Antibodies targeting RANKL and sclerostin and uses thereof

Abstract

Bispecific antibodies that target both human RANKL and sclerostin are provided. Also provided herein are polynucleotides encoding the bispecific antibodies, pharmaceutical compositions comprising the bispecific antibodies, and methods of producing the bispecific antibodies. Medical uses of the bispecific antibodies described herein are also disclosed.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 520,102, filed August 17, 2023, which is incorporated by reference in its entirety.

[0002] Reference Sequence Listing filed electronically

[0003] The instant application incorporates by reference a Sequence Listing in XML file, named “022A005WO02_SL”, which was created on August 14, 2024, and has a size of 84,092 bytes. TECHNICAL FIELD

[0004] The present invention relates to molecular and cellular biology. Provided herein include bispecific antibodies that specifically bind to human RANKL and human sclerostin, and their use, for example, in reducing osteoporosis. BACKGROUND

[0005] Osteoporosis is a debilitating disease in humans characterized by a significant decrease in bone mass and mineral density, deterioration of bone structure including deterioration of bone microarchitecture, and a corresponding increase in bone fragility and susceptibility to fracture in the skeleton of the afflicted individual. Osteoporosis results from a functional imbalance between osteoclasts and osteoblasts. Current strategies for preventing and treating osteoporosis target either osteoclasts or osteoblasts, which can provide some benefit to the individual, but do not ensure resolution of the disorder or are associated with adverse side effects. Thus, there is an urgent need for additional therapeutic options for osteoporosis. The compositions and methods provided in the present disclosure address this need and provide related advantages. SUMMARY

[0006] The bispecific antibodies provided herein are capable of binding to both human RANKL and human sclerostin simultaneously. The antibodies disclosed herein are capable of effectively blocking osteoclast differentiation and promoting osteoblast function.

[0007] The bispecific antibodies provided herein comprise (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1), wherein the VL1 / VH1 pair specifically binds to human RANKL, and wherein the VL1 comprises a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and wherein the VH1 comprises a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and (ii) a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), wherein the VL2 / VH2 pair specifically binds to human sclerostin; and wherein the VL2 comprises a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, and wherein the VH2 comprises a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. In some embodiments, the VL1, VH1, VL2, and VH2 have the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0008] In some embodiments, the bispecific antibodies provided herein comprise (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, a VL1 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, a VH1, a heavy chain constant domain 1 (CH1), and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising a VL2 and a VH2, a linker, and a Hole-Fc region.

[0009] In some embodiments, the bispecific antibody provided herein comprises (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain 1 (CH1), and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL2 and VH2, a linker, and a Knob-Fc region.

[0010] In some embodiments, the scFv comprises, from N-terminus to C-terminus, VL2, a second linker, and VH2. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VH2, a second linker, and VL2.

[0011] In some embodiments, the bispecific antibody provided herein comprises (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1), and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL1 and VH1, a linker, and a Hole-Fc region.

[0012] In some embodiments, the bispecific antibody provided herein comprises (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1), and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL1 and VH1, a linker, and a Knob-Fc region.

[0013] In some embodiments, the scFv comprises, from N-terminus to C-terminus, VL1, a second linker, and VH1. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VH1, a second linker, and VL1.

[0014] In some embodiments of the bispecific antibodies provided herein, the Knob-Fc region is a human IgG2 Fc region variant having up to 10 amino acid substitutions, including a T366W substitution; and the Hole-Fc region is a human IgG2 Fc region variant having up to 10 amino acid substitutions, including T366S, L368A, Y407V substitutions. In some embodiments, the Knob-Fc region further comprises a S354C substitution and the Hole-Fc region further comprises a Y349C substitution. In some embodiments, the Knob-Fc region further comprises a Y349C substitution and the Hole-Fc region further comprises a S354C substitution.

[0015] In some embodiments of the bispecific antibodies provided herein, (i) the CL region is a kappa CL (CK; SEQ ID NO: 19) or a lambda CL (CL; SEQ ID NO: 20) or a variant thereof having up to 10 amino acid substitutions; (ii) the CH1 domain is a human IgG2 CH1 domain (SEQ ID NO: 36) or a variant thereof having up to 10 amino acid substitutions; and / or (iii) the Knob-Fc region and the Hole-Fc region (1) have the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 32, respectively, (2) have the amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 33, respectively, or (3) have the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 34, respectively, or a variant thereof having up to 10 amino acid substitutions. In some embodiments, the CL region, CH1 domain, Knob-Fc region, and Hole-Fc region (1) have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 29, and SEQ ID NO: 32, respectively, (2) have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 30, and SEQ ID NO: 33, respectively, or (3) have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 31, and SEQ ID NO: 34, respectively.

[0016] In some embodiments of the bispecific antibodies provided herein, C1, C2, and C3 have the amino acid sequences of (1) SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 54, respectively, (2) SEQ ID NO: 51, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, (3) SEQ ID NO: 51, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; or (4) SEQ ID NO: 51, SEQ ID NO: 59, and SEQ ID NO: 60, respectively.

[0017] The bispecific antibodies provided herein comprise a VL1 / VH1 pair that specifically binds to human RANKL and a VL2 / VH2 pair that specifically binds to human sclerostin. In some embodiments, the bispecific antibodies provided herein comprise (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant region (CL); and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant region (CH), a linker, and a single chain variable fragment (scFv) comprising VL2 and VH2. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VL2, a second linker, and VH2. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VH2, a second linker, and VL2.

[0018] In some embodiments, the bispecific antibodies provided herein comprise (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant region (CH), a linker, and a single chain variable fragment (scFv) comprising VL1 and VH1. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VL1, a second linker, and VH1. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VH1, a second linker, and VL1.

[0019] In some embodiments of the bispecific antibodies provided herein: (1) the CL region is a kappa CL (CK; SEQ ID NO: 19) or a lambda CL (CL; SEQ ID NO: 20) or a variant thereof having up to 10 amino acid substitutions; or (2) the CH region is a human IgGl CH region (SEQ ID NO: 21), an IgG2 CH region (SEQ ID NO: 22), an IgG3 CH region (SEQ ID NO: 23), or an IgG4 CH region (SEQ ID NO: 24), or a variant thereof having up to 10 amino acid substitutions; or both (1) and (2). In some embodiments, (1) the CL region is a kappa CL (CK; SEQ ID NO: 19); and (2) the CH region is a human IgG2 CH region (SEQ ID NO: 22).

[0020] In some embodiments, C1 and C2 (1) have the amino acid sequence of SEQ ID NO: 51 and SEQ ID NO: 61, respectively, (2) have the amino acid sequence of SEQ ID NO: 51 and SEQ ID NO: 62, respectively. In some embodiments, C1 and C2 (1) have the amino acid sequence of SEQ ID NO: 52 and SEQ ID NO: 63, respectively, (2) have the amino acid sequence of SEQ ID NO: 52 and SEQ ID NO: 64, respectively.

[0021] Also provided herein are pharmaceutical compositions comprising a therapeutically effective amount of the bispecific antibodies described herein and a pharmaceutically acceptable carrier.

[0022] Also provided herein are polynucleotides encoding the peptide chains of the bispecific antibodies described herein. In some embodiments, the polynucleotides provided herein encode all of the peptide chains of the bispecific antibodies. In some embodiments, also provided herein are multiple polynucleotides that collectively encode all of the peptide chains of the bispecific antibodies.

[0023] Also provided herein are vectors comprising the polynucleotides described herein.

[0024] Also provided herein are cells comprising the polynucleotides or multiple polynucleotides described herein.

[0025] Also provided herein are methods of making a bispecific antibody that specifically binds to human RANKL and human sclerostin, comprising culturing the cells described herein under conditions that allow the bispecific antibody to be expressed.

[0026] Also provided herein are methods of preventing or treating osteoporosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody described herein. In some embodiments, the subject has primary osteoporosis. In some embodiments, the subject has postmenopausal osteoporosis (Type I). In some embodiments, the subject has senile osteoporosis (Type II). In some embodiments, the subject has secondary osteoporosis. In some embodiments, the methods provided herein prevent or treat osteoporosis secondary to an endocrine or metabolic cause, a collagen or genetic disease, a drug cause, or a nutritional origin. In some embodiments, the subject is a human.

[0027] Also provided herein are uses of the bispecific antibodies described herein as a medicament. Also provided herein are uses of the bispecific antibodies described herein in the prevention or treatment of osteoporosis. Also provided herein are uses of the bispecific antibodies described herein for the preparation of a medicament for the prevention or treatment of osteoporosis. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figures 1A-1B Provided are schematic diagrams of the bispecific antibodies provided herein ("DRAbs") that specifically bind to RANKL and sclerostin. Figure 1A Provided is an exemplary description of a knobs-into-holes (KIH) configuration of a bispecific antibody. Figure 1B Provided is an exemplary description of an IgG-scFv configuration of a bispecific antibody.

[0029] Figure 2 Provided are ELISA results showing the specific binding of DRAbs DR-P7, DR-P15, and DR-P16 to human RANKL (top panel) and sclerostin (bottom panel).

[0030] Figure 3 Provided are results of a TRACP assay showing that DRAbs DR-P7 and DR-P8 effectively block osteoclast differentiation.

[0031] Figure 4 Provided are assay results measuring the expression of marker genes (MMP9, FATcl, Acp5, CTSK) of osteoclast differentiation showing that DRAbs DR-P7 and DR-P8 effectively block the expression of these marker genes.

[0032] Figure 5 Provided are results of a NanoLuc assay using HEK 293 cells showing that DRAbs DR-P7 and DR-P8 effectively block sclerostin-mediated inhibition of WNT signaling.

[0033] Figure 6 Results in an ovary-removed mouse model are provided, which show that DRAb DR-P7 effectively promotes bone regrowth in mice and a synergistic effect is observed using DRAb compared to antibodies targeting RANKL or sclerostin alone. OVX: ovariectomy; Tb.N: trabecular number; Tb.Sp: trabecular separation. DETAILED DESCRIPTION

[0034] Provided herein are bispecific antibodies ("DRAb") that specifically bind to human RANKL and human sclerostin. Also disclosed herein are pharmaceutical compositions comprising a therapeutically effective amount of such antibodies. Also disclosed herein are uses of such antibodies and pharmaceutical compositions for reducing osteoporosis and / or increasing bone mass.

[0035] Osteoporosis is a skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to increased bone fragility and susceptibility to fracture. It is a systemic disorder of whole bone, leading to decreased bone mineral density and structural integrity. Osteoporosis can be primarily divided into two categories: primary osteoporosis and secondary osteoporosis.

[0036] Primary osteoporosis includes postmenopausal osteoporosis (Type I) or age-related osteoporosis (Type II). Postmenopausal osteoporosis (Type I) usually occurs in postmenopausal women, which is due to the decrease in estrogen levels. Estrogen plays a crucial role in maintaining bone density. The loss of estrogen leads to accelerated bone resorption, resulting in increased bone fragility. Age-related osteoporosis (Type II) or osteoporosis of aging affects both men and women with age. It is a result of the natural aging process and gradual decrease in bone mass over time. Primary osteoporosis also includes idiopathic osteoporosis, which refers to the condition of osteoporosis occurring without apparent cause. It can affect individuals of any age and can have a genetic component.

[0037] Secondary osteoporosis refers to a condition characterized by decreased bone density and strength, which is caused by underlying disorders or external factors. Several factors can contribute to the development of secondary osteoporosis, including endocrine or metabolic causes, collagen / genetic disorders, medications, and nutritional deficiencies.

[0038] Endocrine or metabolic causes are one of the major types of secondary osteoporosis. For example, disorders such as hyperparathyroidism, hypercortisolism, hyperprolactinemia, etc. can disrupt the normal balance of bone remodeling-related hormones. For example, in hyperparathyroidism, the overproduction of parathyroid hormone leads to increased bone resorption, resulting in decreased bone mineral density and increased risk of bone fracture.

[0039] Collagen / genetic diseases are another group of conditions associated with secondary osteoporosis. Diseases such as Marfan syndrome, osteogenesis imperfecta, Ehlers-Danlos syndrome, glycogen storage diseases, and homocystinuria affect the structure or production of collagen, a key component of bone tissue. Alterations in collagen synthesis or structure can weaken the bone matrix, making bones more susceptible to fracture and decreasing bone density.

[0040] Certain medications are associated with secondary osteoporosis due to their effects on bone metabolism. For example, long-term use of glucocorticoids (such as prednisone) can disrupt the balance between bone formation and resorption. Glucocorticoids suppress the activity of osteoblasts, leading to decreased bone formation, while promoting the differentiation and function of osteoclasts, resulting in increased bone resorption. Other medications, such as cyclosporine, methotrexate, and phenobarbital, can also lead to bone loss and increased risk of fractures.

[0041] Nutritional deficiencies and lifestyle factors can also contribute to the development of secondary osteoporosis. For example, alcoholism can have detrimental effects on bone health by interfering with the absorption and metabolism of calcium and other essential nutrients. Chronic liver disease can impair the activation of vitamin D, which is necessary for calcium absorption. Malabsorption syndromes, in which the body cannot properly absorb nutrients, can lead to insufficient levels of calcium and vitamin D, affecting bone health.

[0042] Bone tissue is constantly remodeled through dynamic processes that primarily involve two types of cells: osteoblasts and osteoclasts. Osteoblasts are responsible for bone formation, while osteoclasts are involved in bone resorption. These cells collectively maintain a delicate balance between bone formation and bone resorption, known as bone remodeling. In osteoporosis, this balance is disrupted, leading to increased bone resorption and decreased bone formation. Osteoclasts become overactive and break down bone at a faster rate than osteoblasts can rebuild it. This results in a net loss of bone mass and weakening of the skeletal structure.

[0043] Osteoblasts play a crucial role in bone formation. These cells are responsible for synthesizing and depositing new bone matrix, which is primarily composed of collagen and other proteins. They also promote the mineralization process by facilitating the accumulation of calcium and phosphate ions in the bone tissue. When osteoblast activity is impaired or insufficient, as in osteoporosis, impaired bone formation leads to decreased bone density and increased susceptibility to fractures. Osteoblast function requires activation of bone morphogenetic proteins (BMPs), TGFβ, and canonical WNT signaling. Key regulators of WNT signaling include: sclerostin, Dickkopf 1, and secreted Frizzled-related protein 1. Estrogen promotes osteogenic differentiation of mesenchymal stem cells. Estrogen also stimulates IGF1 and TGFβ production by osteoblasts and procollagen synthesis. Sclerostin is a protein secreted primarily by osteocytes that becomes embedded within mineralized bone tissue. Its primary function is to inhibit osteoblast activity and bone formation. Sclerostin acts by binding to specific receptors on osteoblasts, thereby interfering with intracellular signaling pathways that promote osteoblast differentiation and function. Sclerostin indirectly affects osteoclast activity and bone resorption by inhibiting osteoblast function.

[0044] On the other hand, osteoclasts are multinucleated cells derived from monocytes and are responsible for bone resorption. They secrete enzymes and acids to break down the mineralized bone matrix, allowing the release of calcium and other minerals into the bloodstream. In osteoporosis, osteoclasts are overactive and excessively resorb bone, further contributing to bone mass and density loss. Osteoclast differentiation and bone resorption are controlled by the IL-1, TNF-α, TGFβ, M-CSF, and RANK-RANKL-osteoprotegerin pathways, with RANKL (receptor activator of nuclear factor kappa B ligand) playing a key role. RANKL binds to RANK on osteoclast precursor cells and activates the MAPK and NF-κB pathways through TRAF6 and TAK1. It promotes the differentiation of osteoclast precursor cells into mature osteoclasts. Upon activation, mature osteoclasts are responsible for bone resorption by breaking down the mineralized bone matrix. RANKL stimulates osteoclast formation, activation, and survival, leading to increased bone resorption.

[0045] Osteoporosis treatments include the use of drugs called RANKL inhibitors. These drugs essentially block the interaction between RANKL and its receptor on osteoclasts, preventing the stimulation of osteoclast formation and activation. By reducing osteoclast-mediated bone resorption, RANKL inhibitors help maintain bone mass and reduce the risk of fractures in osteoporosis patients. Another approach to osteoporosis treatment involves the use of drugs that neutralize or inhibit sclerostin. These drugs allow osteoblasts to function more effectively by blocking the inhibitory effects of sclerostin, promoting bone formation and bone mineral density. Increasing osteoblast activity can help counteract the excessive bone resorption found in osteoporosis and improve overall bone health.

[0046] Provided in the present disclosure are bispecific antibodies ("DRAbs") that specifically bind to human RANKL and human sclerostin. By targeting both RANKL and sclerostin, a dual-acting approach is achieved that addresses both the excess bone resorption and impaired bone formation found in osteoporosis. This dual-inhibition approach provides the advantage of a more comprehensive intervention, while addressing multiple aspects of bone remodeling. It not only slows bone loss, but also promotes bone formation, thereby restoring bone strength and reducing the risk of fracture. As shown below, the DRAbs described herein show synergistic effects in enhancing bone formation, which provide greater efficacy in treating osteoporosis compared to targeting RANKL or sclerostin alone.

[0047] Before further description of the disclosure, it should be understood that the disclosure is not limited to the particular embodiments described herein and that the terminology used herein should not be taken to limit the scope of the disclosure unless otherwise stated.

[0048] 1. Definitions

[0049] Unless defined otherwise herein, scientific and technical terms used in this disclosure have the meanings that are commonly understood by one of ordinary skill in the art. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the nomenclature used herein and the techniques described herein are those known and used by those of ordinary skill in the art in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization.

[0050] The term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" is understood to mean one or more antibodies.

[0051] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components and any combination thereof. For example, the term "and / or" when used in the phrases "A and / or B" is to be taken as specific disclosure of each of the following alternatives: A; B; A and B. Likewise, the term "and / or" when used in the phrases "A, B and / or C" is to be taken as specific disclosure of each of the following alternatives: A; B; C; A and B; A and C; B and C; A, B and C. Likewise, the term "and / or" when used in the phrases "at least one of A and / or B" is to be taken as specific disclosure of each of the following alternatives: at least one A; at least one B; and at least one A and at least one B.

[0052] The term "about" as used herein is used to indicate that a value includes the inherent variation of error for the device, method, or apparatus being employed to determine the value, or the variation that exists among the study subjects. The term "about" includes the exact listed value. In some embodiments, "about" means plus or minus 10% of the given value or range. In certain embodiments, "about" means a variation of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value that "about" refers to. In some embodiments, "about" means a variation of ±1%, ±0.5%, ±0.2%, or ±0.1% of the value that "about" refers to.

[0053] The terms "peptide chain," "peptide," "polypeptide," "protein," and their grammatical equivalents, used interchangeably herein, refer to a polymer of amino acids of any length, which can be linear or branched. It can contain non-natural or modified amino acids, or can be interrupted by non-amino acids. Polypeptides, peptides, peptide chains, or proteins can also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

[0054] The terms "polynucleotide," "nucleic acid," and their grammatical equivalents, used interchangeably herein, refer to a polymer of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0055] The term "variant" as used herein with respect to a protein or polypeptide having a particular sequence characteristic ("reference protein" or "reference polypeptide") refers to a different protein or polypeptide having one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions as compared to the reference protein or reference polypeptide. The change in the amino acid sequence can be an amino acid substitution. The change in the amino acid sequence can be a conservative amino acid substitution. A functional fragment or functional variant of a protein or polypeptide retains the essential structural and functional characteristics of the reference protein or reference polypeptide.

[0056] As used herein, the term "specific binding" means that the interaction of a polypeptide or molecule with an epitope, protein, or target molecule is more frequent, faster, longer-lasting, more affinity, or a combination thereof, compared to interactions with surrogate substances (including related and unrelated proteins). The binding moiety (e.g., antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified by, for example, immunoassay, ELISA, biolayer interference (“BLI”), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, the specific response is at least twice the background signal or noise, and can be greater than 10 times the background. See, for example, Paul, ed., 1989. Fundamental Immunology Second Edition Raven Press, New York, at pages 332-336, discusses antibody specificity. In some implementations, "specific binding" means, for example, binding at approximately 0.1 mM or lower K+. D Binding to molecular targets. In some embodiments, "specific binding" means that the peptide or molecule binds at a concentration of about 10 µM or less, or about 1 µM or less. D Binding to the target. In some implementations, "specific binding" means that the peptide or molecule binds at a concentration of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. D Target binding. Due to sequence identity between homologous proteins in different species, specific binding can include peptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology in certain regions of the polypeptide sequences of different proteins, specific binding can include peptides or molecules that recognize more than one protein or target.

[0057] The term "binding affinity" as used in this article generally refers to the total strength of the non-covalent interactions between the binding moiety and the target molecule (e.g., antigen). Binding to the target molecule is a reversible process, and the affinity of binding is typically reported as the equilibrium dissociation constant (K0). D K D It is the dissociation rate (k off or k d ) and binding rate (k on or k a The ratio of ) to K. D The lower the value, the higher the affinity. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. Specific illustrative embodiments include the following. In some embodiments, “K…” D "or "K D The value can be measured experimentally as known in the art, for example, by combining experimental measurements. K DIt can be measured in the radiolabeled antigen binding assay (RIA) (Chen, et al. , (1999) J. Mol Biol 293:865-881). K D or K D The value can also be measured using biological layer interferometry (BLI), such as the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). D or K D The value can also be measured using Biacore surface plasmon resonance (SPR) analysis, such as using the BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). Binding affinity can also be measured using EC. 50 Quantification, EC 50 It is the concentration of ligands when half of the target is in the bound state during the binding assay.

[0058] The terms "identical," percent "identity," and grammatical equivalents thereof, as used herein in the context of two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that when optimally aligned and compared have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, and in some embodiments at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity over the entire aligned length of the two or more sequences or subsequences. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are publicly available for obtaining an alignment of amino acid or nucleotide sequences. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical when optimally aligned and compared, using a sequence comparison algorithm or by visual inspection, that they have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90%, and in some embodiments, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity of nucleotides or amino acid residues over the entire aligned length of the two or more sequences or subsequences. In some embodiments, identity exists over a region that is at least about 10 residues, at least about 20 residues, at least about 40 to 60 residues, at least about 60 to 80 residues, or any integral value therein, in length of an amino acid sequence. In some embodiments, identity exists over a longer region of more than 60 to 80 residues, e.g., at least about 80 to 100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences to be compared, e.g., the coding regions of a target protein or antibody. In some embodiments, identity exists over a region that is at least about 10 residues, at least about 20 residues, at least about 40 to 60 residues, at least about 60 to 80 residues, or any integral value therein, in length of an amino acid sequence. In some embodiments, identity exists over a longer region of more than 60 to 80 residues, e.g., at least about 80 to 100 residues or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences to be compared, e.g., a nucleotide sequence encoding a protein of interest.

[0059] The term "vector" and its grammatical equivalents as used herein refer to an agent for carrying genetic material (e.g., a polynucleotide sequence), which can be introduced into a host cell, where it can be replicated and / or expressed. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into the host cell chromosome. In addition, vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive promoters and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more polynucleotides are to be co-expressed, the two polynucleotides can be inserted, for example, into a single expression vector or separate expression vectors. For single vector expression, the coding polynucleotides can be operably linked to a common expression control sequence, or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art can be used to confirm the introduction of polynucleotides into host cells. It will be understood by those skilled in the art that the polynucleotides are expressed in sufficient amounts to produce the desired product, and it will be further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0060] The term "encode" and its grammatical equivalents as used herein refer to the inherent property of a specific sequence of nucleotides in a polynucleotide or nucleic acid (e.g., a gene, cDNA, or mRNA) to serve as a template for synthesis of other polymers and macromolecules having defined sequences of nucleotides (i.e., rRNA, tRNA, and mRNA) or of amino acids and their production of biological properties. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein, the gene is said to encode the protein. Unless otherwise specified, "a nucleotide sequence that encodes an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

[0061] An "isolated" polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition that exists in a form that does not occur in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to some degree and no longer exist in the form in which they are found in nature. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

[0062] RANGES: Throughout this disclosure, various aspects of the application can be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and that one

[0063] Exemplary genes and polypeptides are described herein with reference to GenBank Accession Numbers, GI Numbers, and / or SEQ ID NOs. It will be appreciated by those skilled in the art that homologous sequences can be readily identified by reference to the sequence source, including but not limited to GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ).

[0064] 2. Bispecific antibodies targeting human RANKL and human sclerostin

[0065] Provided herein are bispecific antibodies that specifically bind to human RANKL and human sclerostin. In some embodiments, the bispecific antibodies provided herein are monoclonal antibodies. In some embodiments, the bispecific antibodies provided herein are isolated. In some embodiments, the bispecific antibodies provided herein are substantially pure.

[0066] 2.1 General provisions

[0067] As used herein and as understood in the art, an “antibody” is an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein) through at least one antigen binding fragment, which is typically within the variable region of the immunoglobulin molecule. An “antibody” can be of various different types and structures. For example, an antibody can be a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, a monospecific antibody, a monovalent antibody, or any other modified immunoglobulin molecule comprising an antigen binding site. Antibodies also include, but are not limited to, mouse antibodies, camelid antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (isotypes) (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), based on the identity of their heavy-chain constant domains, designated a, d, e, g, and m, respectively. Unless explicitly indicated otherwise, the term “antibody” as used herein includes “antigen binding fragments” of intact antibodies. The term “antigen binding fragment” as used herein refers to a portion or fragment of an intact antibody, i.e., the antigenic determining variable region of an intact antibody. Examples of antigen binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy chain antibodies (HCAb), light chain antibodies (LCAb), disulfide-linked scFv (dsscFv), diabodies, triabodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (sdAb; e.g., camelid antibodies, llama antibodies), and single variable domain antibodies of heavy chains (VHH).

[0068] As used herein and as understood in the art, a “bispecific” antibody is an artificial hybrid antibody having two different antigen binding fragments. The two different antigen binding fragments specifically bind to two different target antigens. Bispecific antibodies can be formed from antibody fragments.

[0069] The structure of immunoglobulins is well characterized (see, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Typically, an immunoglobulin comprises two pairs of polypeptide chains, one pair of light (L; low molecular weight) chains and one pair of heavy (H; high molecular weight) chains, all four interconnected by disulfide bonds.

[0070] Each light chain of an immunoglobulin typically comprises a light chain variable region (“VL region”) and a light chain constant region (“CL region”). There are two different types of light chains, designated kappa (K) and lambda (l), based on the amino acid sequence of the CL region. The amino acid sequences of the CL region are well known in the art.

[0071] Each heavy chain typically includes a heavy chain variable region ("VH region") and a heavy chain constant region ("CH region"). Based on amino acid sequence, the VH region can be one of five different types, called alpha (a), delta (5), epsilon (e), gamma (g), and mu (m). These different types of heavy chains give rise to the five well-known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, when combined with a light chain. IgG has four subclasses, IgGl, IgG2, IgG3, IgG4. The amino acid sequences of the CH regions of the different classes of antibodies are well known in the art.

[0072] The CH region of an immunoglobulin comprises more than one domain. For example, the CH region of an IgG antibody comprises three domains: heavy chain constant domain 1 (CH1), heavy chain constant domain 2 (CH2), and heavy chain constant domain 3 (CH3). The highly flexible region between the CH1 and CH2 domains is referred to as the "hinge region." Disulfide bonds of the hinge region are part of the interaction between the two heavy chains in an immunoglobulin. The "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. In the IgG, IgA, and IgD subclasses, the Fc region comprises the CH2 and CH3 domains; the IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4). The amino acid sequences of the Fc regions of human IgG, IgA, IgD, IgM, IgE, and the IgGl, IgG2, IgG3, IgG4 subclasses are known to those of ordinary skill in the art. As used herein, the Fc region of an IgG heavy chain extends from the hinge region to the carboxy-terminus of the heavy chain. The native Fc region can be modified. Modifications of the Fc region are further described below. In some embodiments, the bispecific antibodies provided herein can include a pair of Fc domains comprising a pair of different modifications that promote their binding to each other rather than forming a homodimer.

[0073] Unless otherwise specified or contradicted by context, reference to an amino acid position in a constant region is made according to the EU numbering (Edelman et al. 1969; 63:78-85, Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). A list of exemplary amino acid sequences of the constant domains / regions of human IgG antibodies is provided below. Some exemplary variants are also included, with more variants disclosed in the sections below. et al PNAS . 1969; 63:78-85, Kabat et al ., Sequences of Proteins ofImmunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). A list of exemplary amino acid sequences of the constant domains / regions of human IgG antibodies is provided below. Some exemplary variants are also included, with more variants disclosed in the sections below.

[0074] ​Table 1. Constant regions / domains of natural human IgG.

[0075]

[0076] The term "variable region" refers to a portion of the light or heavy chain of an immunoglobulin, typically located at the amino terminus of the light or heavy chain, and is used for the binding and specificity of each particular antibody to its specific antigen. The variable region of the light chain is called the "light chain variable region" or "VL region," and it includes at least one, typically a "light chain variable domain" or "VL." The variable region of the heavy chain is called the "heavy chain variable region" or "VH region," and it includes at least one, typically a "heavy chain variable domain" or "VH." The sequence differences of the variable domains between different antibodies are significant. A "pair of VL and VH" can bind to each other and form a binding site that specifically binds to the target antigen or epitope.

[0077] VH and VL regions can be further subdivided into highly variable regions (or hypervariable regions, which can be highly variable in sequence and / or structurally defined loop forms), also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called frame regions (FRs). Sequence variability is concentrated in the CDRs, while smaller variable portions within the variable domains are called frame regions (FRs). The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see Chothia and Lesk). J Mol Biol 1987;196:901-17).

[0078] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VL β-sheet framework of an antibody. CDR regions are well known to those skilled in the art and have been defined using various methods / systems. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the most hypervariable region in the antibody variable (V) domain (Kabat...). et al , J. Biol. Chem 252:6609-6616 (1977); Kabat, Adv. Prot. Chem 32: 1-75 (1978)). Software programs (e.g., abYsis) are available and known to those skilled in the art for analyzing antibody sequences and determining CDRs.

[0079] Single-chain Fv (“scFv”) polypeptides are covalently linked VL / VH heterodimers, typically expressed from a gene fusion including VLand VH-encoding genes linked by a peptide-encoding linker. scFv fragments include CDRs held in the appropriate conformation by the use of recombinant DNA techniques. In some embodiments of scFv, the N-terminus of the VL is connected by a linker to the C-terminus of the VH. In some embodiments of scFv, the N-terminus of the VH is connected by a linker to the C-terminus of the VL.

[0080] The term “linker” as used herein refers to one or more amino acid residues inserted between domains (e.g., immunoglobulin domains) to provide sufficient mobility to the domains. The linker can be inserted at the transition between variable domains at the sequence level, or between variable and constant domains, respectively. One of ordinary skill in the art will appreciate that the bispecific antibodies and fusion proteins disclosed herein are not limited by the specified linkers exemplified herein. Any peptide linker having appropriate length and flexibility that allows the correct formation of an antigen binding site by the VL / VH pair can be used.

[0081] Table 2. Exemplary linkers

[0082] The term “humanized antibody” as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulins, chimeric immunoglobulins, or fragments thereof comprising minimal non-human sequences. Typically, a humanized antibody is a human immunoglobulin. In some cases, variable region residues of a human immunoglobulin are replaced by corresponding residues from an antibody of a non-human species. In some cases, residues of CDRs are replaced by residues from CDRs of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and / or binding capacity. A humanized antibody can be further modified by the substitution of additional residues either within the variable region and / or within the replaced non-human residues to improve and optimize antibody specificity, affinity, and / or binding capacity. The term “human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to that produced by a human using techniques known in the art.

[0083] The bispecific antibodies provided herein include a first pair of VL and VH that specifically binds to human RANKL and a second pair of VL and VH that specifically binds to human sclerostin. Exemplary pairs of VL / VH that specifically bind to human RANKL / sclerostin are provided below. In addition to the specified pairs of VL / VH exemplified herein, variants of these pairs of VL / VH that maintain their binding to the respective target antigen are expressly contemplated.

[0084] Table 3A: VL / VH and CDRs of exemplary anti-RANKL antibodies

[0085] Table 3B: VL / VH and CDRs of exemplary anti- sclerostin antibodies

[0086] In some embodiments, the bispecific antibodies provided herein include (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1), wherein the VL1 / VH1 pair specifically binds to human RANKL, and wherein the VL1 comprises a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and wherein the VH1 comprises a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and (ii) a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), wherein the VL2 / VH2 pair specifically binds to human sclerostin; and wherein the VL2 comprises a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, and wherein the VH2 comprises a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. In some embodiments of the bispecific antibodies disclosed herein, VL1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the sequence of SEQ ID NO: 13. In some embodiments of the bispecific antibodies disclosed herein, VH1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the sequence of SEQ ID NO: 14. In some embodiments of the bispecific antibodies disclosed herein, VL2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the sequence of SEQ ID NO: 15. In some embodiments of the bispecific antibodies disclosed herein, VH2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the sequence of SEQ ID NO: 16, respectively.

[0087] In some embodiments of the bispecific antibodies disclosed herein, VL1 and VH1 that specifically bind to human RANKL have the amino acid sequences of SEQ ID NO: 13 (VL1) and SEQ ID NO: 14 (VH1), respectively. In some embodiments of the bispecific antibodies disclosed herein, VL2 and VH2 that specifically bind to human sclerostin have the amino acid sequences of SEQ ID NO: 15 (VL2) and SEQ ID NO: 16 (VH2), respectively.

[0088] 2.2 Heterodimeric Fc (e.g., KIH)

[0089] In some embodiments, the bispecific antibodies provided herein comprise mutations that promote heterodimerization of the Fc region. In some embodiments, the dimerized Fc region of the bispecific antibodies provided herein is formed from Fc regions comprising amino acid mutations, substitutions, additions, or deletions to promote heterodimerization, wherein different polypeptides comprising different Fc regions can dimerize to produce a heterodimeric configuration. In some embodiments, the bispecific antibodies of the present disclosure comprise a first Fc sequence comprising a first CH3 region and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and such that the heterodimeric interaction between the first and second CH3 regions is stronger than each homodimeric interaction of the first and second CH3 regions.

[0090] Methods to promote heterodimerization of Fc regions include amino acid deletions, additions, or substitutions of the Fc region amino acid sequence, such as by comprising a set of “knobs-into-holes” deletions, additions, or substitutions, or comprising amino acid deletions, additions, or substitutions that effect Fc electrostatic steering, to favor attractive interactions between different polypeptide chains. Methods to promote heterodimerization of complementary Fc polypeptides have been previously described, for example, Ridgway et al ., 1996, Protein Eng , 9:617-621; Merchant et al ., 1998, Nature Biotechnol , 16:677-681; Moore et al .,2011, MAbs , 3:546-557; Von Kreudenstein et al ., 2013, 5:646-654; Gunasekaran et al ., 2010, J Biol Chem , 285: 19637-19464; Leaver-Fay et al ., 2016,Structure , 24:641-651; Ha et al ., 2016, Frontiers in Immunology , 7: 1; Davis et. , 2010, P rotein Eng Des Sei , 23: 195-202; WO1996 / 027011; WO1998 / 050431; WO2006 / 028936; WO2009 / 089004; WO2011 / 143545; WO2014 / 067011; WO2012 / 058768; WO2018 / 027025; US2014 / 0363426; US2015 / 0307628; US2018 / 0016354; US2015 / 0239991; US2017 / 0058054; USPN5731168; USPN7183076; USPN9701759; USPN9605084; USPN9650446; USPN8216805; USPN8765412; and USPN8258268.

[0091] Modifications that promote the binding of a pair of Fc domains in a bispecific antibody include modifications called “knobs-into-holes” modifications, which consist of a “knob” modification in one Fc domain and a “hole” modification in the other Fc domain. In some embodiments, the bispecific antibodies provided herein have complementary Fc peptides to form heterodimers in a “knobs-into-holes” configuration or a “KIH” configuration. The “knobs-into-holes” technology is described in references such as US 5,731,168; US 7,695,936, US 8,216,805, US 8,765,412; Ridgway et al ., Prot. Eng. 9,617-621 (1996) and Carter, J Immunol. Meth 248, 7-15 (2001). Typically, this method involves introducing a protrusion (“knob”) at the interface of a first Fc (“Knob-Fc”) and a corresponding pore (“Hole”) at the interface of a second Fc (“Hole-Fc”), such that the protrusion can be located within the pore, thereby promoting the formation of heterodimers and inhibiting the formation of homodimers. The protrusion is constructed by replacing the small amino acid side chain from the interface of the first peptide with a larger side chain (e.g., tyrosine or tryptophan). Compensating pores of the same or similar size as the protrusion are created at the interface of the second peptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0092] Accordingly, "Knob-Fc regions" and "Hole-Fc regions" are designed to form heterodimeric pairs. A Knob-Fc region refers to an Fc region in which the amino acids of the CH3 domain are replaced with amino acid residues having a larger side chain volume, creating a knob within the CH3 domain, which can be positioned in a hole of the CH3 domain of a Hole-Fc region, in which the amino acid residues of the CH3 domain are replaced with amino acid residues having a smaller side chain volume, creating a hole within the CH3 domain, in which the knob within the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The knob and hole can be formed by altering the nucleic acid encoding the polypeptide (e.g. by site-specific mutagenesis), or by peptide synthesis.

[0093] In some embodiments, the threonine residue at position 366 of the Knob-Fc region is replaced with a tryptophan residue (T366W), and the tyrosine residue at position 407 of the Hole-Fc region is replaced with a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A). The introduction of two cysteine residues results in the formation of a disulfide bond between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Thus, in these configurations, the first Fc polypeptide comprises amino acid modifications that form a “knob,” and the second Fc polypeptide comprises amino acid modifications that form a “hole,” thereby forming an Fc heterodimer comprising complementary Fc polypeptides. In some embodiments, the Knob-Fc region additionally has a serine residue at position 354 replaced with a cysteine residue (S354C), or a glutamic acid residue at position 356 replaced with a cysteine residue (E356C), and the Hole-Fc region additionally has a tyrosine residue at position 349 replaced with a cysteine residue (Y349C). In some embodiments, the Hole-Fc region additionally has a serine residue at position 354 replaced with a cysteine residue (S354C), or a glutamic acid residue at position 356 replaced with a cysteine residue (E356C), and the Knob-Fc region additionally has a tyrosine residue at position 349 replaced with a cysteine residue (Y349C). In some embodiments, the Knob-Fc region comprises the amino acid substitutions S354C and T366W, and the Hole-Fc region comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V. In some embodiments, the Knob-Fc region comprises the amino acid substitutions Y349C and T366W, and the Hole-Fc region comprises the amino acid substitutions S354C, T366S, L368A, and Y407V. All amino acid residues are numbered according to the EU Index.

[0094] In some embodiments, the bispecific antibodies provided herein have a “knobs-into-holes” or “KIH” structure (e.g., Figure 1A ). The “KIH” model facilitates the formation of bispecific antibody heterodimers, rather than heavy chain homodimers.

[0095] In some embodiments, the complementary Fc polypeptide of the Fc heterodimer comprises mutations to alter the charge polarity at the Fc dimer interface, such that co-expression of electrostatically matched Fc regions supports favorable attractive interactions, thereby facilitating the formation of the desired Fc heterodimer; whereas unfavorable repulsive charge interactions inhibit the formation of the unwanted Fc homodimers (Guneskaranet al. , 2010, J Biol Chem, 285: 19637-19646). When co-expressed in cells, the polypeptide chains can associate between one another, but due to charge repulsion, the chains do not substantially self-associate.

[0096] Exemplary pairwise amino acid modifications of complementary Fc polypeptides in Fc heterodimeric format are listed in the table below (EU numbering).(Brinkmann and Kontermann, MAbs . 2017 Feb-Mar; 9(2): 182-212.).

[0097] Table 4: Exemplary pairwise Fc modifications of heterodimeric Fc domains

[0098] The bispecific antibodies disclosed herein can include complementary Fc regions having the modifications described in Table 4 above. In some embodiments, the bispecific antibodies disclosed herein include a first CH region and a second CH region comprising a pair of Fc modifications described in Table 4 above. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a T366Y substitution and a Y407T, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a T366W substitution and a T366S / L368W / Y407V substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a T366W substitution and a T366S / L368A / Y407V substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a T366W / S354C substitution and a T366S / L368A / Y407V / Y349C substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a T366W / E356C substitution and a T366S / L368A / Y407V / Y349C substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a T350V / L351Y / F405A / Y407V substitution and a T350V / T366L / K392L / T394W substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a K360D / D399M / Y407A substitution and a E345R / Q347R / T366V / K409V substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a K409D / K392D substitution and a D399K / E356K substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a K360E / K409W substitution and a Q347R / D399V / F405T substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a L360E / K409W / Y349C substitution and a Q347R / D399V / F405T / S354C substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a S364H / F405A substitution and a Y349T / T394F substitution, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have a D221E / P228E / L368E substitution and a D221R / P228R / K409R substitution, respectively, or vice versa.In some embodiments, the first CH region and the second CH region of the bispecific antibody disclosed herein have F405L substitution and K409R substitution, respectively, and vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibody disclosed herein have Y349S / K307Y / T366M / K409V substitution and E356G / E357D / S364Q / Y407A substitution, respectively, and vice versa. In some embodiments, to further reduce homodimer formation, one of the CH regions also has H435R and Y436F substitution. In some embodiments, the second CH region also has H435R and Y436F substitution.

[0099] In some embodiments, the bispecific antibody provided herein comprises: (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1), wherein the VL1 / VH1 pair specifically binds to human RANKL, and (ii) a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), wherein the VL2 / VH2 pair specifically binds to human sclerosingin. Figure 1A As shown, to avoid mismatch, the KIH design includes an scFv on the second heavy chain. Therefore, in some embodiments, the bispecific antibody provided herein may have three peptide chains: (1) a first peptide chain (C1) comprising VL1 and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) a second peptide chain (C2) comprising VH1, a heavy chain constant region 1 (CH1), and a Knob-Fc region from the N-terminus to the C-terminus; and (3) a third peptide chain (C3) comprising a linker, a single-chain variable fragment (scFv) containing VL2 and VH2, a linker, and a Hole-Fc region from the N-terminus to the C-terminus.

[0100] One of ordinary skill in the art will appreciate that in the bispecific antibodies having the KIH configuration described above, the Knob-Fc and Hole-Fc can switch places. In other words, in the C2 and C3 pair of bispecific antibodies employing the KIH design, C2 can include the Knob-Fc region and C3 can include the Hole-Fc region; or, in some embodiments, C2 can include the Hole-Fc region and C3 can include the Knob-Fc region. Accordingly, in some embodiments, the bispecific antibodies provided herein can have three peptide chains: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain 1 (CH1), and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL2 and VH2, a linker, and a Knob-Fc region.

[0101] In the scFv, the VL can be located at the N-terminus of the VH, or vice versa. In some embodiments of the bispecific antibodies disclosed herein, wherein C3 comprises a scFv having VL2 and VH2, the scFv can comprise, from N-terminus to C-terminus, VL2, a second linker, and VH2. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VH2, a second linker, and VL2.

[0102] In some embodiments, the bispecific antibodies provided herein comprise a VL1 / VH1 pair that specifically binds to human RANKL and a VL2 / VH2 pair that specifically binds to human sclerostin. In some embodiments, the bispecific antibodies employ a KIH configuration with a scFv on the second heavy chain. In some embodiments, the scFv comprises a VL2 / VH2 pair that specifically binds to human sclerostin. In some embodiments, the scFv comprises a VL1 / VH1 pair that specifically binds to human RANKL. Thus, in some embodiments, the bispecific antibodies can have three peptide chains: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1), and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL1 and VH1, a linker, and a Hole-Fc region. In some embodiments, the bispecific antibodies provided herein can have three peptide chains: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1), and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL1 and VH1, a linker, and a Knob-Fc region.

[0103] In some embodiments of the bispecific antibodies disclosed herein, wherein C3 comprises a scFv with VL1 and VH1, the scFv can comprise, from N-terminus to C-terminus, VL1, a second linker, and VH1. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VH1, a second linker, and VL1.

[0104] The bispecific antibodies provided herein in KIH configuration include a CL region, a CH1 domain, and two Fc regions (Knob-Fc and Hole-Fc, each comprising a hinge, a CH2 domain, and a CH3 domain). The amino acid sequences of the CH1, CL region, and Fc regions of the bispecific antibodies disclosed herein can be from any suitable source, for example, the constant regions of an antibody, such as IgGl, IgG2, IgG3, or IgG4. Antibody heavy and light chain constant region amino acid sequences are well known in the art, for example, those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0105] In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgGl. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG2. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG3. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG4. In some embodiments, the amino acid sequences of the CHI, CL regions, and Fc regions (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein can comprise one or more amino acid substitutions from the wild-type immunoglobulin. Such substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US8323962, US6737056, and US7416727).

[0106] In some embodiments, the CHI domain of the bispecific antibodies provided herein can be selected from the group consisting of a human IgGl CHI domain (SEQ ID NO: 35), a human IgG2 CHI domain (SEQ ID NO: 36), a human IgG3 CHI domain (SEQ ID NO: 37), and a human IgG4 CHI domain (SEQ ID NO: 38). The CHI domain can be a human IgGl CHI domain (SEQ ID NO: 35) or a variant thereof having up to 10 amino acid substitutions. The CHI domain can be a human IgG2 CHI domain (SEQ ID NO: 36) or a variant thereof having up to 10 amino acid substitutions. The CHI domain can be a human IgG3 CHI domain (SEQ ID NO: 37) or a variant thereof having up to 10 amino acid substitutions. The CHI domain can be a human IgG4 CHI domain (SEQ ID NO: 38) or a variant thereof having up to 10 amino acid substitutions.

[0107] In some embodiments, the CL region of the bispecific antibodies provided herein can be a kappa CL (CK; SEQ ID NO: 19). In some embodiments, the CL region of the bispecific antibodies provided herein can be a lambda CL (CL; SEQ ID NO: 20).

[0108] In some embodiments, the Fc region of the bispecific antibodies provided herein can be a variant of the Fc region of human IgG2. In some embodiments, the Knob-Fc region is a human IgG2 Fc region variant having up to 10 amino acid substitutions, including a T366W substitution; and the Hole-Fc region is a human IgG2 Fc region variant having up to 10 amino acid substitutions, including a Y407V substitution. In some embodiments, the Hole-Fc region can also have T366S and L368A substitutions. In some embodiments, the Knob-Fc and Hole-Fc regions can also comprise S354C and Y349C substitutions, respectively. In some embodiments, the Knob-Fc and Hole-Fc regions can also include Y349C and S354C substitutions, respectively. All amino acid residues are numbered according to the EU Index.

[0109] In some embodiments, the Knob-Fc region and the Hole-Fc region can (1) have the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 32, respectively; (2) have the amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 33, respectively; or (3) have the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 34, respectively. The Knob-Fc region and the Hole-Fc region can have the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 32, respectively. The Knob-Fc region and the Hole-Fc region can have the amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 33, respectively. The Knob-Fc region and the Hole-Fc region can have the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 34, respectively.

[0110] Table 5: Exemplary Fc sequences in KIH model.

[0111]

[0112] The bispecific antibodies in KIH format provided herein also comprise a linker connecting the scFv and the Knob-Fc, specifically, connecting the C-terminus of the scFv and the N-terminus of the Knob-Fc. The linker can be any suitable linker disclosed in the art or otherwise known. For example, the linker can be selected from those identified in Table 2. In some embodiments, the linker is a GS linker, which has the amino acid sequence of SEQ ID NO: 41. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 42. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 43. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 44. In some embodiments, the linker has the amino acid sequence of SEQ ID NO: 45.

[0113] In some embodiments of the scFv, the light chain variable domain (VL) and the heavy chain variable domain (VH) are connected by a second linker. The second linker can be any suitable linker disclosed in the art or otherwise known. For example, the second linker can be selected from those identified in Table 2. In some embodiments, the linker is a GS linker, which has the amino acid sequence of SEQ ID NO: 41. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 42. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 43. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 44. In some embodiments, the linker has the amino acid sequence of SEQ ID NO: 45.

[0114] In some embodiments of the bispecific antibodies provided herein, (i) the CL region is a kappa CL (CK; SEQ ID NO: 19) or a lambda CL (CL; SEQ ID NO: 20) or a variant thereof having up to 10 amino acid substitutions, (ii) the CHI domain is a human IgG2 CHI domain (SEQ ID NO: 36) or a variant thereof having up to 10 amino acid substitutions, and / or (iii) the Knob-Fc region and the Hole-Fc region have (1) the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 32, respectively, (2) the amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 33, respectively, or (3) the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 34, respectively, or a variant thereof having up to 10 amino acid substitutions. In some embodiments, the CL region, CHI domain, Knob-Fc region, and Hole-Fc region have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 29, and SEQ ID NO: 32, respectively. In some embodiments, the CL region, CHI domain, Knob-Fc region, and Hole-Fc region have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 30, and SEQ ID NO: 33, respectively. In some embodiments, the CL region, CHI domain, Knob-Fc region, and Hole-Fc region have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 31, and SEQ ID NO: 34, respectively.

[0115] In some embodiments of the bispecific antibodies provided herein, the CL region is Ckappa (SEQ ID NO: 19), the CH1 domain is a human IgG2 CH1 domain (SEQ ID NO: 36), the Knob-Fc and Hole-Fc have the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 32, respectively. In some embodiments, the CL region is Ckappa (SEQ ID NO: 19), the CH1 domain is a human IgG2 CH1 domain (SEQ ID NO: 36), and the Knob-Fc region and Hole-Fc region have the amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 33, respectively. In some embodiments, the CL region is Ckappa (SEQ ID NO: 19), the CH1 domain is a human IgG2 CH1 domain (SEQ ID NO: 36), and the Knob-Fc region and Hole-Fc region have the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 34, respectively.

[0116] Table 6A provides a chart of the three peptide chains of an exemplary bispecific antibody in KIH format.

[0117] Table 6A: Peptide chains of exemplary bispecific antibody (KIH)

[0118] Note: VL1 / VH1 pair that specifically binds to RANKL (e.g., SEQ ID NO: 13 and SEQ ID NO: 14); VL2 / VH2 that specifically binds to sclerostin (e.g., SEQ ID NO: 15 and SEQ ID NO: 16); Ckappa is kappa CL.(L): linker

[0119] Table 6B: Exemplary bispecific antibody (KIH) sequences

[0120] In some embodiments, provided herein are bispecific antibodies that specifically bind to human RANKL and human sclerostin, wherein the bispecific antibodies have a first peptide chain (Cl), a second peptide chain (C2), and a third peptide chain (C3), wherein Cl, C2, and C3 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences of (1) SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 54, respectively; (2) SEQ ID NO: 51, SEQ ID NO: 55, and SEQ ID NO: 56, respectively; (3) SEQ ID NO: 51, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; (4) SEQ ID NO: 51, SEQ ID NO: 59, and SEQ ID NO: 60, respectively. In some embodiments, Cl, C2, and C3 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 54, respectively. In some embodiments, Cl, C2, and C3 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 55, and SEQ ID NO: 56, respectively. In some embodiments, Cl, C2, and C3 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 57, and SEQ ID NO: 58, respectively. In some embodiments, Cl, C2, and C3 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 59, and SEQ ID NO: 60, respectively.

[0121] 2.3 IgG-scFv

[0122] In some embodiments, the bispecific antibodies provided herein have an “IgG-scFv” structure, as Figure 1BIn some embodiments, the bispecific antibodies provided herein comprise: (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1), wherein the VL1 / VH1 pair specifically binds to human RANKL, and (ii) a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), wherein the VL2 / VH2 pair specifically binds to human sclerostin. In some embodiments, the bispecific antibodies of the IgG-scFv format comprise two peptide chains: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant region (CH), a linker, a single chain variable fragment (scFv) comprising VL2 and VH2. Alternatively, in some embodiments, the bispecific antibodies of the IgG-scFv format comprise two peptide chains: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant region (CH), a linker, a single chain variable fragment (scFv) comprising VL1 and VH1.

[0123] The bispecific antibodies provided herein of the IgG-scFv format comprise a CL region and a CH region. The amino acid sequences of the CL region and the CH region of the bispecific antibodies disclosed herein can be from any suitable source, for example, the constant region of an antibody such as IgGl, IgG2, IgG3, or IgG4. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgGl. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG2. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG3. In some embodiments, the constant region of the bispecific antibodies provided herein is derived from human IgG4. In some embodiments, the amino acid sequences of the CL region and the CH of the bispecific antibodies disclosed herein can comprise one or more amino acid substitutions from the wild-type immunoglobulin. Such substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US8323962, US6737056, and US7416727).

[0124] In some embodiments, the CL region of the bispecific antibodies provided herein can be a kappa CL (CK; SEQ ID NO: 19) or a variant thereof having up to 10 amino acid substitutions. In some embodiments, the CL region of the bispecific antibodies provided herein can be a lambda CL (CL; SEQ ID NO: 20) or a variant thereof having up to 10 amino acid substitutions.

[0125] In some embodiments, the CH region of the bispecific antibodies provided herein can be selected from a human IgGl CH region (SEQ ID NO: 21), a human IgG2 CH region (SEQ ID NO: 22), a human IgG3 CH region (SEQ ID NO: 23), and a human IgG4 CH region (SEQ ID NO: 24), or a variant thereof having up to 10 amino acid substitutions. In some embodiments, the CH region is a human IgG2 CH region (SEQ ID NO: 22) or a variant thereof having up to 10 amino acid substitutions.

[0126] In some embodiments of the bispecific antibodies provided herein, (1) the CL region is CK (SEQ ID NO: 19) or CL (SEQ ID NO: 20), or a variant thereof having up to 10 amino acid substitutions; or (2) the CH region is a human IgGl CH region (SEQ ID NO: 21), an IgG2 CH region (SEQ ID NO: 22), an IgG3 CH region (SEQ ID NO: 23), or an IgG4 CH region (SEQ ID NO: 24), or a variant thereof having up to 10 amino acid substitutions; or both (1) and (2).

[0127] In some embodiments of the bispecific antibodies provided herein, the CL region is CK (SEQ ID NO: 19), and the CH region is a human IgG2 CH region (SEQ ID NO: 22).

[0128] The bispecific antibodies of the IgG-scFv format provided herein also comprise a linker connecting the heavy chain constant region (CH) and the scFv, specifically, connecting the C-terminus of the CH and the N-terminus of the scFv. The linker can be any suitable linker disclosed in the art or otherwise known. For example, the linker can be selected from those identified in Table 2. In some embodiments, the linker is a GS linker, which has the amino acid sequence of SEQ ID NO: 41. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 42. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 43. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 44. In some embodiments, the linker has the amino acid sequence of SEQ ID NO: 45.

[0129] In some embodiments of the scFv containing the VL2 and VH2 pair, the VL2 and VH2 are connected by a second linker. In some embodiments of the scFv containing the VL1 and VH1 pair, the VL1 and VH1 are connected by a second linker. The second linker can be any suitable linker disclosed herein or otherwise known in the art. For example, the second linker can be selected from those identified in Table 2. In some embodiments, the linker is a GS linker, which has the amino acid sequence of SEQ ID NO: 41. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 42. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 43. In some embodiments, the GS linker has the amino acid sequence of SEQ ID NO: 44. In some embodiments, the linker has the amino acid sequence of SEQ ID NO: 45.

[0130] A chart of the two peptide chains of exemplary bispecific antibodies of the IgG-scFv format is provided in Table 7A.

[0131] Table 7A: Exemplary peptide chains of bispecific antibodies (IgG-scFv)

[0132] Note: VL1 / VH1 pair that specifically binds to RANKL (e.g., SEQ ID NO: 13 and SEQ ID NO: 14); VL2 / VH2 that specifically binds to sclerostin (e.g., SEQ ID NO: 15 and SEQ ID NO: 16); Ckappa refers to kappa CL.(L): linker

[0133] Table 7B: Sequences of exemplary bispecific antibodies (IgG-scFv)

[0134] In some embodiments, provided herein are bispecific antibodies that specifically bind to human RANKL and human sclerostin, wherein the bispecific antibodies have a first peptide chain (C1) and a second peptide chain (C2), wherein C1 and C2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity, respectively, to the amino acid sequences of: (1) SEQ ID NO: 51 and SEQ ID NO: 61; (2) SEQ ID NO: 51 and SEQ ID NO: 62; (3) SEQ ID NO: 52 and SEQ ID NO: 63; or SEQ ID NO: 52 and SEQ ID NO: 64. In some embodiments, C1 and C2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity, respectively, to the amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 61. In some embodiments, C1 and C2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity, respectively, to the amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 62. In some embodiments, C1 and C2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity, respectively, to the amino acid sequences of SEQ ID NO: 52 and SEQ ID NO: 63. In some embodiments, C1 and C2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity, respectively, to the amino acid sequences of SEQ ID NO: 52 and SEQ ID NO: 64.

[0135] 2.4 Variants

[0136] The present disclosure further contemplates other variants and equivalents of the bispecific antibodies described herein that have substantially the same function. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermal stability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will appreciate that amino acid changes can alter post-translational processing of the antibody, for example, changing the number or position of glycosylation sites or changing membrane anchor characteristics.

[0137] Also provided herein are antibodies comprising functional variants of the heavy chain, light chain, VL region, VH region, or one or more CDRs of an example antibody. Functional variants of a heavy chain, light chain, VL, VH, or CDR used in the context of an antibody still allow the antibody to retain at least a substantial portion (at least about 90%, 95%, or more) of the functional characteristics of the "reference" and / or "parent" antibody, including affinity and / or specificity / selectivity, Fc inertness, and PK parameters such as half-life, Tmax, Cmax. Such functional variants typically retain significant sequence identity to the parent antibody and / or have substantially similar heavy and light chain lengths. Exemplary variants include those that differ from the heavy and / or light chain, VH and / or VL, and / or CDR regions of the parent antibody sequence by conservative substitutions, e.g., 10 substitutions, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the variant, can be conservative amino acid residue replacements.

[0138] In some embodiments, variants of the bispecific antibodies disclosed herein can retain their binding ability to RANKL and sclerostin to a similar, identical, or higher degree than the parent bispecific antibodies. In some embodiments, the variants are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent antibody or antigen binding fragment. In certain embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parent bispecific antibodies of the disclosure with one or more conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include substitutions of one amino acid for another amino acid having certain physical and / or chemical properties.

[0139] In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parent antibody with one or more non-conservative amino acid substitutions. In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parent binding antibody with one or more non-conservative amino acid substitutions, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant. In certain embodiments, the one or more conservative amino acid substitutions and / or the one or more non-conservative amino acid substitutions can enhance the biological activity of the variant such that the biological activity of the functional variant is increased compared to the parent antibody.

[0140] In some embodiments, the variant can have 1, 2, 3, 4, or 5 amino acid substitutions in the CDRs of the binding moiety (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3).

[0141] In some embodiments, the bispecific antibodies provided herein include modifications in the Fc region thereof. In some embodiments, the modified antibody (e.g., modified Fc region) provides altered effector function, which in turn affects the biological characteristics of the antibody. For example, in some embodiments, deletion or inactivation of the constant region (by point mutation or other methods) reduces Fc receptor binding of the modified antibody when in circulation. In some embodiments, the constant region modification reduces the immunogenicity of the antibody. In some embodiments, the constant region modification increases the serum half-life of the antibody. In some embodiments, the constant region modification decreases the serum half-life of the antibody. In some embodiments, the constant region modification reduces or eliminates ADCC and / or complement dependent cytotoxicity (CDC) of the antibody. In some embodiments, replacing specified amino acids in a human IgG2 Fc region with the corresponding IgG1 or IgG4 residues reduces effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody does not have one or more effector functions (e.g., an “effectorless” antibody). In some embodiments, the antibody does not have ADCC activity and / or CDC activity. In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody does not have effector function. In some embodiments, the constant region modification increases or enhances ADCC and / or CDC of the antibody. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified to add / replace one or more amino acids to provide one or more cytotoxins, oligosaccharides, or carbohydrate attachment sites.

[0142] In some embodiments of the bispecific antibodies provided herein, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor. In some embodiments of the bispecific antibodies provided herein, the Fc domain comprises one or more amino acid substitutions that reduce effector function. The effector function can be complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), cytokine secretion, or any combination thereof. In some embodiments, the effector function is ADCC.

[0143] In some embodiments, the variants can include the addition of amino acid residues at the amino and / or carboxyl terminus of the antibody. The additional amino acid residues can range in length from one residue to one hundred or more residues. In some embodiments, the variants comprise an N-terminal methionyl residue. In some embodiments, the variants are designed to be detectable and can include a detectable tag and / or protein (e.g., a fluorescent tag or enzyme).

[0144] The variant antibodies described herein can be produced using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0145] In some embodiments, the bispecific antibodies disclosed herein can be chemically modified, either naturally or by intervention. In some embodiments, the bispecific antibodies are chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, and / or linkage to a cellular ligand or other protein. Any of a number of chemical modifications can be carried out by known techniques. The bispecific antibodies provided herein can include one or more analogs of an amino acid (including, e.g., unnatural amino acids) and other modifications known in the art.

[0146] The physical, chemical, and / or biological properties of the bispecific antibodies of the present disclosure can be analyzed by various methods known in the art. In some embodiments, the bispecific antibodies provided herein are tested for their ability to bind human RANKL and sclerostin. Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore), ELISA, and FACS. In addition, the solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency of the antibodies can be assessed.

[0147] In some embodiments, the bispecific antibodies disclosed herein can be conjugated to a detectable substance or molecule that allows for detection of the agent. Detectable substances can include, but are not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin, flavin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials; positron emitting metals; and magnetic metal ions positron emitting metals; and magnetic metal ions.

[0148] The anti-RANKL / osteopontin bispecific antibodies disclosed herein can be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized bispecific antibodies are used in immunoassays. In some embodiments, the immobilized bispecific antibodies are used for purification.

[0149] 3 Polynucleotides, Vectors, and Cells

[0150] Provided herein are polynucleotides encoding at least one light chain or one heavy chain of the anti-RANKL / osteopontin bispecific antibodies disclosed herein. In some embodiments, the polynucleotides provided herein encode one polypeptide, e.g., a light chain or a heavy chain of a bispecific antibody. In some embodiments, the polynucleotides provided herein encode more than one polypeptide. In some embodiments, the polynucleotides provided herein can encode, e.g., a light chain and a heavy chain of a bispecific antibody provided herein, respectively. For example, a cistron can be separated by, e.g., an internal ribosome entry site (IRES) or a 2A element. IRES known in the art refer to nucleotide sequences in an expression cassette that, when transcribed into mRNA, can directly recruit ribosomes without the need for ribosomes to scan the mRNA’s untranslated region in advance. 2A elements known in the art encode self-cleaving short 2A peptides (about 20 amino acids) that provide a mechanism for the subsequent isolation of polypeptides of interest in equimolar amounts. Families of self-cleaving 2A peptides have been described in the art (see, e.g., Kim, J.H. et al. (2011) Biotechnol. Lett. 33: 1243-1252; and Kim, J.H. et al. (2011) Biotechnol. Lett. 33: 1243-1252). The skilled person will understand that other art-recognized linkers can be suitable for use in the constructs of the present disclosure (e.g., encoded by the nucleic acids of the present disclosure). The skilled person will also understand that other multi-cistronic constructs can be suitable for use in the uses provided herein. et al . (2011) PLoS ONE 6:el8556). The skilled person will understand that other art-recognized linkers can be suitable for use in the constructs of the present disclosure (e.g., encoded by the nucleic acids of the present disclosure). The skilled person will also understand that other multi-cistronic constructs can be suitable for use in the uses provided herein.

[0151] In some embodiments, provided herein are polynucleotides encoding the peptide chain C1, C2, C3, or any combination thereof, of the anti-RANKL / osteopontin bispecific antibodies disclosed herein having a KIH structure. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both, of the anti-RANKL / osteopontin bispecific antibodies disclosed herein having an IgG-scFv structure.

[0152] In some embodiments, provided herein is a polynucleotide encoding C1, C2, C3, or any combination thereof, of the bispecific antibody designated DR-P7. In some embodiments, provided herein are a plurality of polynucleotides that collectively encode C1, C2, and C3, of the bispecific antibody designated DR-P7. In some embodiments, provided herein is a polynucleotide encoding C1, C2, C3, or any combination thereof, of the bispecific antibody designated DR-P8. In some embodiments, provided herein are a plurality of polynucleotides that collectively encode C1, C2, and C3, of the bispecific antibody designated DR-P8. In some embodiments, provided herein is a polynucleotide encoding C1, C2, C3, or any combination thereof, of the bispecific antibody designated DR-P15. In some embodiments, provided herein are a plurality of polynucleotides that collectively encode C1, C2, and C3, of the bispecific antibody designated DR-P15. In some embodiments, provided herein is a polynucleotide encoding C1, C2, C3, or any combination thereof, of the bispecific antibody designated DR-P16. In some embodiments, provided herein are a plurality of polynucleotides that collectively encode C1, C2, and C3, of the bispecific antibody designated DR-P16.

[0153] In some embodiments, provided herein is a polynucleotide encoding LC, HC, or both, of the bispecific antibody designated DR-P25. In some embodiments, provided herein are a first polynucleotide and a second polynucleotide that respectively encode LC and HC of the bispecific antibody designated DR-P25. In some embodiments, provided herein is a polynucleotide encoding LC, HC, or both, of the bispecific antibody designated DR-P26. In some embodiments, provided herein are a first polynucleotide and a second polynucleotide that respectively encode LC and HC of the bispecific antibody designated DR-P26. In some embodiments, provided herein is a polynucleotide encoding LC, HC, or both, of the bispecific antibody designated DR-P27. In some embodiments, provided herein are a first polynucleotide and a second polynucleotide that respectively encode LC and HC of the bispecific antibody designated DR-P27. In some embodiments, provided herein is a polynucleotide encoding LC, HC, or both, of the bispecific antibody designated DR-P28. In some embodiments, provided herein are a first polynucleotide and a second polynucleotide that respectively encode LC and HC of the bispecific antibody designated DR-P28.

[0154] The term "polynucleotide encoding a polypeptide" encompasses both the polynucleotide that includes only those linksages of the polypeptide which is encoded as well as polynucleotides which include additional coding and / or non-coding sequences. The polynucleotides of the present disclosure can be in the form of RNA or DNA. The DNA can be cDNA, genomic DNA or synthetic DNA, and can be double-stranded or single-stranded. Single-stranded DNA can be the coding strand or the non- coding (anti-sense) strand. The polynucleotides of the present disclosure can be mRNA.

[0155] The present disclosure also provides variants of the polynucleotides described herein, wherein the variants have a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a polynucleotide sequence encoding at least one polypeptide chain of an anti-RANKL / osteopontin bispecific antibody described herein. The phrase "a polynucleotide has a nucleotide sequence that is at least about 95% identical to a polynucleotide sequence" as used herein means that, out of every 100 nucleotides in the reference nucleotide sequence, the polynucleotide can include up to 5 point mutations, and the nucleotide sequence of the polynucleotide is otherwise identical to the reference sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with other nucleotides or a number of nucleotides up to 5% of the total number of nucleotides in the reference sequence can be inserted into the reference sequence. The mutations in the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those positions, they can be interspersed by one or more contiguous groups of the nucleotides in the reference sequence.

[0156] Polynucleotide variants can include changes in the coding region, non-coding region, or both. In some embodiments, a polynucleotide variant includes changes that result in silent substitutions, additions, or deletions, but which do not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant includes silent substitutions that do not result in changes to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be generated for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., changing codons in a human mRNA to those preferred by a bacterial host such as E. coli (http: / / www.kazusa.orjp / codon / )). E. coli

[0157] ​In some embodiments, a polynucleotide variant is generated to modulate or alter expression (or level of expression) of a polypeptide encoded thereby. In some embodiments, a polynucleotide variant is generated to increase expression of a polypeptide encoded thereby. In some embodiments, a polynucleotide variant is generated to decrease expression of a polypeptide encoded thereby. In some embodiments, a polynucleotide variant increases expression of a polypeptide encoded thereby as compared to a parent polynucleotide sequence. In some embodiments, a polynucleotide variant decreases expression of a polypeptide encoded thereby as compared to a parent polynucleotide sequence.

[0158] In some embodiments, a polynucleotide comprises a coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a polynucleotide that facilitates expression and secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretion sequence to control transport of the polypeptide). The polypeptide can have the leader sequence cleaved off by the host cell to form the "mature" form of the polypeptide.

[0159] In some embodiments, a polynucleotide comprises a coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a marker or tag sequence. For example, in some embodiments, the marker sequence is a hexa-histidine tag (HIS tag) that allows efficient purification of the polypeptide fused to the marker. In some embodiments, when a mammalian host (e.g., COS-7 cells) is used, the marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG™ tag. In some embodiments, the marker can be used in conjunction with other markers or tags.

[0160] In some embodiments, a polynucleotide is isolated. In some embodiments, a polynucleotide is substantially pure.

[0161] In some embodiments, also provided herein are vectors comprising the polynucleotides disclosed herein. As used herein, the term "vector" and its grammatical equivalents refer to a vehicle for carrying genetic material (e.g., a polynucleotide sequence), which can be introduced into a host cell, where it can be replicated and / or expressed. Useful vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into the host cell chromosome. In addition, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive promoters and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more polynucleotides are to be co-expressed, the two polynucleotides can be inserted into, for example, a single expression vector or separate expression vectors. For single vector expression, the coding polynucleotides can be operably linked to one common expression control sequence, or to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of the polynucleotides into a host cell can be confirmed using methods well known in the art. It will be understood by those skilled in the art that the polynucleotides are expressed in sufficient amounts to produce the desired product, and it will be further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0162] In some embodiments, the vectors provided herein can be expression vectors. In some embodiments, the vectors provided herein comprise a polynucleotide that encodes at least one polypeptide chain of an anti-RANKL / osteopontin bispecific antibody described herein. In some embodiments, provided herein are recombinant expression vectors that can be used to amplify and express a polynucleotide that encodes at least one polypeptide chain of an anti-RANKL / osteopontin bispecific antibody described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes a synthetic or cDNA-derived DNA segment that encodes at least one polypeptide chain of an anti-RANKL / osteopontin bispecific antibody described herein operably linked to suitable transcriptional and / or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. In some embodiments, viral vectors are used. DNA segments are "operably linked" when they are functionally connected. For example, a promoter is operably linked to a coding sequence if the promoter controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned for purposes of permitting translation. In some embodiments, structural elements intended for use in certain expression systems include a leader sequence enabling extracellular secretion of translated protein by host cells. In some embodiments, when a recombinant protein is expressed without a leader or transit sequence, the polypeptide can include an N-terminal methionine residue.

[0163] Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Useful expression vectors for bacterial hosts include known bacterial plasmids, e.g., plasmids from E. coli, including pCRl, pBR322, pMB9 and derivatives thereof, and more widely applicable vectors derived from plasmids such as M13 and other filamentous single stranded DNA bacteriophages. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC)), bacteriophages (e.g., lambda phage or M13 phage), and animal viruses. Examples of classes of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Exemplary transposon systems can be used, e.g., Sleeping Beauty and PiggyBac, which can stably integrate into the genome (e.g., Ivics et al. , Cell, 91 (4): 501-510 (1997); Cadiñanos et al. , (2007) Nucleic Acids Research. 35 (12): e87).

[0164] In some embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that is capable of replication without integration into the chromosomal DNA of the host and without gradual loss from dividing host cells, and also means that the vector replicates extrachromosomally or episomally. The vector is engineered to carry a sequence encoding a DNA replication origin or "ori" from a lymphotropic herpes virus or gamma-herpes virus, adenovirus, SV40, bovine papilloma virus, or yeast, specifically a replication origin of a lymphotropic herpes virus or gamma-herpes virus corresponding to oriP of EBV. In some embodiments, the lymphotropic herpes virus can be Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes saimiri (HS), or Marek's disease virus (MDV). Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of gamma herpes viruses. Typically, the host cell includes an activated viral transactivator protein that activates replication.

[0165] "Expression control sequences," "control elements," or "regulatory sequences" present in the expression vector are those non-translated regions of the vector, i.e., origins of replication, selectable marker, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence) intron, polyadenylation sequence, 5' and 3' untranslated regions that interact with host cellular proteins to carry out transcription and translation. The strength and specificity of such elements can vary. Depending upon the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters, can be used.

[0166] Exemplary ubiquitous expression control sequences useful in the present disclosure include, but are not limited to: cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) promoter (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pll promoters from vaccinia virus, elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP 70), beta-kinesin (beta-KIN), human ROSA 26 locus (Irions et al. , Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, and beta-actin promoter.

[0167] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as the promoters of genes encoding glucocorticoid or estrogen receptors (induced by treatment with the corresponding hormone), metallothionein promoters (induced by treatment with various heavy metals), MX-1 promoter (induced by interferons), the "GeneSwitch" mifepristone-regulated system (Sirin et al. , 2003, Gene , 323:67), cumate-inducible GeneSwitch (WO 2002 / 088346), tetracycline-dependent regulatory systems, and the like. The bispecific antibodies described herein can be produced by any method known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise indicated, the practice of the present application employs conventional techniques of molecular biology, microbiology, gene analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis et al.Sambrook, E.F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. Sambrook, E.F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. Sambrook, E.F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. Sambrook, E.F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al.(eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc; each of which is incorporated by reference in its entirety.

[0168] The present disclosure also provides cells comprising a polynucleotide disclosed herein encoding at least one polypeptide chain of an anti-RANKL / osteopontin bispecific antibody described herein. In some embodiments, the cells provided herein comprise a polynucleotide encoding C1, C2, and C3 of an anti-RANKL / osteopontin bispecific antibody disclosed herein having a KIH structure. In some embodiments, the cells provided herein comprise a plurality of polynucleotides collectively encoding C1, C2, and C3 of an anti-RANKL / osteopontin bispecific antibody disclosed herein having a KIH structure.

[0169] In some embodiments, the cells provided herein comprise a polynucleotide encoding both LC and HC of an anti-RANKL / osteopontin bispecific antibody disclosed herein having an IgG-scFv structure. In some embodiments, the cells provided herein comprise a first polynucleotide encoding LC of an anti-RANKL / osteopontin bispecific antibody disclosed herein having an IgG-scFv structure and a second polynucleotide encoding HC of the bispecific antibody.

[0170] Cells comprising a vector disclosed herein are also contemplated. In some embodiments, host cells comprising a vector comprising a polynucleotide disclosed herein are provided herein. In some embodiments, the host cells provided herein comprise a vector or a plurality of vectors collectively comprising polynucleotides encoding polypeptide chains of an anti-RANKL / osteopontin bispecific antibody described herein. In some embodiments, the host cells provided herein produce an anti-RANKL / osteopontin bispecific antibody described herein.

[0171] Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (of monkey kidney origin), L-929 (of murine fibroblast origin), C127 (of murine mammary

[0172] 4. Methods of production

[0173] Also provided herein are methods of producing the anti-RANKL / osteopontin bispecific antibodies disclosed herein. In some embodiments, the bispecific antibodies disclosed herein comprise more than one polypeptide chain, which can be produced separately or together. In some embodiments, the methods provided herein produce at least one polypeptide chain of the bispecific antibodies disclosed herein. In some embodiments, the methods provided herein produce all of the polypeptide chains of the bispecific antibodies disclosed herein.

[0174] The bispecific antibodies or polypeptides described herein can be produced and isolated using methods known in the art. The polypeptides can be synthesized, in whole or in part, using chemical methods (e.g., see Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, A.K., Therapeutic Peptides And Proteins, Formulation, Processing And Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be performed using a variety of solid phase techniques (e.g., see Roberge, S. et al., Peptide Research 6: 125-131 (1993); Sipe, M. B., Current Opinion in Biotechnology 4: 548-558 (1993); and Science 269:202 (1995); Merrifield, Methods. Enzymol.289:3 (1997)) and can be accomplished using an automated synthesizer according to the manufacturer's instructions, e.g., using an ABI 431 A peptide synthesizer (Perkin Elmer). Peptides can also be synthesized using combinatorial methods. Synthetic residues and polypeptides can be synthesized using various procedures and methods known in the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, J. D., et al. (Eds) John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification methods (see, e.g., Belousov, A. V., et al. (1999) J. Am. Chem. Soc. 121 : 3344-3345; and Zhang, Z. et al. (1991) Biochem. 30: 5428-5434). Peptide sequence variants, derivatives, substitutions, and modifications can also be made using methods such as oligonucleotide-directed (site- directed) mutagenesis, alanine scanning, and PCR-based mutagenesis, among others. Site-directed mutagenesis can be performed on cloned DNA (Carter, P. J., et al. (1989) Biochem. Soc. Trans. 17: 212-213; Saiki, R. K., et al. (1988) Nature 334: 677- 680; and Kunkel, T. A., et al. (1987) Proc. Natl. Acad. Sci. USA 82: 488-492). et al . (Eds) John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification methods (see, e.g., Belousov, Nucleic Acids Res . 25:3440 (1997); Frenkel, Free Radic. Biol. Med . 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994))).. Peptide sequence variants, derivatives, substitutions, and modifications can also be made using methods such as oligonucleotide-directed (site- directed) mutagenesis, alanine scanning, and PCR-based mutagenesis, among others. Site-directed mutagenesis can be performed on cloned DNA (Carter et al ., Nucl. Acids Res ., 13:4331 (1986); Zoller et al ., Nucl. Acids Res . 10:6487 (1987)) expression cassette mutagenesis (Wells et al ., Gene 34:315(1985)) restriction selection mutagenesis (Wells et al ., Philos. Trans. R. Soc. London SerA 317:415 (1986)) and other techniques to produce the inventive peptide sequences, variants, fusions and chimeras and variants, derivatives, substitutions and modifications thereof.

[0175] Various host expression vector systems can be used to recombinantly express the bispecific antibodies described herein or one or more polypeptide chains thereof. Suitable host cells for expression include prokaryotic, yeast, insect, or higher eukaryotic cells under the control of appropriate promoters. Suitable cloning and expression vectors, and methods for their use, are well known in the art, as are methods for the production of proteins, including antibodies, using bacterial, fungal, yeast, and mammalian cell hosts. Such host expression systems represent vehicles by which the coding sequences that can produce the bispecific antibodies described herein can be produced and subsequently purified, but also represent cells that, upon transformation or transfection with the appropriate polynucleotide coding sequences, can express the bispecific antibodies described herein in situ. These include but are not limited to bacteria (e.g., E. coli and B. subtili B. subtilis )) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequences of the compounds described herein; yeast (e.g., Pichia pastoris Saccharomyces pichia )) transformed with recombinant yeast expression vectors containing the sequences encoding the compounds described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the sequences encoding the compounds described herein; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the sequences encoding the molecular compounds described herein; or mammalian cell systems (e.g., COS cells, CHO cells, BHK cells, 293 cells, 293T cells, 3T3 cells, lymphoma cells) (see U.S. Pat. No. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell), transformed with recombinant expression constructs containing promoters derived from mammalian cell genes (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0176] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of the bispecific antibodies described herein, vectors which direct the expression of high levels of protein products suitable for pharmaceutical applications are preferable. Among these, suitable vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al . (1983), EMBO J. 2: 1791-1794); pIN vectors (Inouye et al. (1985), Nucleic Acids Res. 13:3101-3110; Van Heeke et al . (1989),J. Biol. Chem. 24:5503-5509); and the like. The pGEX vectors can also be used to express polypeptides as fusion proteins with glutathione S-transferase (GST). Usually, such proteins are soluble and can be purified under reducing conditions of a renaturation step after elution from a matrix glutathione-agarose bead on which the proteins are adsorbed and bound. The pGEX vectors are designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

[0177] Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. In mammalian host cells, a number of viral-based expression systems can be utilized. Examples of suitable mammalian host cell lines include but are not limited to COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical carcinoma-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Expression of recombinant proteins in insect cell culture systems, such as baculovirus, also provides a robust method for production of correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well known to those skilled in the art. Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.

[0178] Furthermore, host cell strains can be chosen which modulate the expression of inserted sequences, or which modify and process the genetic product in the desired particular fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. For example, in certain embodiments, the antibodies described herein can be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a polyprotein precursor), requiring proteolytic cleavage by natural or recombinant cellular mechanisms to form the individual polypeptides of the bispecific antibodies described herein. Accordingly, the present disclosure includes engineered nucleic acid sequences to encode polyprotein precursor molecules comprising the polypeptides of the bispecific antibodies described herein, including coding sequences capable of directing post-translational cleavage of the polyprotein precursor. Post-translational cleavage of the polyprotein precursor results in the polypeptides of the bispecific antibodies described herein. Post-translational cleavage of the precursor molecules of the polypeptides comprising the compounds described herein can occur in vivo (i.e., by natural or recombinant cellular systems / mechanisms within a host cell, e.g., furin cleavage at appropriate sites), or can occur in vitro (e.g., incubation of the polypeptide chains in a composition comprising a known protease or peptidase and / or in a composition comprising an environment or reagent that can facilitate the desired proteolysis). Purification and modification of recombinant proteins are well known in the art, such that the design of polyprotein precursors can include a number of embodiments readily understood by the skilled artisan. Any known protease or peptidase known in the art can be used for the modification of the precursor molecules described.

[0179] Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells can be used, which have the requisite cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO cells, VERY cells, BHK cells, HeLa cells, COS cells, MDCK cells, 293 cells, 293T cells, 3T3 cells, WI38 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, and T47D cells, CRL7030 cells, and Hs578Bst cells.

[0180] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the compounds described herein can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid

[0181] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al (1977), Cell 11: 223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al (1992) Bioessays 14: 495- 500), and adenine phosphoribosyltransferase (Lowy et al (1980), Cell 22: 817-823) genes can be employed in tk-, hgprt-, or aprt-cells, respectively. Likewise, drug resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al (1980) PNAS 77:3567-3570; O'Hare et al (1981) PNAS , 78: 1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al (1981) PNAS , 78: 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tolstoshev (1993), Ann. Rev. Pharmacol. Toxicol . 32:573-596;Mulligan (1993), Science 260:926-932; and Morgan et al (1993), Ann. Rev. Biochem. 62: 191-217) and hygro, which confers resistance to hygromycin (Santerre et al . (1984) Gene 30: 147-156). Methods known in the art of recombinant DNA technology that can be used are described in the following references: Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY, Chapters 12 and 13. et al . (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY, Chapters 12 and 13. et al . (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY, Chapters 12 and 13.

[0182] The expression level of the bispecific antibodies described herein or their polypeptide chains can be increased by vector amplification (see review by Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987). When the marker in the vector system described herein is amplifiable, increases in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene, and thus the number of copies of the desired protein, since the amplified region is associated with the nucleotide sequence of the desired protein (Crouse et al . (1983) Mol. Cell. Biol. 3:257-266).

[0183] The host cell can be co-transfected with more than one expression vector, each expression vector encoding a polypeptide chain of the bispecific antibodies described herein. The vectors can contain the same selectable marker, so that expression of all polypeptides is equalized. Alternatively, a single vector encoding two or more polypeptides can be used. The coding sequences for the polypeptides of the compounds described herein can include cDNA or genomic DNA.

[0184] When the bispecific antibodies described herein or the polypeptides described herein have been recombinantly expressed, then the bispecific antibodies described herein and the polypeptides described herein can be purified by any method known in the art for purifying polypeptides, multi-proteins, or antibodies (e.g., similar to antibody purification protocols based on antigen selectivity), for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography with a specific antigen (optionally following protein A selection (in the case of a compound comprising an Fc domain (or portion thereof)) and size exclusion chromatography), centrifugation, differential solubility, or by any other standard technique for purifying polypeptides and antibodies.

[0185] Provided herein are methods of producing the anti-RANKL / osteopontin bispecific antibodies described herein or the polypeptide chains of the bispecific antibodies described herein, the methods comprising obtaining the cells described herein and expressing the polynucleotides described herein in the cells. In some embodiments, the methods provided herein comprise culturing the cells under conditions that allow for expression of the bispecific antibodies. In some embodiments, the methods further comprise isolating and purifying the bispecific antibodies or polypeptide chains described herein.

[0186] The bispecific antibodies described herein can be tested for binding to human RANKL and / or osteopontin, for example, by standard ELISA. Briefly, microtiter plates are coated with purified antigen and then blocked with bovine serum albumin. Antibody dilutions are added to each well and incubated. Plates are washed and incubated with a secondary reagent (e.g., for human antibodies, goat anti-human IgG Fc specific polyclonal reagent) coupled to horseradish peroxidase (HRP). After washing, plates can be developed and analyzed by spectrophotometry. The ability of the antibodies to bind to cell lines expressing human RANKL and / or osteopontin, but not to control cell lines that do not express the target antigen, can be further tested by flow cytometry. Briefly, binding of the antibodies can be assessed by incubating CHO cells expressing RANKL and / or osteopontin with the bispecific antibodies provided herein. Cells are washed and bound detected with an anti-human IgG Ab. Flow cytometry analysis can be performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

[0187] The reactivity of the anti-RANKL / osteopontin bispecific antibodies provided herein with target antigens can be further tested by Western blotting and other methods known in the art for analyzing the binding affinity, cross-reactivity, and binding kinetics of the various anti-RANKL / osteopontin bispecific antibodies described herein, including, for example, using Bio-Layer Interferometry (BLI) with a Gator system (Probe Life) or an Octet-96 system (Sartorius AG), or BIACORE™ surface plasmon resonance (SPR) analysis using a BIACORE™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

[0188] 5. Pharmaceutical compositions

[0189] Also provided herein are pharmaceutical compositions comprising the anti-RANKL / osteopontin bispecific antibodies disclosed herein. In some embodiments, the pharmaceutical compositions comprise a therapeutically effective amount of the bispecific antibodies disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are useful in treating an inflammatory disease or an autoimmune disease.

[0190] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a material that is suitable for use with an active agent in administering to an individual without causing an adverse biological effect or interacting in a deleterious manner with any of the other components of the pharmaceutical composition. In some embodiments, the pharmaceutical compositions disclosed herein can comprise one or more of a buffering agent, a preservative, a tonicity agent, a chelating agent, a stabilizer, and / or a surfactant, and various combinations thereof. Preservatives, isotonic agents, chelating agents, stabilizers, and surfactants used in the pharmaceutical compositions are well known to those skilled in the art. Reference can be made to Remington: The Science and Practice of Pharmacy, 19thedition, 1995. th

[0191] ​In some embodiments, the pharmaceutical compositions provided herein comprise an anti-RANKL / osteopontin bispecific antibody provided herein. The anti-RANKL / osteopontin bispecific antibody can be present in various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise an anti-RANKL / osteopontin bispecific antibody provided herein at a concentration of 1 mg / mL to 1000 mg / mL. In some embodiments, the pharmaceutical compositions comprise an anti-RANKL / osteopontin bispecific antibody provided herein at a concentration of 10 mg / mL to 500 mg / mL, 10 mg / mL to 400 mg / mL, 10 mg / mL to 300 mg / mL, 10 mg / mL to 200 mg / mL, 10 mg / mL to 100 mg / mL, 20 mg / mL to 100 mg / mL, or 50 mg / mL to 100 mg / mL. In some embodiments, the pharmaceutical compositions provided herein comprise an anti-RANKL / osteopontin bispecific antibody provided herein at a concentration of about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 120 mg / mL, about 150 mg / mL, about 180 mg / mL, about 200 mg / mL, about 300 mg / mL, about 500 mg / mL, about 800 mg / mL, or about 1000 mg / mL. One of skill in the art can readily adjust the dosage; for example, a decrease in purity can require an increase in dosage.

[0192] Pharmaceutically acceptable carriers that can be used in the compositions provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., the anti-RANKL / osteopontin bispecific antibody) can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

[0193] Also provided herein are pharmaceutical compositions or formulations that improve the stability of anti-RANKL / osteopontin bispecific antibodies to allow for long-term storage. In some embodiments, the pharmaceutical compositions or formulations disclosed herein comprise: (a) an anti-RANKL / osteopontin bispecific antibody disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a bulking agent; and / or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or more. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4°C, 25°C, or 40°C.

[0194] The buffer used in the pharmaceutical compositions or formulations disclosed herein can be a weak acid or a weak base that serves to bring the acidity (pH) of the solution close to a selected value upon the addition of another acid or base. Suitable buffers can maximize the stability of the pharmaceutical formulation by maintaining the pH of the formulation. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties can also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) and an isotonic agent, and can contain a pH adjusting agent known in the art. In certain embodiments, the buffer is L-histidine. In certain embodiments, the pH of the formulation is maintained at about 2 to about 10, or at about 4 to about 8.

[0195] Stabilizers are added to pharmaceutical products to stabilize the product. Such agents can stabilize proteins in different ways. Common stabilizers include, but are not limited to, amino acids (e.g., glycine, alanine, lysine, arginine, or threonine), sugar classes (e.g., dextrose, sucrose, trehalose, raffinose, or maltose), polyols of any type or molecular weight (e.g., glycerol, mannitol, sorbitol, cyclodextrin, or dextran), or PEG. In some embodiments, the stabilizer is selected to maximize the stability of the antibody in the lyophilized formulation. In certain embodiments, the stabilizer is sucrose and / or arginine.

[0196] Bulking agents can be added to the pharmaceutical compositions or formulations to add bulk and mass to the pharmaceutical product, thereby facilitating its accurate metering and handling. Common bulking agents include, but are not limited to, lactose, sucrose, dextrose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0197] Surfactants are amphiphilic substances having a lyophilic group and a lyophobic group. Surfactants can be anionic, cationic, zwitterionic, or non-ionic surfactants. Examples of non-ionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols (e.g., cetyl alcohol or oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethyl amine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0198] In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but can also include colloids, dispersions, emulsions, and multiple phase materials. The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w water.

[0199] In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized, to which a solvent and / or diluent is added by a physician or patient prior to use.

[0200] The pharmaceutical compositions disclosed herein can also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0201] Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.

[0202] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0203] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. In some embodiments, the pharmaceutical compositions provided herein comprise an anti-RANKL / osteopontin bispecific antibody or cell provided herein, wherein the composition is for local administration.

[0204] The pharmaceutical compositions or formulations must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. In many cases, the compositions can include isotonic agents, for example, sugars, polyalcohols, (for example, mannitol, sorbitol) or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, monostearate salts and gelatin.

[0205] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with one or more of the ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders, some methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional ingredient present in the previously sterile-filtered solution.

[0206] The amount of active ingredient that can be combined with the carrier materials, in the pharmaceutical compositions or formulations disclosed herein, can vary. In some embodiments, the amount of active ingredient that can be combined with the carrier materials is an amount that produces therapeutic effects. Generally, the amount of active ingredient that can be combined with a pharmaceutical carrier material will be determined by the solubility and chemical properties of the active ingredient in the carrier, chosen route of administration, and appropriate dosage range.

[0207] The pharmaceutical compositions disclosed herein can be prepared with carriers that protect the active ingredient against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations have been patented and are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0208] Also provided herein are kits for preparing a pharmaceutical composition having an anti-RANKL / dickkopf bispecific antibody disclosed herein. In some embodiments, the kits include one or more containers of an anti-RANKL / dickkopf bispecific antibody disclosed herein and a pharmaceutically acceptable carrier. In another embodiment, the kits can include an anti-RANKL / dickkopf bispecific antibody disclosed herein for administration to a subject. In particular embodiments, the kits include instructions for preparing and / or administering the anti-RANKL / dickkopf bispecific antibody.

[0209] 6. Methods and Uses

[0210] The anti-RANKL / osteopontin bispecific antibodies provided herein are useful in medicine. Also provided herein are methods of preventing or treating osteoporosis in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of an anti-RANKL / osteopontin bispecific antibody disclosed herein. Also provided herein are methods of promoting bone growth in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-RANKL / osteopontin bispecific antibody disclosed herein. Also provided herein are methods of increasing bone mass, comprising administering to the subject in need thereof a therapeutically effective amount of an anti-RANKL / osteopontin bispecific antibody disclosed herein. In some embodiments, the subject is a human. In some embodiments, the subject has primary osteoporosis. In some embodiments, the subject has secondary osteoporosis. In some embodiments, the subject is at risk of developing osteoporosis.

[0211] Provided herein are uses of an anti-RANKL / osteopontin bispecific antibody disclosed herein as a medicament. Also provided herein are uses of an anti-RANKL / osteopontin bispecific antibody disclosed herein for preventing or treating osteoporosis. Also provided herein are uses of an anti-RANKL / osteopontin bispecific antibody disclosed herein for promoting bone growth or increasing bone mass. Also provided herein are uses of an anti-RANKL / osteopontin bispecific antibody disclosed herein for the manufacture of a medicament for preventing or treating osteoporosis. Also provided herein are uses of an anti-RANKL / osteopontin bispecific antibody disclosed herein for the manufacture of a medicament for promoting bone growth or increasing bone mass.

[0212] The term "treatment" and its grammatical equivalents as used herein in connection with a disease or disorder, or a subject having a disease or disorder, means an action, intervention and / or measure that inhibits, eliminates, reduces and / or ameliorates a symptom, severity of a symptom and / or frequency of a symptom associated with the disease or disorder being treated. Treatment of osteoporosis can inhibit, eliminate, reduce or ameliorate symptoms associated with osteoporosis. These symptoms include primarily bone loss, decrease in bone density and increased risk of bone fracture, and treatment aims to reduce the rate of bone loss, strengthen the bone, and reduce the likelihood of bone fracture.

[0213] The term "prevention" and its grammatical equivalents as used herein in connection with a disease or disorder, or a subject at risk of a disease or disorder, means an action taken to hinder, impede or alleviate the occurrence, development or progression of a disease or disorder. The aim of prevention is to reduce the risk, delay the onset, or completely avoid the manifestation of the disease or disorder in an individual predisposed to the disease or disorder.

[0214] The term "administering" and its grammatical equivalents as used herein refer to the act of delivering or causing the delivery of a therapeutic agent or a pharmaceutical composition to the body of a subject by a method described herein or a method known in the art. The therapeutic agent can be a compound, a polypeptide, an antibody, a cell, or a population of cells. Administering a therapeutic agent or a pharmaceutical composition includes prescribing a therapeutic agent or a pharmaceutical composition to be delivered into the body of a subject. Exemplary administration forms include oral dosage forms such as tablets, capsules, syrups, suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); transdermal dosage forms including creams, gels, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0215] The term "effective amount," "therapeutically effective amount," and their grammatical equivalents as used herein refer to an amount of an agent, alone or as part of a pharmaceutical composition, that when administered to a subject, is capable of having any detectable positive effect on any symptom, aspect, or feature of a disease, disorder, or condition. A therapeutically effective amount can be determined by measuring the relevant physiological effect. The exact amount required will vary from subject to subject, depending on the age, body mass, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate "effective amount" in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0216] The term "subject" as used herein refers to any animal (e.g., a mammal), including, but not limited to, a human, a non-human primate, a canine, a feline, a rodent, and the like, who is the recipient of a particular treatment. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment can be a human subject who has a disease, who is at risk of having a disease, or who is suspected of having a disease. A subject who has a disease can be identified by a routine health examination (e.g., physical examination), laboratory test, organ function test, CT scan, or ultrasound examination. A subject who is suspected of having such a disease can show one or more symptoms of the disease. A subject who is at risk of having a disease can be a subject who has one or more risk factors for the disease. The subject can be a human. The subject can have a particular disease or condition.

[0217] In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for treating or preventing osteoporosis. In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for treating or preventing primary osteoporosis. In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for treating postmenopausal osteoporosis (Type I). In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for treating age-related osteoporosis (Type II). In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for treating idiopathic osteoporosis. In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for preventing postmenopausal osteoporosis (Type I). In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for preventing age-related osteoporosis (Type II). In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for preventing idiopathic osteoporosis.

[0218] In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for treating secondary osteoporosis. In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein are useful for preventing secondary osteoporosis. It will be appreciated by those of ordinary skill in the art that the bispecific antibodies and pharmaceutical compositions provided herein are capable of treating or preventing secondary osteoporosis without being limited to any particular cause. For example, secondary osteoporosis of endocrine or metabolic origin, or osteoporosis associated with collagen / genetic diseases, drugs, and / or nutritional deficiencies, can be treated using the bispecific antibodies and pharmaceutical compositions provided herein.

[0219] Accordingly, subjects that can be treated using the bispecific antibodies and pharmaceutical compositions provided herein can have, or be at risk of having, osteoporosis. In some embodiments, the subject is diagnosed with osteoporosis. In some embodiments, the subject is diagnosed with type I osteoporosis. In some embodiments, the subject is diagnosed with type II osteoporosis. In some embodiments, the subject is diagnosed with secondary osteoporosis. In some embodiments, the subject is at risk of having osteoporosis. In some embodiments, the subject at risk of having osteoporosis is a postmenopausal woman. In some embodiments, the subject at risk of having osteoporosis is 50 years of age or older. In some embodiments, the subject at risk of having osteoporosis has a family history of osteoporosis or bone fracture. In some embodiments, the subject at risk of having osteoporosis has hormonal imbalances, such as hypogonadism, hyperthyroidism, and / or adrenal disorders. In some embodiments, the subject at risk of having osteoporosis has certain medical conditions, such as rheumatoid arthritis, celiac disease, kidney disease, and hormone-related disorders. In some embodiments, the subject at risk of having osteoporosis uses long-term medications such as corticosteroids (prednisone), anticonvulsants, and some cancer treatments that weaken the bones. In some embodiments, the subject at risk of osteoporosis has poor nutrition, low calcium and vitamin D intake, a sedentary lifestyle, excessive alcohol consumption, smoking, and / or lack of weight-bearing exercise.

[0220] In some embodiments, the methods provided herein prevent or treat osteoporosis in a subject. In some embodiments, the methods provided herein prevent bone fracture in a subject. In some embodiments, the methods provided herein increase bone density. In some embodiments, the methods provided herein increase bone mass. In some embodiments, the methods provided herein slow or stop bone loss. In some embodiments, the methods provided herein prevent further deterioration of bone density. In some embodiments, the methods provided herein alleviate symptoms associated with osteoporosis, including chronic pain. In some embodiments, the methods provided herein enhance the mobility and functionality of a subject. In some embodiments, the methods provided herein minimize the risk of osteoporosis complications (e.g., spinal deformity and loss of height).

[0221] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein (i.e., the anti-RANKL / osteopontin bispecific antibodies disclosed herein) can be varied to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0222] The anti-RANKL / osteopontin bispecific antibodies disclosed herein can be administered as sustained-release formulations, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the anti-RANKL / osteopontin bispecific antibodies in the patient. In therapeutic applications, the anti-RANKL / osteopontin bispecific antibodies are supplied as a relatively high initial dose followed by one or more supplemental or lesser doses, until the desired treatment regimen is achieved. Alternatively, the anti-RANKL / osteopontin bispecific antibodies can be supplied in a continuous infusion regimen.

[0223] The anti-RANKL / osteopontin bispecific antibodies or pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, lymph node administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, e.g., by injection or infusion, or directly to the thymus. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinally, epidural, and intrasternal injection and infusion. In some embodiments, subcutaneous administration is employed. In some embodiments, intravenous administration is employed. In some embodiments, oral administration is employed. In one embodiment, the antibodies or antigen-binding fragments provided herein can be delivered locally. In another embodiment, the antibodies or antigen-binding fragments provided herein can be administered systemically.

[0224] The anti-RANKL / osteopontin bispecific antibodies or pharmaceutical compositions provided herein can be administered with medical devices known in the art. For example, in some embodiments, needle-free hypodermic injection devices, such as those disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556 can be used. Examples of known implants and modules for use in the uses described herein include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapy administration set for administering medication through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at precise infusion rates; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic pharmaceutical delivery system having multi-chambers; and U.S. Patent No. 4,475,196, which discloses an osmotic pharmaceutical delivery system. These patents are incorporated herein by reference. Numerous other such implants, delivery systems, and modules are known to those of skill in the art.

[0225] In some embodiments, the anti-RANKL / osteopontin bispecific antibodies or pharmaceutical compositions provided herein can be administered with additional therapies. The additional therapies can be administered prior to, concurrently with, or subsequent to administration of the bispecific antibodies or pharmaceutical compositions described herein. The combined administration can include coadministration, in a single pharmaceutical formulation, or in separate formulations, or sequential administration, with generally continuous therapy with all active agents. One of skill in the art can readily determine appropriate regimens for administration of the pharmaceutical compositions described herein and the additional therapies, including timing and dosages for additional agents in combination therapy, based on the needs of the subject being treated.

[0226] 7. Exemplary Embodiments

[0227] Embodiment 1 : A bispecific antibody comprising (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1), wherein the VL1 / VH1 pair specifically binds to human RANKL, and wherein the VL1 comprises a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and wherein the VH1 comprises a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and (ii) a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), wherein the VL2 / VH2 pair specifically binds to human sclerostin; and wherein the VL2 comprises a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, and wherein the VH2 comprises a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0228] Embodiment 2: The bispecific antibody of embodiment 1, wherein VL1, VH1, VL2, and VH2 have the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0229] Embodiment 3: The bispecific antibody of embodiment 1 or 2, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain 1 (CH1), and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL2 and VH2, a linker, and a Hole-Fc region.

[0230] Embodiment 4: The bispecific antibody of embodiment 1 or 2, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain 1 (CH1), and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL2 and VH2, a linker, and a Knob-Fc region.

[0231] Embodiment 5: The bispecific antibody of embodiment 3 or 4, wherein the scFv comprises, from N-terminus to C-terminus, VL2, a second linker, and VH2.

[0232] Embodiment 6: The bispecific antibody of embodiment 3 or 4, wherein the scFv comprises, from N-terminus to C-terminus, VH2, a second linker, and VL2.

[0233] Embodiment 7: The bispecific antibody of embodiment 1 or 2, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1), and a Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL1 and VH1, a linker, and a Hole-Fc region.

[0234] Embodiment 8: The bispecific antibody of embodiment 1 or 2, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1), and a Hole-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL1 and VH1, a linker, and a Knob-Fc region.

[0235] Embodiment 9: The bispecific antibody of embodiment 7 or 8, wherein the scFv comprises, from N-terminus to C-terminus, VL1, a second linker, and VH1.

[0236] Embodiment 10: The bispecific antibody of embodiment 7 or 8, wherein the scFv comprises, from N-terminus to C-terminus, VH1, a second linker, and VL1.

[0237] Embodiment 11: The bispecific antibody of any one of embodiments 3 to 10, wherein the Knob-Fc region is a human IgG2 Fc region variant having up to 10 amino acid substitutions, including a T366W substitution; and the Hole-Fc region is a human IgG2 Fc region variant having up to 10 amino acid substitutions, including T366S, L368A, Y407V substitutions.

[0238] Embodiment 12: The bispecific antibody of embodiment 11, wherein the Knob-Fc region further comprises a S354C substitution, and the Hole-Fc region further comprises a Y349C substitution.

[0239] Embodiment 13: The bispecific antibody of embodiment 11, wherein the Knob-Fc region further comprises a Y349C substitution, and the Hole-Fc region further comprises a S354C substitution.

[0240] Embodiment 14: The bispecific antibody of embodiment 11, wherein (i) the CL region is a kappa CL (CK; SEQ ID NO: 19) or a lambda CL (CL; SEQ ID NO: 20) or a variant thereof having up to 10 amino acid substitutions; (ii) the CH1 domain is a human IgG2 CH1 domain (SEQ ID NO: 36) or a variant thereof having up to 10 amino acid substitutions; and / or (iii) the Knob-Fc region and the Hole-Fc region (1) have the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 32, respectively, or a variant thereof having up to 10 amino acid substitutions, (2) have the amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 33, respectively, or a variant thereof having up to 10 amino acid substitutions, or (3) have the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 34, respectively, or a variant thereof having up to 10 amino acid substitutions.

[0241] Embodiment 15: The bispecific antibody of embodiment 14, wherein the CL region, CH1 domain, the Knob-Fc region, and the Hole-Fc region (1) have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 29, and SEQ ID NO: 32, respectively, (2) have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 30, and SEQ ID NO: 33, respectively, or (3) have the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 31, and SEQ ID NO: 34, respectively.

[0242] Embodiment 16: The bispecific antibody of embodiment 3, wherein the C1, C2, and C3 (1) have the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 54, respectively, (2) have the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, (3) have the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 57, and SEQ ID NO: 58, respectively; or (4) have the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 59, and SEQ ID NO: 60, respectively.

[0243] Embodiment 17: The bispecific antibody of embodiment 1 or 2, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL1 and a light chain constant region (CL); and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH1, a heavy chain constant region (CH), a linker, and a single chain variable fragment (scFv) comprising VL2 and VH2.

[0244] Embodiment 18: The bispecific antibody of embodiment 17, wherein the scFv comprises, from N-terminus to C-terminus, VL2, a second linker, and VH2.

[0245] Embodiment 19: The bispecific antibody of embodiment 17, wherein the scFv comprises, from N-terminus to C-terminus, VH2, a second linker, and VL2.

[0246] Embodiment 20: The bispecific antibody of embodiment 1 or 2, comprising: (1) a first peptide chain (C1) comprising, from N-terminus to C-terminus, VL2 and a light chain constant region (CL); and (2) a second peptide chain (C2) comprising, from N-terminus to C-terminus, VH2, a heavy chain constant region (CH), a linker, and a single chain variable fragment (scFv) comprising VL1 and VH1.

[0247] Embodiment 21: The bispecific antibody of embodiment 20, wherein the scFv comprises, from N-terminus to C-terminus, VL1, a second linker, and VH1.

[0248] Embodiment 22: The bispecific antibody of embodiment 20, wherein the scFv comprises, from N-terminus to C-terminus, VH1, a second linker, and VL1.

[0249] Embodiment 23: The bispecific antibody of any one of embodiments 17-22, wherein (1) the CL region is kappa CL (CK; SEQ ID NO: 19) or lambda CL (CL; SEQ ID NO: 20) or a variant thereof having up to 10 amino acid substitutions; and / or (2) the CH region is a human IgGl CH region (SEQ ID NO: 21), an IgG2 CH region (SEQ ID NO: 22), an IgG3 CH region (SEQ ID NO: 23), or an IgG4 CH region (SEQ ID NO: 24), or a variant thereof having up to 10 amino acid substitutions.

[0250] Embodiment 24: The bispecific antibody of embodiment 23, wherein (1) the CL region is kappa CL (CK; SEQ ID NO: 19); and (2) the CH region is a human IgG2 CH region (SEQ ID NO: 22).

[0251] Embodiment 25: The bispecific antibody of embodiment 17, wherein the C1 and C2 (1) have the amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 61, respectively, (2) have the amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 62, respectively.

[0252] Embodiment 26: The bispecific antibody of embodiment 20, said C1 and C2 have (1) the amino acid sequences of SEQ ID NO: 52 and SEQ ID NO: 63, respectively, (2) the amino acid sequences of SEQ ID NO: 52 and SEQ ID NO: 64, respectively.

[0253] Embodiment 27: A pharmaceutical composition comprising a therapeutically effective amount of the bispecific antibody of any one of embodiments 1 to 26 and a pharmaceutically acceptable carrier.

[0254] Embodiment 28: A polynucleotide encoding a peptide chain of the bispecific antibody of any one of embodiments 3 to 26.

[0255] Embodiment 29: The polynucleotide of embodiment 28, encoding all peptide chains of the bispecific antibody.

[0256] Embodiment 30: A plurality of polynucleotides of embodiment 28, collectively encoding all peptide chains of the bispecific antibody.

[0257] Embodiment 31 : A vector comprising the polynucleotide of embodiment 28 or 29.

[0258] Embodiment 32: A cell comprising the polynucleotide or plurality of polynucleotides of any one of embodiments 28 to 30, or the vector of embodiment 31.

[0259] Embodiment 33: A method of making a bispecific antibody that specifically binds to human RANKL and human sclerostin, comprising culturing the cell of embodiment 32 under conditions that allow expression of the bispecific antibody.

[0260] Embodiment 34: A method of preventing or treating osteoporosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific antibody of any one of embodiments 1 to 26.

[0261] Embodiment 35: The method of embodiment 34, wherein the subject has primary osteoporosis.

[0262] Embodiment 36: The method of embodiment 35, wherein the subject has postmenopausal osteoporosis (Type I).

[0263] Embodiment 37: The method of embodiment 35, wherein the subject has age-related osteoporosis (Type II).

[0264] Embodiment 38: The method of embodiment 34, wherein the subject has secondary osteoporosis.

[0265] Implementation Scheme 39: The method according to Implementation Scheme 38, wherein the osteoporosis is secondary to endocrine or metabolic causes, collagen or genetic disorders, drug causes, or nutritional causes.

[0266] Implementation Scheme 40: The method according to any one of Implementation Schemes 34 to 39, wherein the object is a human being.

[0267] Implementation Scheme 41: The use of the bispecific antibody as a drug as described in any one of Implementation Schemes 1 to 26.

[0268] Implementation Scheme 42: The use of the bispecific antibody described in any one of Implementation Schemes 1 to 26 for the prevention or treatment of osteoporosis.

[0269] Implementation Scheme 43: The use of the bispecific antibody described in any one of Implementation Schemes 1 to 26 for the preparation of a medicament for the prevention or treatment of osteoporosis.

[0270] 8 Experiments

[0271] Unless otherwise stated, the embodiments provided below are for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not be construed as being limited to the following embodiments, but should be construed as including any and all variations that become apparent from the teachings provided herein.

[0272] 8.1 Example 1: Production of asymmetric bispecific antibody (DRAb)

[0273] Bispecific IgG2 antibodies with a knobs-in-the-holes conformation that specifically bind to human RANKL and human sclerosingin were produced. Figures 1A-1B The heavy chains (DRAbs) include DR-P7, DR-P8, DR-P15, and DR-P16 (Table 6B). Point mutations are introduced into the CH3 domain of the heavy chain. Specifically, S354C and T366W are introduced into the "knob" chain; and Y349C, T366S, L368A, and Y407V are introduced into the "hole" chain. The "knob" chain contains an antibody-binding fragment (Fab) targeting RANKL, while the "hole" chain contains a single-chain variable fragment (scFv) targeting sclerosingin. Therefore, mismatches in the light chains should be eliminated.

[0274] 8.2 Example 2: Binding of DRAb to its ligand

[0275] The binding affinity of some of the candidate bispecific antibodies to human RANKL protein and sclerostin was detected using ELISA. Briefly, His-tagged human RANKL at a concentration of 5 pg / mL was used to coat ELISA plates overnight at 4°C. The coated plates were washed 3 times with PBST buffer and blocked with PBS in the presence of 1% BSA for 2 hours at room temperature. After washing the blocked plates 3 times with PBST, a series of concentrations (10 pg / mL as the highest concentration, 5-fold dilution, 7-point concentrations) of the bispecific antibodies (50 pL) were added to the wells of the His-tagged human RANKL protein-coated ELISA plates and incubated for 1 hour at room temperature. The ELISA plates were washed 3 times with PBST and the binding of the bispecific antibodies to the His-tagged RANKL was revealed using HRP-conjugated goat anti-human Fc specific antibody (Abeam, ab97225) diluted 1:5000 in PBS with 1% BSA. For binding to sclerostin, His-tagged human sclerostin protein was used for coating at a concentration of 5 pg / mL and incubated overnight at 4°C. The ELISA was performed as described previously. Figure 2 Exemplary results are provided. As shown, all three DRAb: DR-P7, DR-P15, and DR-P16 specifically bind to human RANKL and human sclerostin. Denosumab and Romosozumab were used as reference antibodies.

[0276] 8.3 Example 3: DRAb effectively block osteoclast differentiation induced by RANKL

[0277] DRAb were tested for their blocking effect on osteoclast differentiation. The murine macrophage precursor cell line RAW24.7 was activated with human RANKL for osteoclast differentiation. Briefly, 100 ng / mL human RANKL was incubated with RAW264.7 for 7 days. The medium containing human RANKL was changed every 2 to 3 days. Thereafter, the cells were fixed with 4% paraformaldehyde for 5 minutes at room temperature and washed once with PBS. The cells were stained according to the TRAcP kit procedure (Wako, Fujifilm) and photographed using a standard inverted microscope (Olympus, Japan). A multinucleated cell was counted as a mature osteoclast when more than 3 nuclei were present in the stained cell. Denosumab was used as a reference antibody. Figure 3 Exemplary results are provided. As shown, both the bispecific antibodies DR-P7 and DR-P8 disclosed herein effectively inhibit osteoclast differentiation.

[0278] 8.4 Example 4: DRAb effectively inhibit osteoclast marker gene expression induced by RANKL

[0279] Expression of osteoclast differentiation marker genes was detected to confirm the inhibitory effect of DRAb. Murine macrophage precursor cell line RAW24.7 was activated with human RANKL to undergo osteoclast differentiation. Briefly, 100 ng / mL human RANKL was incubated with RAW264.7 for 7 days. The medium containing human RANKL was changed every 2 to 3 days. Thereafter, RNA was collected with 500 μL RNAzol (MRC Inc., Ohio) and RNA was extracted according to the manufacturer’s protocol. 0.5 μg of RNA was reverse-transcribed into cDNA using PrimeScript RT Reagent Kit (Takara, Shiga). Quantitative PCR was performed using GoTaq® qPCR Master Mix with the following cycling conditions: polymerase activation, 95°C, 2 minutes; denaturation, 95°C, 15 seconds; annealing and extension, 60°C, 1 minute. Forty cycles of denaturation, annealing, and extension were performed. Selected genes essential for osteoclast differentiation were detected. Denosumab was used as a reference antibody. Figure 4 Exemplary results are provided. As shown in the figures, both DR-P7 and DR-P8 effectively inhibited the expression of osteoclast marker genes.

[0280] 8.5 Example 5: DRAb effectively blocked sclerostin-mediated inhibition of WNT signaling in HEK 293 cells

[0281] HEK 293 cells were used to evaluate the activity of DRAb in blocking sclerostin-mediated inhibition of WNT signaling. Cells were seeded at a density of 6000 cells / cm2in 24-well culture plates pre-coated with 50 μg / mL poly-D-lysine (Merck, A-003-E) for 1 hour. Cells were placed in complete growth medium (Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) with 10% fetal bovine serum at 37°C with 5% CO2. After 24 hours, HEK 293 cells were transfected with pNL3.3 minP nanoluc TCE-LEF reporter plasmid (Promega) and human WNT1 expression plasmid (Genscript). The TCE-LEF reporter can monitor WNT signaling pathway activity. Twenty-four hours after transfection, cells were treated with human sclerostin (Sino Biological) and the indicated concentrations of antibodies overnight. The next day, nanoluciferase assay (Promega) was performed to measure the level of nanoluciferase in the medium, which indicates the level of activation of the canonical WNT signaling pathway.

[0282] Figure 5Example results are provided. As shown, romosozumab inhibited the hardinectin-mediated repression of the WNT signaling pathway with an IC50 of 18.94 nM. DRAb (DR-P7 and DR-P8) also inhibited hardinectin activity with IC50 values of 26.88 nM and 24.92 nM, respectively. These results indicate that DRAb can effectively block hardinectin-mediated repression of the WNT signaling pathway in HEK 293 cells.

[0283] 8.6 Example 6: Bone formation in ovariectomized mice after antibody treatment

[0284] Transgenic mice expressing human RANKL (B6-hRANKL) were obtained from GemPharmatech Co., Ltd. Experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of GemPharmatech. Female mice were housed in cages and placed under a strict 12-hour light: 12-hour dark cycle at 22°C, fed with standard chow pellets, and mice had free access to tap water. After one week of acclimation, 8-week-old female mice were randomly divided into the following groups: control group (ovary intact + vehicle (saline) intravenous injection), ovariectomized (OVX) group + IgG2 subtype (OVX + IgG), OVX + 5 mg / kg denosumab group (OVX + Deno), OVX + 5 mg / kg romosozumab group (OVX + Romo), OVX + 4 mg / kg DRAb group (OVX + DR-P7). Ovariectomy was performed on day 0 in a surgery room that had been exposed to ultraviolet light overnight. After anesthesia, the bilateral ovaries were exposed and removed to prepare OVX animals; in the control group, the ovaries were exposed but remained intact. Starting on day 0, the antibodies were administered intravenously 4 times per week. The mice were sacrificed 28 days after ovariectomy and their femurs were collected and fixed in 4% paraformaldehyde for 2 days. The samples were scanned using a Scano micro-CT 40 system (Scanco Medical, Brütisellen, Switzerland) with a power of 70 kVp, a current of 113 μΑ, and an integration time of 200 ms.

[0285] Gaussian filtering (sigma = 0.8, support = 1) was applied to reduce background noise. To analyze the distal femur trabecular bone, the distal femur metaphysis was scanned with a nominal resolution of 10 μιη / voxel. The region of interest analyzed extended proximally 0.9 mm starting 0.03 mm proximal to the growth plate. As Figure 6As shown, OVX treatment decreased trabecular number (Tb.N) and increased trabecular separation (Tb.Sp). Denosumab or romosozumab treatment partially restored Tb.N and Tb.Sp. However, the group treated with DRAb showed a significant increase in Tb.N and a more significant decrease in Tb.Sp, indicating a synergistic effect in promoting bone regeneration by targeting both RANKL and sclerostin compared to targeting either of them alone.

Claims

1. Bispecific antibodies, which contain (i) a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1), wherein the VL1 / VH1 pair specifically binds to human RANKL, and wherein the VL1 comprises VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 have amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 have amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and (ii) a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), wherein the VL2 / VH2 pair specifically binds to human sclerosingin; and wherein the VL2 comprises VLCDR1, VL CDR2, and VL CDR3, wherein the VL... CDR1, VL CDR2, and VL CDR3 have the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, and wherein the VH2 comprises VH CDR1, VH CDR2, and VH CDR3, and the VH CDR1, VH CDR2, and VH CDR3 have the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

2. The bispecific antibody according to claim 1, wherein VL1, VH1, VL2 and VH2 have the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively.

3. The bispecific antibody according to claim 1 or 2, comprising... (1) A first peptide chain (C1), wherein the first peptide chain (C1) includes a VL1 and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) A second peptide chain (C2), comprising VH1, heavy chain constant domain 1 (CH1), and a Knob-Fc region from the N-terminus to the C-terminus; and (3) The third peptide chain (C3) comprises, from the N-terminus to the C-terminus, a single-chain variable fragment (scFv) containing VL2 and VH2, a linker, and a Hole-Fc region.

4. The bispecific antibody according to claim 1 or 2, comprising... (1) A first peptide chain (C1), wherein the first peptide chain (C1) includes a VL1 and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) A second peptide chain (C2), comprising VH1, heavy chain constant domain 1 (CH1), and a Hole-Fc region from the N-terminus to the C-terminus; and (3) The third peptide chain (C3) includes, from the N-terminus to the C-terminus, a single-chain variable fragment (scFv) containing VL2 and VH2, a linker, and a Knob-Fc region.

5. The bispecific antibody according to claim 3 or 4, wherein the scFv comprises VL2, a second linker, and VH2 from the N-terminus to the C-terminus.

6. The bispecific antibody according to claim 3 or 4, wherein the scFv comprises VH2, a second linker, and VL2 from the N-terminus to the C-terminus.

7. The bispecific antibody according to claim 1 or 2, comprising: (1) A first peptide chain (C1), wherein the first peptide chain (C1) includes VL2 and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) A second peptide chain (C2), comprising VH2, heavy chain constant domain 1 (CH1), and a Knob-Fc region from the N-terminus to the C-terminus; and (3) The third peptide chain (C3) includes, from the N-terminus to the C-terminus, a single-chain variable fragment (scFv) containing VL1 and VH1, a linker, and a Hole-Fc region.

8. The bispecific antibody according to claim 1 or 2, comprising: (1) A first peptide chain (C1), wherein the first peptide chain (C1) includes VL2 and a light chain constant region (CL) from the N-terminus to the C-terminus; (2) A second peptide chain (C2), comprising VH2, a heavy chain constant domain 1 (CH1), and a Hole-Fc region from the N-terminus to the C-terminus; and (3) The third peptide chain (C3) includes, from the N-terminus to the C-terminus, a single-chain variable fragment (scFv) containing VL1 and VH1, a linker, and a Knob-Fc region.

9. The bispecific antibody according to claim 7 or 8, wherein the scFv comprises VL1, a second linker, and VH1 from the N-terminus to the C-terminus.

10. The bispecific antibody according to claim 7 or 8, wherein the scFv comprises VH1, a second linker, and VL1 from the N-terminus to the C-terminus.

11. The bispecific antibody according to any one of claims 3 to 10, wherein the Knob-Fc region is a human IgG2 Fc region variant having up to 10 amino acid substitutions, including T366W substitution; and the Hole-Fc region is a human IgG2 Fc region variant having up to 10 amino acid substitutions, including T366S, L368A, and Y407V substitution.

12. The bispecific antibody of claim 11, wherein the Knob-Fc region further comprises an S354C substitution, and the Hole-Fc region further comprises a Y349C substitution.

13. The bispecific antibody of claim 11, wherein the Knob-Fc region further comprises a Y349C substitution, and the Hole-Fc region further comprises an S354C substitution.

14. The bispecific antibody according to claim 11, wherein... (i) The CL region is kappa CL (Cκ; SEQ ID NO: 19) or lambda CL (Cλ; SEQ ID NO: 20) or a variant thereof having up to 10 amino acid substitutions; (ii) The CH1 domain is a human IgG2 CH1 domain (SEQ ID NO: 36) or a variant thereof having up to 10 amino acid substitutions; and / or (iii) The Knob-Fc region and the Hole-Fc region (1) have amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 32, respectively, or variants thereof having up to 10 amino acid substitutions; (2) have amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 33, respectively, or variants thereof having up to 10 amino acid substitutions; or (3) have amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 34, respectively, or variants thereof having up to 10 amino acid substitutions.

15. The bispecific antibody according to claim 14, wherein the CL region, CH1 domain, Knob-Fc region and Hole-Fc region (1) have amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 29 and SEQ ID NO: 32 respectively, (2) have amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 30 and SEQ ID NO: 33 respectively, or (3) have amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 36, SEQ ID NO: 31 and SEQ ID NO: 34 respectively.

16. The bispecific antibody according to claim 3, wherein C1, C2 and C3 (1) have amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 54 respectively, (2) have amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 55 and SEQ ID NO: 56 respectively, (3) have amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 57 and SEQ ID NO: 58 respectively; or (4) have amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 59 and SEQ ID NO: 60 respectively.

17. The bispecific antibody according to claim 1 or 2, comprising: (1) A first peptide chain (C1), wherein the first peptide chain (C1) comprises a VL1 and a light chain constant region (CL) from the N-terminus to the C-terminus; and (2) Second peptide chain (C2), which includes VH1, heavy chain constant region (CH), linker and single-chain variable fragment (scFv) containing VL2 and VH2 from N-terminus to C-terminus.

18. The bispecific antibody of claim 17, wherein the scFv comprises VL2, a second linker, and VH2 from the N-terminus to the C-terminus.

19. The bispecific antibody of claim 17, wherein the scFv comprises VH2, a second linker, and VL2 from the N-terminus to the C-terminus.

20. The bispecific antibody according to claim 1 or 2, comprising: (1) A first peptide chain (C1), wherein the first peptide chain (C1) comprises VL2 and a light chain constant region (CL) from the N-terminus to the C-terminus; and (2) Second peptide chain (C2), which includes VH2, heavy chain constant region (CH), linker and single-chain variable fragment (scFv) containing VL1 and VH1 from N-terminus to C-terminus.

21. The bispecific antibody of claim 20, wherein the scFv comprises VL1, a second linker, and VH1 from the N-terminus to the C-terminus.

22. The bispecific antibody of claim 20, wherein the scFv comprises VH1, a second linker, and VL1 from the N-terminus to the C-terminus.

23. The bispecific antibody according to any one of claims 17 to 22, wherein (1) The CL region is kappa CL (Cκ; SEQ ID NO: 19) or lambda CL (Cλ; SEQ ID NO: 20) or a variant thereof having up to 10 amino acid substitutions; and / or (2) The CH region is a human IgG1 CH region (SEQ ID NO: 21), IgG2 CH region (SEQ ID NO: 22), IgG3 CH region (SEQ ID NO: 23) or IgG4 CH region (SEQ ID NO: 24), or a variant thereof having up to 10 amino acid substitutions.

24. The bispecific antibody according to claim 23, wherein (1) the CL region is kappa CL (Cκ; SEQ ID NO: 19); and (2) the CH region is the human IgG2 CH region (SEQ ID NO: 22).

25. The bispecific antibody according to claim 17, wherein C1 and C2 (1) have amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 61, respectively, and (2) have amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 62, respectively.

26. The bispecific antibody according to claim 20, wherein C1 and C2 (1) have amino acid sequences of SEQ ID NO:52 and SEQ ID NO:63, respectively, and (2) have amino acid sequences of SEQ ID NO:52 and SEQ ID NO:64, respectively.

27. A pharmaceutical composition comprising a therapeutically effective amount of any one of claims 1 to 26 of the bispecific antibody and a pharmaceutically acceptable carrier.

28. A polynucleotide encoding a peptide chain of the bispecific antibody according to any one of claims 3 to 26.

29. The polynucleotide of claim 28, wherein all peptide chains of the bispecific antibody are encoded.

30. The polynucleotides of claim 28, wherein the polynucleotides collectively encode all peptide chains of the bispecific antibody.

31. A vector comprising the polynucleotide of claim 28 or 29.

32. A cell comprising any one of the polynucleotides or multiple polynucleotides according to claims 28 to 30, or the carrier according to claim 31.

33. A method for preparing a bispecific antibody that specifically binds to human RANKL and human sclerosingin, comprising culturing the cells of claim 32 under conditions that allow expression of the bispecific antibody.

34. A method for preventing or treating osteoporosis in a subject of need, comprising administering to the subject a therapeutically effective amount of the bispecific antibody as described in any one of claims 1 to 26.

35. The method of claim 34, wherein the subject suffers from primary osteoporosis.

36. The method of claim 35, wherein the subject suffers from postmenopausal osteoporosis (type I).

37. The method of claim 35, wherein the subject suffers from osteoporosis of the elderly (type II).

38. The method of claim 34, wherein the subject suffers from secondary osteoporosis.

39. The method of claim 38, wherein the osteoporosis is secondary to endocrine or metabolic causes, collagen or genetic disorders, drug-related causes, or nutritional causes.

40. The method according to any one of claims 34 to 39, wherein the object is a human being.

41. Use of the bispecific antibody according to any one of claims 1 to 26 as a medicament.

42. Use of the bispecific antibody according to any one of claims 1 to 26 in the prevention or treatment of osteoporosis.

43. Use of the bispecific antibody according to any one of claims 1 to 26 in the preparation of a medicament for the prevention or treatment of osteoporosis.

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