-Dio) and untargeted (anti-Scl) versions of the anti-(mouse)-sclerostin antibody were synthesized, incorporating murine kappa and IgGia constant regions and Dio-affinity ligands fused to the C-terminus of each heavy chain. The targeted anti-sclerostin antibody was selected to be a bivalent antibody (e.g. containing Dio affinity ligands fused to both heavy chains), as supported by the data above indicating superior properties of the bivalent IgG compared to the monovalent IgG. Throughout the application and figures, the abbreviations “Scl” and “SOST” are used interchangeably herein to denote the sclcrostin antibody. The purity of anti-Scl-Dw and anti-Scl was confirmed by HPLC (FIG. 11 A) and the structure determined by western blot (FIG. 1 IB), demonstrating the successful development of pure anti-sclerostin and anti-sclerosin-Dio antibodies having the expected size and components.

After confirming structure and purity of bone-targeted and untargeted antibodies, binding to recombinant mouse sclerostin was tested. Equivalent affinities were observed (Figure 5B). Next, antibodies were radiolabeled and mice were injected intravenously at a dose of 5mg/kg. Overall, the blood PK mirrored that of mCherry-Fc fusion proteins, with both proteins having prolonged circulation time and the bone-targeted antibody showing significantly decreased blood levels at later time points due to target-mediated disposition (Figure 5C). Consistent with this notion, anti-Scl-Dio demonstrated an order of magnitude increase in bone targeting at 24 hours post-injection, as compared to untargeted anti-Scl (14.47 ± 3.19 vs. 2.41 ± 0.44 %ID/g on femur, p<0.001, 13.4 ± 2.96 vs. 1.45 ± 0.21 %ID/g on vertebrae, /? < 0.001) (Figure 5D, E). Moreover, bone uptake of anti-Scl-Dio continued to increase from 1 to 3 to 7 days (14.47 ± 3.19, 16.63 ± 2.14, 20.90 ± 2.5 %ID/g on femur, 13.4 ± 2.96, 16.47 ± 2.36, 19.5 ± 2.48 %ID/g on vertebrae), whereas the bone uptake of anti-Scl was no longer detectable at these later time points.

Having confirmed bone targeting of anti-Scl-DlO vs. anti-Scl, the therapeutic efficacy in a murine model of ovariectomy-induced (OVX) osteoporosis was next evaluated. A bone- targeted but non-functional antibody (i.e., IgG-Dio) was used as an additional control. As shown in FIG. 6, 14-week-old healthy C57BL/6 wild-type mice were subjected to a bilateral ovariectomy and treated with antibodies or vehicle control weekly, starting one week after surgery. A 5 mg/kg dose, similar to the monthly dose in humans, but considerably lower than the 25mg/kg dose previously tested in rodents (J Bone Miner Res. 2009 Apr;24(4):578-88; J Clin Med. 2021 Feb 16; 10(4):787; U.S. Patent No. 7,592,429 B2), was chosen. A pilot study with n=4 sham and vehicle-treated OVX mice was performed to generate data for a sample size calculation. Based on the effect size of 21.2% absolute difference in BV/TV on microcomputed tomography analysis (sham = 26.6+3.5%; OVX = 5.4+3%), a sample size of n-8 mice per group was determined to be sufficient to detect a 35% decrease in bone loss with 0.80 power. The results of the therapeutic experiment are shown in FIG. 7. Volumetric analysis of the femur trabecular compartment in the defined region of interest demonstrated that OVX-induccd mice treated with anti-Scl-DlO had a significantly greater recovery of bone mass (BV/TV) than OVX mice treated with anti-Scl, IgG-DlO, or vehicle control (Fig 6B, Table 1). Anti-Scl-DlO also increased connectivity density (FIG. 6B), and the structure model index (FIG. 6D), two clinically relevant measures of bone quality, and bone mineral density (Fig 6H). These clinical measures reflect the evaluation that the bone in anti-Scl-DlO-treated OVX-mice is well organized and morphometrically normal relative to healthy control mice. Both Anti-Scl-DlO (0.199 ± 0.01198) and Anti-Scl (0.07471 ± 0.01839) improved BV/TV compared to vehicle treated control (0.04194 ± 0.01031). Similarly connective density and SMI were normalized to be nonsignificantly different in Anti-Scl-DlO treatment (Conn Dens: 112.8 ± 24.5; SMI: 1.986 ± 0.1833) from sham (healthy) mice (Conn Dens: 114.9 ± 27.32; SMI: 1.733 ± 0.2999) compared to vehicle-treated OVX (Conn Dens: 13.63 ± 5.912; SMI: 3.3 ± 0.2689) and Anti-Scl (Conn Dens: 35.48 ± 15.17; SMI: 2.896 ± 0.281). Anti-Scl treatment significantly improved these measures compared to OVX but was significantly less improves than Anti-Scl-DlO. Metrics of trabecular morphology — Tb.N, Tb.Th, and Tb.Sp - were significantly improved in both Anti-Scl (Tb.N: 3.315 ± 0.3273; Tb.Th: 0.05159 + 0.003628; Tb.Sp: 0.3011 + 0.03161) and Anti-Scl-DlO (Tb.N: 3.423 + 0.2579; Tb.Th: 0.05391 + 0.003456; Tb.Sp: 0.2878 + 0.2410) treated mice, compared to OVX (Tb.N: 2.926 ± 0.252; Tb.Th: 0.04631 ± 0.003897; Tb.Sp: 0.3453 ± 0.03378). Both BMD and TMD were significantly improved compared to vehicle-treated OVX mice (BMD: 41.86 + 9.923; TMD: 854.2 + 41.04), while Anti-Scl-DlO (BMD: 105.6 + 10.89; TMD: 911.2 ± 25.29) treatment significantly improved outcomes compared to Anti-Scl (BMD: 71.71 ± 17.48 ; TMD: 878.5 + 23.58).

Table 1: Descriptive statistics for microcomputed tomography of femur trabecular bone corresponding to Figure 6. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 8 mice (n = 16 femurs) for each condition and time point.

The impacts of these reagents for bone loss of the cortical compartments of femora was also investigated (midshaft, Fig. 8A). Relative to vehicle-treated OVX-micc, both anti-Scl and anti-Scl-DlO significantly improved cortical bone quality, without significantly affecting total mineral density of the cortex (Fig 8B-D, Table 2). Both Anti-Scl-DlO (0.4384 ± 0.02138) and Anti-Scl improved BV/TV (0.4578 ± 0.02119), with an insignificant difference between the two, compared to vehicle treated control (0.429 ± 0.01126). Both BMD and TMD were significantly improved compared to vehicle-treated OVX mice (BMD: 530.9 ± 22.54; TMD: 1258 ± 23.32), while Anti-Scl-DlO (BMD: 571.1 ± 25.25; TMD: 1268 ± 16.81 ) treatment significantly improved outcomes compared to Anti-Scl (BMD: 586.4 ± 21.54; TMD: 1250 ± 24.49).

Table 2: Descriptive statistics for microcomputed tomography of femur cortical bone corresponding to Figure 7, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 8 mice (n = 16 femurs) for each condition and time point.

Consistent with the anabolic effects on femoral bone maintenance, anti-Scl-DlO treatment promotes anabolism of vertebral trabecular bone in multiple clinically relevant outcomes (Fig 9, Table 3). Both Anti-Scl-DlO (0.2584 ± 0.01652) and Anti-Scl (0.2125 ± 0.0161) improved BV/TV compared to vehicle treated control (0.1691 ± 0.01078). Similarly connective density and SMI were normalized to be non-significantly different in Anti-Scl-DlO treatment (Conn Dens: 126.6 ± 23.34; SMI: 0.5098 ± 0.1858) from sham (healthy) mice (Conn Dens: 140.5 ± 9.468; SMI: 0.5065 ± 0.2537) compared to vchiclc-trcatcd OVX (Conn Dens: 83.92 ± 12.59; SMI: 1.237 ± 0.1244) and Anti-Scl (Conn Dens: 114 ± 12.27; SMI: 1.061 ± 0.1881). Anti-Scl treatment significantly improved these measures compared to OVX but was significantly less improves than Anti-Scl-DlO. Metrics of trabecular morphology — Tb.N, Tb.Th, and Tb.Sp - were significantly improved in both Anti-Scl (Tb.N: 3.944 ± 0.4431; Tb.Th: 0.05869 + 0.003908; Tb.Sp: 0.2491 + 0.03022) and Anti-Scl-DlO (Tb.N: 4.034 + 0.3484; Tb.Th: 0.06253 ± 0.002525; Tb.Sp: 0.2428 ± 0.01757) treated mice, compared to OVX (Tb.N: 3.441 ± 0.2008; Tb.Th: 0.05389 + 0.002785; Tb.Sp: 0.2885 + 0.01794). Both BMD and TMD were significantly improved compared to vehicle-treated OVX mice (BMD: 137.7 ± 10.37; TMD: 857.2 ± 47.37), while Anti-Scl-DlO (BMD: 210.8 ± 15.56; TMD: 873.8 ± 35.2) treatment significantly improved outcomes compared to Anti-Scl (BMD: 175.9 ± 13.48; TMD: 861.4 ± 22.96).

Table 3: Descriptive statistics for microcomputed tomography of L4 vertebral trabecular bone corresponding to Figure 8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 8 mice (n = 16 femurs) for each condition and time point.

Three-dimensional reconstructions of microcomputed tomography scans support the quantitative increase in bone in OVX mice treated with anti-Scl-DlO compared to other interventions in OVX mice and healthy control mice (Fig 7A, 8A, 9A). Treatment with anti-Scl- D10 significantly increased femoral and vertebral bone mass and recapitulated the morphometric characteristics of healthy control mice. Based on these findings, anti-Scl-DlO antibody therapy represents a novel, efficacious method for increasing bone formation and mitigating the degenerative effects of osteoporosis, superior to anti-Scl. Additional therapeutic conjugates were generated using anti-RANKL antibodies conjugating to the DIO bone targeting moiety. The bivalent conjugate anti-RANKL-DlO-DlO was generated and purified. Successful production was confirmed by western blot and HPLC, as shown in FIG. 16A and FIG. 16B, respectively. Adult mice were injected with non-functional IgG as a control, bivalent IgG-DlO, bivalent anti-RANKL-DlO, or anti-RANKL antibody. Blood pharmacokinetics and biodistribution in lung, liver, kidney, heart, spleen, brain, vertebrae, and femur was evaluated. Results are shown in FIG. 17. As shown in FIG. 17A and FIG. 17B, distribution of bone targeted anti-RANKL-DlO-DlO (e.g. bivalent anti-RANKL-DlO) was significantly higher in the vertebrae and femur co pared to non-targeted anti-RANKL. Biodistribution in other organs was not significantly different between groups. As shown in FIG. 17C, blood pharmacokinetics was similar’ amongst groups. Binding to recombinant RANKL was evaluated by ELISA. Results are shown in FIG. 17D. No differences in binding to recombinant RANKL was seen between antk- RANKL and bond targeted bivalent anti-RANKL (anti-RANKL-DlO-DlO).

Additional therapeutic conjugates were generated using anti-TGBp antibodies conjugated to the DIO bone targeting moiety. The bivalent conjugate anti- TGBp -D10-D10 was generated and purified. Successful production was confirmed by western blot and HPLC, as shown in FIG. 18A and FIG. 18B, respectively.

Adult mice were injected with non-functional IgG as a control, bivalent IgG-DlO, bivalent anti- TGBP-D10 (anti- TGBP-D10-DI0), or anti- TGBP antibody. Blood pharmacokinetics and biodistribution in lung, liver, kidney, heart, spleen, brain, vertebrae, and femur was evaluated. Results are shown in FIG. 19. As shown in FIG. 19A and FIG. 19B, distribution of bone targeted anti- TGBP-D10-D10 (e.g. bivalent TGBP-D10) was higher in the vertebrae and femur compared to non-targeted anti-TGBp. Biodistribution in other organs was not significantly different. As shown in FIG. 19C, blood pharmacokinetics was similar amongst groups.

Discussion:

Bone-targeted therapeutics are of great interest to localize therapeutic efficacy specific to the bone microenvironment. Unlike other tissues, such as vasculature, where cell surface markers enable efficient tissue- specific targeting, targeting the bone mineral surface requires targeting via electrostatic interactions. Bisphosphonates (such as ALD), acidic oligopeptides including decaaspartate (Dio, SEQ ID NO: 1) and icosa-aspartate (D20, SEQ ID NO: 8)affinity ligands are possible bone targeting moieties for therapeutics investigated herein. However, only one bone- targeted clinical therapeutic exists in the market (Bowden, S. A. & Foster, B. L. Profile of asfotasc alfa in the treatment of hypophosphatasia: design, development, and place in therapy. Drug Des. Devel. Ther. Volume 12, 3147-3161 (2018)). The current study is the first to compare the ALD, DIO (SEQ ID NO: 1), D20 (SEQ ID NO: 8) , and E10 (SEQ ID NO: 48) targeting moieties to establish the critical parameters in localizing protein therapeutics to bone. Literature reports of ALD-modification rely on nonspecific EDC/NHS chemistry whereby the formation of an NHS-ester with accessible carboxylic acid residues in the protein allows for the nucleophilic substitution by the primary amine of alendronate to form an amide bond. This bioconjugation method works well for small molecules with a single carboxylic acid residue, but it indiscriminately targets all accessible residues, limiting control over degree of modification. Furthermore the relationship between stoichiometric molar equivalents and actual degree of modification are unclear. Here a site-specific labelling method was used by fusing a LPXTGG motif (“sortag”) which acts as a linker to enable transpeptidation by Sortase A and site-specific modification of an azide-tag, containing the fluorescent molecule FITC, allowing for convenient purification and identification, and orthogonal Click conjugation to DBCO-ALD (FIG. 10). This method has a minimal effect on affinity, and therefore was the ideal method to enable a comparison between single-modified DIO and ALD proteins.

Compared to the EDC/NHS method, the sortag-sortase method enables a high degree of control to add a bisphosphonate affinity ligand to a protein with control over position and stoichiometry. Interestingly, a single modification with ALD is sufficient to enable robust targeting to mineral (Fig 2). Both DIO and ALD-modified mCherry demonstrate bone targeting, albeit modest (Fig 3). In both cases, less than 2% of the injected dose remains in circulation past four hours in mice, and approximately 1% of the injected dose is retained at the bone surface. Rather, both mCherry-DlO and mCherry- ALD are rapidly cleared via renal filtration and excreted in the urine. Given these findings, it was hypothesized that circulation time, rather than choice of targeting ligand, may be a major driver of localization to the bone microenvironment. Compared to other highly vascularized tissues, bone is composed of a dense matrix with a lesser vascular supply; this limited blood flow hinders the timely delivery of therapeutics to the bone surface, advocating for sustained exposure to effectively reach the bone. A longer circulation time may also minimize the likelihood of off-target effects or interactions with non-bone tissues. To overcome this limitation of rapid clearance, mCherry fused to the Fc fragment of human IgG, was fused to which the DIO or ALD targeting moiety was attached. Due to the diminishing yield of ALD conjugation to mCherry-Fc and insignificant difference between ALD and DIO ligands reported in Fig 3, the pharmacokinetic properties and biodistribution of mCherry-Fc-DlO were characterized. The Fc fragment of human IgG (immunoglobulin G) is the antibody molecule's constant region, which mediates various effector functions. Additionally, the Fc region is responsible for IgG antibodies' long half-life and extended circulation time in the bloodstream. The neonatal Fc receptor (FcRn) is a receptor that binds to IgG in an acidic environment (like in endosomes) and protects the antibody from lysosomal degradation. This mechanism rescues IgG from degradation pathways, allowing it to recycle back into the bloodstream and extend its circulating half-life. Additionally, the Fc region's size and structure help prevent the filtration of IgG antibodies through the glomeruli in the kidney, reducing their elimination through urine. Following a single intravenous administration, the circulation time of mCherry-Fc is extended significantly compared to mCherry, from ~4 hours to beyond 24 hours, and the same is true of mCherry-Fc-DlO and mCherry-DlO (Fig 4C). In the case of mCherry-Fc and mCherry-Fc-DlO, there is a significant and increasing difference in their blood concentration over time, indicative of bone uptake. Compared to mCherry-DlO, after four hours there is a 5- fold increase in mCherry-Fc-DlO localized to the bone surfaces, which continues to increase up to 24 hours to nearly 15% of the injected dose localizing to bone (Fig 4D). Thus, adding the Fc fragment, increasing circulation time and avidity through bivalent decaaspartate ligand, significantly shifts the biodistribution of mCherry-Fc-DlO towards the femur and vertebrae, and away from renal excretion. Interestingly, even nontargeted mCherry-Fc shows some localization to the bone, although significantly less than mCherry-Fc-DlO (Fig 4F). Fluorescence imaging of histologic sections unveils its localization in the marrow space, but not to the trabecular surface, which may be attributed to Fc receptors in the marrow. Fc receptors (FcRs) are cell surface receptors that specifically bind to the Fc region of antibodies, including immunoglobulin G (IgG), immunoglobulin E (IgE), and immunoglobulin A (IgA). While Fc receptors are predominantly found on various circulating immune cells, it is worth noting that the bone marrow is the site of B cell development. B cells mature in the bone marrow and express Fc receptors, which are involved in B cell activation and other functions. Additionally, some myeloid immune cells, like macrophages and dendritic cells, can originate from progenitor cells in the bone marrow and express Fc receptors once they mature and enter the bloodstream or tissues.

Given the human clinical use of Romosozumab (Evenity), a monoclonal anti-sclerostin antibody approved to treat osteoporosis in postmenopausal women at high risk for fracture, the effect of bone targeting on the therapeutic index of a mouse anti-sclerostin antibody was evaluated. Additionally, bone-targeted TBFp and bone-targeted anti-RANLK antibodies were generated and tested herein. Bone targeted antibodies demonstrated increased biodistribution to the femur and vertebrae compare to non-targeted versions. Bivalent bone-targeted antibodies had superior localization to the femur and vertebrae compared to monovalent bone-targeted antibodies. Specifically, bivalent bone-targeted antibodies containing either DIO (SEQ ID NO: 1) or D20 (SEQ ID NO: 8) as the bone targeting moiety had the greatest increase in localization to the femur and vertebrae compared to untargeted antibody versions.

Similar to mCherry-Fc-DlO, both anti-Scl-DlO and IgG-DlO circulated in the blood up to seven days in mice (Fig 5A). Both IgG-DlO and anti-Scl-DlO target to the femur and vertebrae efficiently at 24 hours, increasing at 72 hours and their concentration at the intended site is maintained at 7 days. Sclerostin is a soluble factor that plays a negative regulatory role in bone maintenance by inhibiting bone formation. In osteoporosis, the overexpression of sclerostin leads to increased bone resorption and decreased bone formation, contributing to reduced bone density and increased fracture risk. It was investigated whether localizing sclerostin sequestration and inactivation to the bone surface would increase its therapeutic efficacy. In a mouse model faithfully recapitulating postmenopausal osteoporosis, the osteoanabolic effects of anti-Scl-DlO were found to significantly outperform anti-Scl in recovering and halting the loss of bone mass resulting from surgical ovariectomy (Fig 7, 8, 9). Furthermore, the results herein suggest that therapeutic inactivation of sclerostin is required locally within the bone microenvironment, rather than inactivation of circulating sclerostin, in order to exert its osteoclast inhibitory effects.

Bisphosphonates have been historically used to treat osteoporosis due to their potent anti- resorptive effects that inhibit bone breakdown by osteoclasts. They also possess anti angiogenic properties that can be beneficial in conditions like cancer. However, long-term use of bisphosphonates can lead to Medication-Related Osteonecrosis of the Jaw (MRONJ), a rare but serious condition characterized by jawbone tissue death. This has raised concerns, prompting the development of guidelines to manage MRONJ risk and highlight the importance of careful monitoring during bisphosphonate treatment.

Non-bisphosphonate bone anabolic agents are a group of medications used to treat osteoporosis by promoting bone formation and increasing bone density. Unlike bisphosphonates, which are anti-resorptive agents that inhibit bone breakdown, non-bisphosphonate bone anabolic agents work by stimulating bone-building cells (osteoblasts) to increase bone formation. Teriparatide is a recombinant form of parathyroid hormone (PTH) that is analogous to the naturally occurring hormone. It stimulates bone formation by activating osteoblasts and increasing the number of bone-forming cells. Romosozumab enhances bone formation by blocking and inactivating sclerostin to decrease bone resorption without causing osteoclast cell death. Both teriparatide and romosozumab have been shown to effectively increase bone mineral density and reduce the risk of fractures in individuals with osteoporosis. Teriparatide is usually prescribed for a limited duration (up to two years) due to concerns about its long-term safety, particularly an increased risk of osteosarcoma, a rare form of bone cancer, seen in animal studies. A potential concern with romosozumab is an increased risk of cardiovascular events, particularly in those with a history of cardiovascular disease. In Phase 2 clinical trials, romosozumab demonstrated significant bone mineral density (BMD) increases in postmenopausal women with low bone mass. Subsequent Phase 3 trials, including the FRAME (J. Bone Miner. Res. 33, 1219— 1226 (2018)) and ARCH (J. Bone Miner. Res. 36, 2139-2152 (2021) studies, showed that romosozumab treatment significantly reduced the risk of vertebral and non-vertebral fractures compared to placebo and the anti-resorptive drug alendronate. These findings highlight romosozumab's potential as an effective therapeutic option for postmenopausal osteoporosis, especially in patients at high risk of fractures. However, there have been concerns about cardiovascular safety, particularly in the ARCH trial, where an increased risk of cardiovascular events was observed in some participants. As a result, the US Food and Drug Administration (FDA) issued a boxed warning for romosozumab, cautioning against its use in individuals with a history of myocardial infarction or stroke. As a result, romosozumab carries a boxed warning from the FDA about potential cardiovascular risks.

The results presented herein demonstrate that affinity targeting to bone increases the potency of a therapeutic protein drug and its safety profile by achieving lower required doses and decreasing dosing interval. The original trials of anti-sclerostin antibody in mice demonstrated efficacy at a dose of 25 mg/kg dosed intravenously every two days, which out-performed PTH (1-34) (/. Bone Miner. Res. 24, 578-588 (2009)). Herein, anti-Scl-DlO, a bone-targeted version, is shown to be efficacious at 5 mg/kg, dosed weekly, which may be attributed to its increased circulation time and affinity targeting. Other literature reports suggest rodent dosing of anti-Scl in excess of 50 mg/kg to maintain efficacy at biweekly administration (Sci. Transl. Med. 5, (2013), Bone 96, 63-75 (2017), Bone 87, 161-168 (2016)). This reduced clinically effective dose seen in the bone-targeted conjugates provided herein pennits safe use in subjects for treatment of bone disease, such as osteoporosis, while minimizing undesirable side effects including risk of cardiovascular events such as myocardial infarction of stroke.

In sum, bone targeted therapeutics have wide potential clinical applications for various resorptive and catabolic bone diseases by locally modulating cellular crosstalk and the balance between bone resorption and formation, while simultaneously decreasing the risk of off-site accumulation. Bone-targeted therapeutics provide a unique opportunity to study the role of local and circulating factors in bone metabolism and homeostasis. By specifically targeting agents to the bone microenvironment, it is possible to investigate the effects of manipulating local factors like osteocytes, osteoblasts, and osteoclasts in isolation from systemic influences. This approach allows for a deeper understanding of bone biology and the interplay between local and systemic factors in maintaining bone health and responding to various pathological conditions, such as osteoporosis and bone metastases.

Provided herein are bone-targeted therapeutics for the treatment of osteoporosis and other bone-related disorders. The study investigates the use of bisphosphonate, deca-aspartate, and icosa-aspartate as affinity ligands for bone targeting, probing at their targeting efficacy in site- specifically modified protein cargo. While both ALD,D10 (SEQ ID NO: 1), and D20 (SEQ ID NO: 8), effectively target the bone, these molecules when conjugated to fluorescent proteins were compromised by their limited circulation time and rapid renal clearance, ultimately limiting their ability to locate to the bone compartment. By incorporating the Fc fragment of human IgG into bone-targeted therapeutics, an increase in circulation time and bone-specific accumulation was observed. This enhancement in bone-targeting efficiency leads to potential lower dosing regimens and extended dosing intervals, which could improve therapeutic outcomes and reduce off-target effects.

Based on these findings, the therapeutic efficacy of a multiple bone-targeted therapeutic antibodies were evaluated. Specifically, bone-targeted ant-sclerostin, bone-targeted TBF0, and bone-targeted anti-RANKL antibodies were generated and tested herein. All bone targeted antibodies were shown to have improved localization to the bone (e.g. femur and vertebrae) compared to non-targeted antibodies, with the greatest improvement in localization seen in bivalent bone-targeted antibodies containing either DIO (SEQ ID NO: 1) or D20 (SEQ ID NO: 8) as the bone-targeting moiety conjugated to each antibody heavy chain.

The bone-targeted anti-sclerostin antibody targeted to the bone surface using the DIO moiety (SEQ ID NO: 1) or the D20 moiety (SEQ ID NO: 8) was evaluated compared to an untargeted anti-sclerostin antibody, the clinically available drug Romosozumab. This bone- targeted approach outperforms the non-targeted anti-sclerostin antibody in recovering bone mass and mitigating the effects of osteoporosis. Accordingly, provided herein are improved treatments for osteoporosis and other bone disorders using affinity targeted therapeutics with sufficient circulation time to reach the bone surface effectively.

Materials: All chemical reagents were purchased from Sigma Aldrich unless specifically mentioned in the below methods. Reagents were used as received unless otherwise specified. Bovine bone chips were obtained from X. Hydroxyapatite disks were obtained from Y.

Expression vector pTT5 and mammalian cell line (HEK293-6E) were licensed from the National Research Council of Canada (NRC).

Animals: Animal studies were conducted following guidance for the care and use of laboratory animals as adopted by the NIH, approved by the institutional animal care and use committee (IACUC) of the University of Michigan. C57BL/6J male or female mice (Jackson Laboratory, 000664), aged 12-16 weeks, were used for all animal experiments.

Bacterial protein production and purification: The amino acid sequences of the fluorescent proteins mCherry, eGFP, and mCardinal were obtained from the UniProt database. N- and C- terminal sorttags or deca-aspartate peptides were appended to the coding sequences. cDNAs encoding each protein were cloned into the pRSET vector (Thermo Fisher, CAT# V35120) and transformed to BL21 (DE3) competent E Coli (Theromfisher, CAT# 600003) for production. Bacteria were grown in Terrific Broth (TB) medium to early log phase, as judged by OD600, induced with IPTG, and then incubated for 24h at 18 degree. Bacterial cell pellets were lysed by sonication and proteins were purified using Ni-NTA agarose His tag affinity resin (Qiagen, CAT# 30210). > 95% purity was verified by SDS-PAGE gel electrophoresis and size exclusion (SEC)-HPLC. All proteins were stored at -80°C in aliquots, until their use, to avoid repeated freeze-thaw cycles.

Mammalian protein production and purification: For production of mCherry-Fc fusion proteins, the fluorescent protein was codon optimized for production in human cells using an online tool (www.idtdna.com/CodonOpt), fused to the Fc fragment of human IgGl, and cloned into the pTT5 mammalian expression vector (National Research Council of Canada). For production of anti-(mouse)-sclerostin antibodies, the sequences of heavy and light chain variable regions (U.S. Patent No. 7,592,429 B2) were cloned into the pTT5 vector with mouse kappa and IgG2a constant regions. The non-functional control IgG is an antibody generated by rat immunization using phosphorylated tau (pS422), which has been shown to have no target in mouse. In each case, bone-targeted versions were created by fusing a sequence encoding the amino acids DDDDDDDDDD (SEQ ID NO: 1) (the Dio peptide) to the C-terminus of the Fc fragment. Protein production was performed by transfecting 25 mL of mammalian cell culture (2xl0⁶ cells/mL) with appropriate plasmids. In each conical tube, 15 pg of DNA was mixed with 45 pF of 40 kDa polyethylenimine (1 mg/mE) and 3 mL of F17 media (Gibco CAT# A13835- 01). The mixture was added to HEK293-6E cells and transferred back to a 5% CO2 incubator @ 37C. 750pL of yeastolate (20% w/w) (Thermo Fisher, CAT# 292804) was added to each tube of transfected cells 24h post transfection. Cells were harvested 5-7 days after transfection and centrifuged at 3500 rpm for 40 min. Protein was purified from the cell supernatant via Protein A agarose (Thermo Fisher, CAT: 20334). Proteins were stored at -80°C in aliquots to avoid repeated freeze-thaw cycles.

Site-specific modification of fluorescent proteins: Sortase modification of N- and C-terminal fluorescent proteins was performed as previous described (PMID: 34386959, 29200285). In brief, LPET (SEQ ID NO: 2) -tagged proteins were reacted with GGG peptide-azide at a 1:5 ratio (5 mol. GGG peptide-azide per 1 mol. LPET-tagged protein) for C-terminal modification in TBS buffer with presence of calcium-dependent sortase (IpM) and Ca²⁺ (ImM) overnight at room temperature. For N-terminal modification, GGG-proteins were reacted with LPET (SEQ ID NO: 2) peptide-azide under the same conditions. Unmodified proteins more removed using Ni-NTA agarose and excess peptide-azide were removed using a lOkDa filter (Amicon, CAT# UFC501096). Azide-modified fluorescent proteins were then reacted with an 5-fold excess of BP-DBCO at room temperature overnight to yield protein-BP conjugates. BP-modified protein was then dialyzed in a lOkDa MWCO cassette (Thermo scientific, CAT# 87729) to remove excess BP-DBCO.

Anti-Scl and anti-Scl-Dio binding affinity ELISA assay: 96- well plate was coated with 0.05 pg recombinant murine sclerostin (R&D systems, CAT# 1589-ST) per well in lOOpl PBS overnight at 4C. Next day, plate was washed 3 times with PBS + 0.05% Tween20 (PBS-T), and then blocked with 200ul PBS+3% BSA at RT for Ih. Post blocking, plate was washed 3 times with PBS-T, followed by adding lOOul of anti-Scl and anti-Scl-Dio at various concentrations and incubated at RT or Ih. Afterwards, plate was washed 3 times with PBS-T and incubated with anti-mouse-HRP (Jackson Immunoresearch, CAT# 115035003) at RT for 30min followed by PBS-T wash. The plate was developed using TMB substrate (Thermo scientific, CAT: 34021) following the manufacture instructions, and the result was read by plate-reader under absorbance of 450nm.

In vitro bone targeting assays: For in vitro assessment of function, bone-targeted and untargeted fluorescent proteins were incubated with hydroxyapatite disks or bovine bone chips at 2pM concentration for 1 hour at room temperature, prior to washing five times with PBS. A Nikon Eclipse C2 confocal laser microscope was used to visualize fluorescence signal. All experiments are repeated at n>3, with representative images shown.

Quantitative radiotracing of protein biodistribution: Proteins were directly radioiodinated with [125I]NaI (Perkin Elmer) using Pierce iodination reagent (Thermo Fisher, 28601) and purified using Zeba desalting columns. Radiochemical purity was assessed via thin layer chromatography (TLC) performed using aluminum TLC silica gel 60 F254 plates (Millipore Sigma, CAT #105554) and a 75%:25% mixture of methanol and 1 M sodium acetate (pH 6.8) as a mobile phase, as previously reported (PMID: 32738178). For radiotracing experiments, a tracer dose (1 or 2 pg) of ¹²⁵I-labeled protein was added to the appropriate mass of non-radioactive protein to give the desired dose (e.g., 2 or 5mg/kg). Doses were administered intravenously via retro-orbital injection. Blood was collected at designated time points from the retro-orbital plexus or from the inferior vena cava at the time of euthanasia. Animals were transcardially perfused with 15mL of phosphate buffered saline (PBS) to flush residual blood content from organs. Radioactivity of blood and organs were measured via gamma counter (PerkinElmer, 2470 Automatic Gamma Counter).

Fluorescence imaging of ex vivo bone tissue:

To determine localization and retention of bone-targeted proteins in vivo, mice were administered fluorescent proteins intravenously via retro-orbital injection. Animals were transcardially perfused at the appropriate time point and bones were harvested for fixation in 4% PFA (48 hours). Bones were embedded in OCT (vendor info) for cryosectioning and imaged using confocal microscopy.

Ovariectomy-Induced Osteoporosis Model: 13-week-old healthy female C57BL/6 mice were received from Jackson Laboratories (000664). Surgical ovariectomy (OVX) was performed as previously described. Briefly, mice were acclimated for 1 week and then anesthetized with a constant plane of isoflurane. Pre-operative carprofen (5 mg/kg) was given via subcutaneous injection. Hair was removed from the dorsal midline and the surgical site was scrubed with alternating betadine and saline, three times. A 1 cm skin incision was made along the dorsal midline to expose the dorsolateral abdominal muscles in the lower back just below the rib cage. A 0.25 cm incision was made bilaterally to locate the ovaries. Holding the edge of the incision, the fat pad attached to the ovary was retracted from the abdominal cavity to expose the oviduct. In “sham control” mice, the ovary and fat pad were immediately returned to the peritoneal cavity. In “OVX” mice that received the full surgery, the exposed ovary and the oviduct were carefully removed using sterile scissors, and hemostasis was achieved. The uterus and remaining oviduct were placed back into the abdominal cavity. The incision in the abdominal wall was closed with a resorbable suture (5-0 vicryl). The remaining skin incision was closed with 4-0 Ethilon monofilament sutures or suture clips. Post-operative carprofen was administered at 24 hours (5 mg/kg subcutaneous injection) and animals were monitored daily for the first 10 days and then twice weekly until euthanasia. Bone-targeted and untargeted sclerostin antibodies and targeted control (non-functional) antibodies (5mg/kg) were administered weekly via retroorbital injection, starting at 7 days post-surgery. After four weeks, mice were euthanized by CO2 asphyxiation and bilateral pneumothorax. Long bones and vertebrae were harvested at the endpoint, fixed with 4% paraformaldehyde at 4°C for 48 hours, and then kept in PBS at 4°C for subsequent analysis.

Micro-computed Tomography Analysis (micro-CT): Micro-CT analysis was performed. The samples were placed in a 19 mm diameter specimen holder and scanned over the entire length of the tibia using a micro-CT system (pCTlOO Scanco Medical, Bassersdorf, Switzerland) with voxel size 10 pm, 70 kVp, 114 pA, 0.5 mm AL filter, and integration time 500 ms. For femur analysis: A 1.0 mm region of trabecular compartment was analyzed immediately below the growth plate using a fixed global threshold of 18%; and a 0.3 mm region of cortical compartment at the midpoint was analyzed using a fixed global threshold of 28% (280 on a grayscale of 0- 1,000). For vertebral analysis: the L4 vertebrae was identified; the trabecular compartment was analyzed using a fixed global threshold of 18%. Trabecular' bone volume fraction (BV/TV), connectivitydensity (Conn Dens), structure model index (SMI), trabecular thickness (Tb. Th), trabecular number (Tb. N), trabecular separation (Tb. Sp), cortical bone volume fraction (BV/TV), cortical porosity, cortical thickness, bone mineral density (BMD), tissue mineral density (TMD), sub-periosteal area and sub-endosteal area were analyzed using an evaluation software from the manufacture.

Statistical Analysis: Statistical analyses were done using one-way analysis of variance (ANOVA) among experimental groups and followed by a Tukey test. All experiments were done with at least three biological replicates or more per group. The results are expressed as the mean ± SD. Statistical significance is considered p < 0.05. For OVX experiments, a sample size calculation was performed using data from a pilot study and G*Power 3 software (Behav Res Methods. 2007 May;39(2): 175-91.

Each of the various references, presentations, publications, provisional and/or nonprovisional U.S. patent applications, U.S. patents, non-U. S. patent applications, and/or non-U. S. patents that have been identified herein, is incorporated herein in its entirety by this reference. Although the various aspects and features of the present invention have been described with respect to various embodiments and specific examples herein, all of which may be made or carried out conventionally, it will be understood that the invention is entitled to protection within the full scope of the appended claims.

Example 2 Table 4 shows representative sequences were used in the development and testing of the conjugates provided herein.

Table 4.

Claims

CLAIMS We claim:

1. A bone targeting therapeutic conjugate, the conjugate comprising: a) a therapeutic antibody for treatment of a bone disease; and b) at least one bone targeting moiety.

2. The conjugate of claim 1, wherein the therapeutic antibody for treatment of a bone disease is an anti-sclerostin antibody, an anti-RANKL antibody, or an anti-TGF antibody.

3. The conjugate of claim 1 or claim 2, wherein the at least one bone targeting moiety is a bisphosphonate or a bone targeting peptide.

4. The conjugate of claim 3, wherein the at least one bone targeting moiety is alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate, or zoledronate.

5. The conjugate of claim 4, wherein the bisphosphonate is alendronate.

6. The conjugate of claim 3, wherein the bone targeting peptide is an acidic oligopeptide.

7. The conjugate of claim 6, wherein the bone targeting peptide is an acidic oligopeptide comprising 6-25 combined aspartic acid and/or glutamic acid residues.

8. The conjugate of claim 7, wherein the bone targeting peptide is deca-aspartate (Dio (SEQ ID NO: 1) or icosa-aspartate (D20) (SEQ ID NO: 8).

9. The conjugate of any one of claims 1-8, wherein the at least one bone targeting moiety is conjugated to a heavy chain of the therapeutic antibody for treatment of a bone disease.

10. The conjugate of claim 9, wherein the at least one bone targeting moiety is directly conjugated to the heavy chain.

11. The conjugate of claim 9, wherein the at least one bone targeting moiety is conjugated to the heavy chain by a linker.

12. The conjugate of claim 11, wherein the linker is a glycine-rich linker.

13. The conjugate of claim 12, wherein the linker comprises one or more repeating GGGGS (SEQ ID NO: 9) units.

14. The conjugate of claim 13, wherein the linker comprises 1-4 repeating GGGGS (SEQ ID NO: 9) units.

15. The conjugate of any one of claims 9-14, wherein a first bone targeting moiety is conjugated to a first heavy chain of the therapeutic antibody for treatment of a bone disease and a second bone targeting moiety is conjugated to a second heavy chain of the therapeutic antibody for treatment of a bone disease.

16. The conjugate of claim 15, wherein the first bone targeting moiety and the second bone targeting moiety are each a bisphosphonate.

17. The conjugate of claim 16, wherein the first bone targeting moiety and the second bone targeting moiety are each alendronate.

18. The conjugate of claim 15, wherein the first bone targeting moiety and the second bone targeting moiety are each an acidic oligopeptide.

19. The conjugate of claim 18, wherein the first bone targeting moiety and the second bone targeting moiety arc each an acidic oligopeptide comprising 6-25 combined aspartic acid and/or glutamic acid residues.

20. The conjugate of claim 19, wherein the first bone targeting moiety and the second bone targeting moiety are each deca-asparate (Dio (SEQ ID NO: 1) or icosa-aspartate (D20) (SEQ ID NO: 8).

21. The conjugate of any one of claims 1-20, wherein the bone disease is osteoporosis.

22. A composition comprising the conjugate of any one of claims 1-21.

23. The conjugate of any one of claims 1-21 or the composition of claim 22, for use in a method of treating a bone disease in a subject.

24. The conjugate of claim 23, wherein the bone disease is osteoporosis.

25. A method of treating a bone disease in a subject, the method comprising providing the conjugate of any one of claims 1-21 or the composition of claim 22 to a subject having or suspected of having a bone disease.

26. The method of claim 25, wherein the bone disease is osteoporosis.

27. The method of claim 25 or claim 26, wherein a therapeutically effective dose of the conjugate is less than a therapeutically effective dose of the therapeutic antibody for treatment of the bone disease.

28. The method of any one of claims 25-27, wherein the subject is a human.

29. The method of claim 28, wherein the therapeutically effective dose of the conjugate is less than 3 mg/kg when the conjugate is provided to the subject monthly.

30. The method of any one of claims 24-29, wherein the conjugate is provided to the subject by injection.

31. Use of the conjugate of any one of claims 1-21 or the composition of claim 22 in a method of treating a bone disease in a subject.

32. Use of claim 31, wherein the bone disease is osteoporosis.

33. Use of claim 31 or claim 32, wherein the subject is a human.