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WO2025036493A1 - Anticorps ciblant les protéines rankl et sclérostine et leurs utilisations - Google Patents

Anticorps ciblant les protéines rankl et sclérostine et leurs utilisations Download PDF

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Publication number
WO2025036493A1
WO2025036493A1 PCT/CN2024/112831 CN2024112831W WO2025036493A1 WO 2025036493 A1 WO2025036493 A1 WO 2025036493A1 CN 2024112831 W CN2024112831 W CN 2024112831W WO 2025036493 A1 WO2025036493 A1 WO 2025036493A1
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region
seq
bispecific antibody
nos
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WO2025036493A9 (fr
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Chi Ho CHONG
Shui On LEUNG
Pang Sum CHIU
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Sinomab Bioscience Ltd
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Sinomab Bioscience Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to molecular biology and cell biology.
  • Provided herein include bispecific antibodies that specifically bind to both human RANKL and human sclerostin and uses thereof in, for example, reducing osteoporosis.
  • Osteoporosis is a debilitating disease in humans characterized by marked decreases in skeletal bone mass and mineral density, structural deterioration of bone including degradation of bone microarchitecture and corresponding increases in bone fragility and susceptibility to fracture in afflicted individuals. Dysregulated function of both osteoclast and osteoblast contribute to osteoporosis. Current strategies for the prevention and treatment of osteoporosis target either osteoclast or osteoblast, which may offer some benefit to individuals but either cannot ensure resolution of the condition or are associated with undesirable side effects. As such, there is an urgent unmet need for additional therapeutic treatment options for osteoporosis.
  • the compositions and methods provided in this disclosure address this need and provide related advantages.
  • bispecific antibodies that can simultaneously bind to human RANKL and human sclerostin.
  • the antibodies disclosed herein can effectively block osteoclast differentiation and promote osteoblast functions.
  • 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 VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 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 VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH2 comprises VH CDR1, V
  • 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) ; (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.
  • C1 comprising, from N-terminus to C-terminus, VL1, and a light chain constant region
  • C2 comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain 1 (CH1) , and a Knob-Fc region
  • C3 comprising, from N-terminus to C-terminus, a single chain variable fragment
  • 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) ; (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.
  • C1 comprising, from N-terminus to C-terminus, VL1, and a light chain constant region (CL)
  • C2 comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain 1 (CH1) , and a Hole-Fc region
  • C3 comprising, from N-terminus to C-terminus, a single
  • 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.
  • 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) ; (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.
  • C1 first peptide chain
  • C2 comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1)
  • CH1 heavy chain constant domain 1
  • Knob-Fc region a Knob-Fc region
  • 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) ; (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.
  • C1 first peptide chain
  • C2 comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1) , and a Hole-Fc region
  • C3 comprising, from N-terminus to C-terminus, a single chain variable fragment (scFv) comprising VL1 and VH1, a linker and
  • 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.
  • the Knob-Fc region is a human IgG2 Fc region variant having up to ten amino acids substitutions, including a T366W substitution; and the Hole-Fc region is a human IgG2 Fc region variant having up to ten amino acids substitutions, including T366S, L368A, Y407V substitutions.
  • the Knob-Fc region further comprises S354C substitution, and the Hole-Fc region further comprises Y349C substitution.
  • the Knob-Fc region further comprises Y349C substitution, and the Hole-Fc region further comprises S354C substitution.
  • the CL region is kappa CL (C ⁇ ; SEQ ID NO: 19) or lambda CL (C ⁇ ; SEQ ID NO: 20) , or a variant thereof having up to ten amino acids substitutions;
  • the CH1 domain is human IgG2 CH1 domain (SEQ ID NO: 36) or a variant thereof having up to ten amino acids substitutions; and/or
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of (1) SEQ ID NOs: 29 and 32, respectively, (2) SEQ ID NOs: 30 and 33, respectively, or (3) SEQ ID NOs: 31 and 34, respectively, or a variant thereof having up to ten amino acids substitutions.
  • the CL region, CH1 domain, the Knob-Fc region and the Hole-Fc region have the amino acid sequences of (1) SEQ ID NOs: 19, 36, 29 and 32, respectively, (2) SEQ ID NOs: 19, 36, 30 and 33, respectively, or (3) SEQ ID NOs: 19, 36, 31 and 34, respectively.
  • the C1, C2, and C3 have amino acid sequences of (1) SEQ ID NOs: 51, 53, and 54, respectively, (2) SEQ ID NOs: 51, 55, and 56, respectively, (3) SEQ ID NOs: 51, 57, and 58, respectively; or (4) SEQ ID NOs: 51, 59 and 60, respectively.
  • bispecific antibodies comprising a VL1/VH1 pair specifically binds to human RANKL and a VL2/VH2 pair that specifically binds to human sclerostin.
  • 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.
  • 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.
  • 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.
  • the scFv comprises, from N-terminus to C-terminus, VL1, a second linker, and VH1.
  • the scFv comprises, from N-terminus to C-terminus, VH1, a second linker, and VL1.
  • the CL region is kappa CL (C ⁇ ; SEQ ID NO: 19) or lambda CL (C ⁇ ; SEQ ID NO: 20) , or a variant thereof having up to ten amino acids substitutions; or (2) the CH region is 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 ten amino acids substitutions; or both (1) and (2) .
  • the CL region is kappa CL (C ⁇ ; SEQ ID NO: 19)
  • the CH region is human IgG2 CH region (SEQ ID NO: 22) .
  • the C1 and C2 have amino acid sequences of (1) SEQ ID NOs: 51 and 61, respectively, (2) SEQ ID NOs: 51 and 62, respectively. In some embodiments, the C1 and C2 have amino acid sequences of (1) SEQ ID NOs: 52 and 63, respectively, (2) SEQ ID NOs: 52 and 64, respectively.
  • compositions comprising a therapeutically effective amount of the bispecific antibody described herein and a pharmaceutically acceptable carrier.
  • polynucleotides encoding a peptide chain of the bispecific antibody described herein.
  • the polynucleotide provided herein encodes all peptide chains of the bispecific antibody.
  • provided herein are also a plurality of polynucleotides that collectively encode all peptide chains of the bispecific antibody.
  • vectors comprising the polynucleotide described herein.
  • cell comprising the polynucleotide or plurality of polynucleotides described herein.
  • bispecific antibody that specifically binds to human RANKL and human sclerostin comprising culturing the cell described herein under conditions that allow expression of the bispecific antibody.
  • the subject has primary osteoporosis.
  • the subject has postmenopausal osteoporosis (Type I) .
  • the subject has senile osteoporosis (Type II) .
  • the subject has secondary osteoporosis.
  • methods provided herein prevent or treat osteoporosis that is secondary to an endocrine or metabolic cause, a collagen or genetic disorder, a medication cause, or a nutritional cause.
  • the subject is a human.
  • bispecific antibodies described herein as a medicament.
  • bispecific antibodies described herein in preventing or treating osteoporosis.
  • bispecific antibodies described herein for the preparation of a medicament for preventing or treating osteoporosis.
  • FIGs. 1A-1B provide schematic diagrams of bispecific antibodies provided herein that specifically bind to RANKL and sclerostin ( “DRAbs” ) .
  • FIG. 1A provides exemplary depictions of the bispecific antibody in knobs-into-holes (KIH) configuration.
  • FIG. 1B provides an exemplary depiction of the bispecific antibody in IgG-scFv configuration.
  • FIG. 2 provides ELISA results showing the specific binding of the DRAbs DR-P7, DR-P15 and DR-P16 to human RANKL (upper panel) and sclerostin (lower panel) .
  • FIG. 3 provides the results of a TRACP assay demonstrating that the DRAbs DR-P7 and DR-P8 effectively blocked osteoclast differentiation.
  • FIG. 4 provides the results of an assay measuring the expression of marker genes (MMP9, FATc1, Acp5, CTSK) for osteoclast differentiation, which demonstrated that the DRAbs DR-P7 and DR-P8 effectively blocked expression of these marker genes.
  • marker genes MMP9, FATc1, Acp5, CTSK
  • FIG. 5 provides the results of a nanoluciferase assay using HEK293 cells, which demonstrated that the DRAbs DR-P7 and DR-P8 effectively blocked sclerostin-mediated inhibition on WNT signaling.
  • FIG. 6 provides the results in a mouse model with ovariectomy showing that the DRAb DR-P7 effectively promoted bone re-growth in mice, and synergistic effect was observed with the DRAb as compared to antibody that targeted RANKL or sclerostin alone.
  • OVX ovariectomy
  • Tb. N trabecular number
  • Tb. Sp trabecular separation.
  • bispecific antibodies that specifically bind to both human RANKL and to human sclerostin ( “DRAbs” ) .
  • Pharmaceutical compositions comprising a therapeutically effective amount of such antibodies are also disclosed herein. Also disclosed herein are uses of such antibodies and pharmaceutical compositions for reducing osteoporosis and/or for increasing bone mass.
  • Osteoporosis is a skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to increased bone fragility and susceptibility to fractures. It is a systemic condition that affects the entire skeleton, resulting in a reduction in bone mineral density and structural integrity. Osteoporosis can be classified into two main types: primary osteoporosis and secondary osteoporosis.
  • Primary osteoporosis mainly includes postmenopausal osteoporosis (Type I) or senile osteoporosis (Type II) .
  • Postmenopausal osteoporosis Type I
  • Estrogen plays a vital role in maintaining bone density. The loss of estrogen leads to accelerated bone resorption, causing increased bone fragility.
  • Senile osteoporosis Type II
  • Primary osteoporosis also includes idiopathic osteoporosis, which refers to cases of osteoporosis that occur without an apparent cause. It can affect individuals of any age and may have a genetic component.
  • Secondary osteoporosis refers to a condition characterized by decreased bone density and strength as a result of underlying medical conditions 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.
  • Endocrine or metabolic causes are one of the main categories of secondary osteoporosis.
  • Conditions such as hyperparathyroidism, hyperadrenocorticism, and hyperprolactinemia can disrupt the normal balance of hormones involved in bone remodeling.
  • excessive production of parathyroid hormone in hyperparathyroidism leads to increased bone resorption, resulting in decreased bone mineral density and increased fracture risk.
  • Collagen/genetic disorders are another group of conditions associated with secondary osteoporosis. Disorders like 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. Altered collagen synthesis or structure can weaken the bone matrix, making the bones more susceptible to fractures and reducing bone density.
  • Certain medications have been linked to secondary osteoporosis due to their effects on bone metabolism. For instance, long-term use of glucocorticoids like prednisone can disrupt the balance between bone formation and resorption. Glucocorticoids inhibit 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 contribute to bone loss and increased fracture risk.
  • Nutritional deficiencies and lifestyle factors can also contribute to the development of secondary osteoporosis.
  • Alcoholism for example, can have a detrimental effect 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, necessary for calcium absorption.
  • Malabsorption syndromes where the body is unable to absorb nutrients properly, can lead to inadequate calcium and vitamin D levels, affecting bone health.
  • Bone tissue is constantly being remodeled through a dynamic process involving two main cell types: osteoblasts and osteoclasts. Osteoblasts are responsible for bone formation, while osteoclasts are involved in bone resorption. Together, these cells maintain a delicate balance between bone formation and bone resorption, known as bone remodeling. In osteoporosis, this balance is disrupted, leading to an increase in bone resorption and a decrease in 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 a weakening of the skeletal structure.
  • Osteoblasts play a crucial role in bone formation. These cells are responsible for synthesizing and depositing new bone matrix, consisting primarily of collagen and other proteins. They also facilitate the mineralization process by promoting the accumulation of calcium and phosphate ions within the bone tissue. When osteoblast activity is impaired or inadequate, as in osteoporosis, compromised bone formation leads to reduced bone density and increased susceptibility to fractures. Osteoblastic functions require the activation of bone morphogenetic protein (BMP) , TGF ⁇ , and canonical WNT signaling. Key regulators for WNT signaling include sclerostin, Dickkopf 1 and secreted Frizzled-related protein 1. Estrogen promotes osteogenic differentiation of mesenchymal stem cells.
  • BMP bone morphogenetic protein
  • TGF ⁇ transforming growth factor
  • WNT signaling Key regulators for WNT signaling include sclerostin, Dickkopf 1 and secreted Frizzled-related protein 1. Estrogen promotes osteogenic differentiation of mesenchymal stem cells.
  • Estrogen also stimulates the production of IGF1 and TGF ⁇ by osteoblasts as well as pro-collagen synthesis.
  • Sclerostin is a protein secreted primarily by osteocytes, which are embedded within the mineralized bone tissue. Its main function is to inhibit the activity of osteoblasts and bone formation. Sclerostin acts by binding to specific receptors on osteoblasts, thereby interfering with the intracellular signaling pathways that promote osteoblast differentiation and function. By inhibiting osteoblast function, sclerostin indirectly affects osteoclast activity and bone resorption.
  • Osteoclasts are multinucleated cells derived from monocytes and are responsible for bone resorption. They secrete enzymes and acids that break down the mineralized bone matrix, allowing for the release of calcium and other minerals into the bloodstream. In osteoporosis, osteoclasts become hyperactive and excessively resorb bone, further contributing to the loss of bone mass and density. Osteoclast differentiation and bone resorption are controlled by IL-1, TNF- ⁇ , TGF ⁇ , M-CSF and RANK-RANKL-osteoprotegerin pathway, in which RANKL (Receptor Activator of Nuclear Factor ⁇ B Ligand) plays a pivotal role.
  • RANKL binds to RANK on osteoclast precursor cell and activates MAPK and NF- ⁇ B pathway via TRAF6 and TAK1. It promotes the differentiation of osteoclast precursor cells into mature osteoclasts. Once activated, 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.
  • Osteoporosis treatment includes the use of medications called RANKL inhibitors. These drugs essentially block the interaction between RANKL and its receptor on osteoclasts, thereby preventing the stimulation of osteoclast formation and activity. By decreasing osteoclast-mediated bone resorption, RANKL inhibitors help to preserve bone mass and reduce the risk of fractures in individuals with osteoporosis.
  • Another approach in osteoporosis treatment involves the use of drugs that neutralize or inhibit sclerostin. By blocking the inhibitory effects of sclerostin, these medications allow osteoblasts to function more effectively, promoting bone formation and bone mineral density. Augmenting osteoblast activity can help offset the excessive bone resorption seen in osteoporosis and improve overall bone health.
  • bispecific antibodies that specifically bind to both human RANKL and to human sclerostin ( “DRAbs” ) .
  • DRAbs human sclerostin
  • a dual-action approach is achieved, addressing both excessive bone resorption and impaired bone formation seen in osteoporosis.
  • This dual inhibition approach offers the advantage of a more comprehensive intervention, targeting multiple aspects of bone remodeling simultaneously. It not only slows down bone loss but also promotes bone formation, leading to the restoration of bone strength and reducing fracture risk.
  • the DRAbs described herein show synergistic effect in enhancing bone formation, which provide greater efficacy in treating osteoporosis compared to targeting either RANKL or sclerostin alone.
  • an entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies.
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .
  • the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
  • the term “about” encompasses the exact number recited.
  • “about” means within plus or minus 10%of a given value or range.
  • “about” means that the variation is ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, ⁇ 0.2%, or ⁇ 0.1%of the value to which “about” refers.
  • “about” means that the variation is ⁇ 1%, ⁇ 0.5%, ⁇ 0.2%, or ⁇ 0.1%of the value to which “about” refers.
  • peptide chain refers to polymers of amino acids of any length, which can be linear or branched. It can include unnatural or modified amino acids or be interrupted by non-amino acids.
  • a polypeptide, peptide, polypeptide chain, peptide chain, or protein can also be modified with, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polynucleotide, ” “nucleic acid, ” and their grammatical equivalents as used interchangeably herein mean polymers 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.
  • variant refers to a different protein or polypeptide having one or more (such as, for example, 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 changes to an amino acid sequence can be amino acid substitutions.
  • the changes to an amino acid sequence can be conservative amino acid substitutions.
  • a functional fragment or a functional variant of a protein or polypeptide maintains the basic structural and functional properties of the reference protein or polypeptide.
  • binding moiety e.g., antibody
  • target molecule e.g., antigen
  • a binding moiety e.g., antibody
  • BBI Bio-Layer Interferometry
  • SPR e.g., Biacore
  • a specific reaction will be at least twice background signal or noise and can be more than 10 times background.
  • “specifically binds” means, for instance, that a binding moiety binds a molecule target with a K D of about 0.1 mM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a K D of at about 10 ⁇ M or less or about 1 ⁇ M or less.
  • “specifically binds” means that a polypeptide or molecule binds a target with a K D of at about 0.1 ⁇ M or less, about 0.01 ⁇ M or less, or about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target.
  • binding affinity generally refers to the strength of the sum total of noncovalent interactions between a binding moiety and a target molecule (e.g., antigen) .
  • the binding of a binding moiety and a target molecule is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (K D ) .
  • K D is the ratio of a dissociation rate (k off or k d ) to the association rate (k on or k a ) .
  • K D is the ratio of a dissociation rate (k off or k d ) to the association rate (k on or k a ) .
  • the lower the K D of a binding pair the higher the affinity.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following.
  • the “K D ” or “K D value” can be measured by assays known in the art, for example by a binding assay.
  • the K D may be measured in a radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293: 865-881) .
  • the K D or K D value can also be measured by using biolayer interferometry (BLI) using, for example, the Gator system (Probe Life) , or the Octet-96 system (Sartorius AG) .
  • the K D or K D value can also be measured by using surface plasmon resonance assays (SPR) by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 BIAcore, Inc., Piscataway, NJ) .
  • SPR surface plasmon resonance assays
  • the binding affinity can also be quantified with EC 50 , which is the concentration of ligand at which half of the target is present in the bound state in a binding assay.
  • nucleotide, % “identity, ” and their grammatical equivalents as used herein in the context of two or more polynucleotides or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art.
  • two polynucleotides or polypeptides provided herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between.
  • identity exists over a longer region than 60-80 bases, such as at least about 80-100 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
  • vector refers to a vehicle that is used to carry genetic material (e.g., a polynucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed.
  • vectors applicable for use 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 a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selection control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • both polynucleotides can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding polynucleotides can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • polynucleotides into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotides are expressed in a sufficient amount to produce a desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • the term “encode” and its grammatical equivalents refer to the inherent property of specific sequences of nucleotides in a polynucleotide or a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein.
  • nucleotide sequence encoding 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.
  • a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • GenBank numbers GI numbers and/or SEQ ID NOS. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .
  • bispecific antibodies that specifically bind to both human RANKL and to human sclerostin.
  • the bispecific antibodies provided herein are monoclonal antibodies.
  • the bispecific antibodies provided herein are isolated.
  • the bispecific antibodies provided herein are substantially pure.
  • an “antibody” is an immunoglobulin molecule that recognizes and specifically binds 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 many different types and structures. For example, antibodies can be polyclonal antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, or any other modified immunoglobulin molecule comprising an antigen-binding site. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
  • An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) , based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the term “antibody” as used herein include “antigen-binding fragment” of intact antibodies.
  • the term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigenic determining variable region of an intact antibody.
  • 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 (HCAbs) , light chain antibodies (LCAbs) , disulfide-linked scFv (dsscFv) , diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD) , single variable domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , and single variable domain of heavy chain antibodies (VHH) .
  • scFv single chain antibody molecules
  • HCAbs heavy chain antibodies
  • LCAbs light chain antibodies
  • dsscFv disulfide-linked scFv
  • diabodies tribodies
  • tetrabodies minibodies
  • DVD dual variable domain antibodies
  • sdAbs single variable domain antibodies
  • bispecific antibody is an artificial hybrid antibody having two different antigen binding fragments.
  • the two different antigen binding fragments specifically bind two different target antigens.
  • Bispecific antibodies can be formed from antibody fragments.
  • immunoglobulins have been well characterized (see, e.g., FUNDAMENTAL IMMUNOLOGY Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989) ) .
  • immunoglobulins comprise 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 inter-connected by disulfide bonds.
  • Each light chain of an immunoglobulin typically includes a light chain variable region ( “VL region” ) and a light chain constant region ( “CL region” ) .
  • VL region variable region
  • CL region light chain constant region
  • ⁇ kappa
  • ⁇ lambda
  • the amino acid sequences of the CL regions are well known in the art.
  • Each heavy chain typically includes a heavy chain variable region (a “VH region” ) and a heavy chain constant region (a “CH region” ) .
  • the VH region can be one of five distinct types, referred to as alpha ( ⁇ ) , delta ( ⁇ ) , epsilon ( ⁇ ) , gamma ( ⁇ ) and mu ( ⁇ ) , based on the amino acid sequence.
  • these distinct types of heavy chains give rise to five well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively.
  • There are four subclasses of IgG namely, IgG1, IgG2, IgG3 and IgG4.
  • the amino acid sequences of the CH regions of different classes of antibodies are well known in the art.
  • the CH region of immunoglobulins comprise more than one domain.
  • the CH region of an IgG antibody is comprised of three domains, heavy chain constant domain 1 (CH1) , heavy chain constant domain 2 (CH2) , and heavy chain constant domain 3 (CH3) .
  • CH1 and CH2 domains The highly flexible region between the CH1 and CH2 domains is referred to as the “hinge region. ” Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an immunoglobulin.
  • the “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the Fc region is comprised of the CH2 domain and the CH3 domain; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2–4) .
  • the amino acid sequences of the Fc region of human IgG, IgA, IgD, IgM and IgE, and subtypes IgG1, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art.
  • the Fc region of an IgG heavy chain extends from the hinge region to the carboxyl-terminus of the heavy chain.
  • the native Fc regions can be modified. Modification of the Fc regions are further described below.
  • a bispecific antibody provided herein can comprise paired Fc domains comprising paired different modifications that promote their association with each other, instead of forming homodimers.
  • variable region refers to a portion of the light or heavy chains of an immunoglobulin that is generally located at the amino-terminal of the light or heavy chain and used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable region of a light chain is referred to as a “light chain variable region” or “VL region, ” which includes at least one, typically one, “light chain variable domain” or “VL. ”
  • the variable region of a heavy chain is referred to as a “heavy chain variable region” or “VH region, ” which includes at least one, typically one, “heavy chain variable domain” or “VH. ”
  • the variable domains differ extensively in sequence between different antibodies.
  • a “pair of VL and VH” can associate with each other and form a binding site that specifically binds the target antigen or epitope.
  • the VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops) , also termed complementarity determining regions (CDRs) , interspersed with regions that are more conserved, termed framework regions (FRs) .
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR) .
  • the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen.
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, J Mol Biol 1987; 196: 901-17) .
  • a CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH ⁇ -sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL ⁇ -sheet framework.
  • CDR regions are well known to those skilled in the art and have been defined by a variety of methods/systems. These systems and/or definitions have been developed and refined over years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the regions of most hypervariability within the antibody variable (V) domains (Kabat et al, J. Biol. Chem.
  • a single chain Fv ( “scFv” ) polypeptide is a covalently linked VL/VH heterodimer which is usually expressed from a gene fusion including VL and VH-encoding genes linked by a peptide-encoding linker.
  • the scFv fragment includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques.
  • the N-terminus of VL is linked to the C-terminus of the VH via a linker.
  • the N-terminus of VH is linked to the C-terminus of the VL via a linker.
  • linker refers to one or more amino acid residues inserted between domains (e.g., immunoglobulin domains) to provide sufficient mobility for the domains.
  • a linker can be inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level.
  • humanized antibody refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences.
  • humanized antibodies are human immunoglobulins.
  • the variable region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species.
  • residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and/or binding capability.
  • humanized antibody can be further modified by the substitution of additional residues either in the variable region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability.
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.
  • Bispecific antibodies provided herein comprise a first VL and VH pair that specifically bind to human RANKL and a second VL and VH pair that specifically binds to human sclerostin.
  • Exemplary VL/VH pairs that specifically bind to human RANKL/sclerostin are provided below.
  • expressly contemplated also include variants of these VL/VH pairs that retain their bindings to the respective target antigens.
  • bispecific antibodies 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 VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 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 VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH2 comprises VH
  • the VL1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 13. In some embodiments of the bispecific antibodies disclosed herein, the VH1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 14. In some embodiments of the bispecific antibodies disclosed herein, the VL2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 15. In some embodiments of the bispecific antibodies disclosed herein, the VH2 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 16, respectively.
  • the 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.
  • the 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.
  • the bispecific antibodies provided herein comprise a mutation to promote heterodimerization of Fc regions.
  • a dimerized Fc region of a bispecific antibody provided herein is formed by Fc regions that contain amino acid mutations, substitutions, additions, or deletions to promote heterodimerization in which different polypeptides comprising different Fc regions can dimerize to yield a heterodimer configuration.
  • a bispecific antibody of the present disclosure comprises 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 are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions.
  • Methods to promote heterodimerization of Fc regions include amino acid deletions, additions, or substitutions of the amino acid sequence of the Fc region, such as by including a set of “knobs-into-holes” deletions, additions, or substitutions or including amino acid deletions, additions, or substitutions to effect electrostatic steering of the Fc to favor attractive interactions among different polypeptide chains.
  • the modification promoting the association of a pair of Fc domains in a bispecific antibody includes the so-called “knobs-into-holes” modification, comprising a “knob” modification in one Fc domain and a “hole” modification in the other one.
  • the bispecific antibodies provided herein have complementary Fc polypeptides to form heterodimer of the “knobs-into-holes” configurations or “KIH” configuration.
  • the “knobs-into-holes” technology is described e.g., in 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.
  • the method involves introducing a protuberance ( “knob” ) at the interface of a first Fc (the “Knob-Fc” ) and a corresponding cavity ( “hole” ) in the interface of a second Fc (the “Hole-Fc” ) , such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • a “Knob-Fc region” and a “Hole-Fc region” are designed to form heterodimer pair.
  • the Knob-Fc region refers to the Fc region in which an amino acid of the CH3 domain is replaced with an amino acid residue having a larger side chain volume, generating a protuberance within the CH3 domain positionable in a cavity within the CH3 domain of the Hole-Fc region, in which an amino acid residue of the CH3 domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain within which the protuberance within the CH3 domain of the first subunit is positionable.
  • said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R) , phenylalanine (F) , tyrosine (Y) , and tryptophan (W) .
  • said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A) , serine (S) , threonine (T) , and valine (V) .
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.
  • the threonine residue at position 366 of the Knob-Fc region is replaced with a tryptophan residue (T366W)
  • the tyrosine residue at position 407 of the Hole-Fc region is replaced with a valine residue (Y407V)
  • 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) .
  • a first Fc polypeptide comprises amino acid modifications to form the “knob” and a second Fc polypeptide comprises amino acid modifications to form the “hole” thus forming an Fc heterodimer comprising complementary Fc polypeptides.
  • the Knob-Fc region additionally has the serine residue at position 354 replaced with a cysteine residue (S354C) , or the glutamic acid residue at position 356 replaced with a cysteine residue (E356C) , and the Hole-Fc region additionally has the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) .
  • the Hole-Fc region additionally has the serine residue at position 354 replaced with a cysteine residue (S354C) , or the glutamic acid residue at position 356 replaced with a cysteine residue (E356C)
  • the Knob-Fc region additionally has the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C)
  • the Knob-Fc region contains the amino acid substitutions S354C and T366W
  • the Hole-Fc region contains the amino acid substitutions Y349C, T366S, L368A and Y407V.
  • the Knob-Fc region contains the amino acid substitutions Y349C and T366W, and the Hole-Fc region contains the amino acid substitutions S354C, T366S, L368A and Y407V. All amino acid residues are numbered according to the EU index.
  • the bispecific antibodies provided herein have the “knobs-into-holes” or “KIH” structure (e.g., FIG. 1A) .
  • the “KIH” model promotes formation of heterodimers of the engineered bispecific antibody instead of heavy chain homodimers.
  • complementary Fc polypeptides of an Fc heterodimer include a mutation to alter charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc regions support favorable attractive interactions, thereby promoting desired Fc heterodimer formation; whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation (Guneskaran et al., 2010, J Biol Chem, 285: 19637-19646) . When co-expressed in a cell, association between the polypeptide chains is possible but the chains do not substantially self-associate due to charge repulsion.
  • the bispecific antibodies disclosed herein can comprise complementary Fc regions having the modifications described in Table 4 above.
  • the bispecific antibodies disclosed herein comprises a first CH region and a second CH region comprising paired Fc modification described in Table 4 above.
  • the first CH region and the second CH region of the bispecific antibodies disclosed herein have T366Y substitution and Y407T, respectively, or vice versa.
  • the first CH region and the second CH region of the bispecific antibodies disclosed herein have T366W substitution and T366S/L368W/Y407V substitutions, respectively, or vice versa.
  • the first CH region and the second CH region of the bispecific antibodies disclosed herein have T366W substitution and T366S/L368A/Y407V substitutions, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have T366W/S354C substitutions and T366S/L368A/Y407V/Y349C substitutions, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have T366W/E356C substitutions and T366S/L368A/Y407V/Y349C substitutions, respectively, or vice versa.
  • first CH region and the second CH region of the bispecific antibodies disclosed herein have T350V/L351Y/F405A/Y407V substitutions and T350V/T366L/K392L/T394W substitutions, respectively, or vice versa.
  • first CH region and the second CH region of the bispecific antibodies disclosed herein have K360D/D399M/Y407A substitutions and E345R/Q347R/T366V/K409V substitutions, respectively, or vice versa.
  • the first CH region and the second CH region of the bispecific antibodies disclosed herein have K409D/K392D substitutions and D399K/E356K substitutions, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have K360E/K409W substitutions and Q347R/D399V/F405T substitutions, respectively, or vice versa. In some embodiments, the first CH region and the second CH region of the bispecific antibodies disclosed herein have L360E/K409W/Y349C substitutions and Q347R/D399V/F405T/S354C substitutions, respectively, or vice versa.
  • the first CH region and the second CH region of the bispecific antibodies disclosed herein have S364H/F405A substitutions and Y349T/T394F substitutions, respectively, or vice versa.
  • the first CH region and the second CH region of the bispecific antibodies disclosed herein have D221E/P228E/L368E substitutions and D221R/P228R/K409R substitutions, respectively, or vice versa.
  • the first CH region and the second CH region of the bispecific antibodies disclosed herein have F405L substitution and K409R substitution, respectively, or vice versa.
  • the first CH region and the second CH region of the bispecific antibodies disclosed herein have Y349S/K307Y/T366M/K409V substitutions and E356G/E357D/S364Q/Y407A substitutions, respectively, or vice versa.
  • one of the CH regions further has H435R and Y436F substitutions.
  • the second CH region further has H435R and Y436F substitutions.
  • bispecific antibodies 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 (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.
  • VL1 first light chain variable domain
  • VH1 first heavy chain variable domain
  • VL2 second light chain variable domain
  • VH2 second heavy chain variable domain
  • 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 Knob-Fc region; and (3) a third peptide chain (C3) comprising, from N-terminus to C-terminus, a linker, a single chain variable fragment (scFv) comprising VL2 and VH2, a linker, and a Hole-Fc region.
  • the Knob-Fc and Hole-Fc can switch places.
  • C2 in the C2 and C3 pair of the bispecific antibodies that adopts the KIH design, C2 can includes a Knob-Fc region and the C3 can include a Hole-Fc region; alternatively, in some embodiments, the C2 can include a Hole-Fc region and the C3 can include a Knob-Fc region.
  • 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.
  • C1 comprising, from N-terminus to C-terminus, VL1, and a light chain constant region
  • C2 comprising, from N-terminus to C-terminus, VH1, a heavy chain constant domain 1 (CH1) , and a Hole-Fc region
  • C3 comprising, from N-terminus to C-terminus
  • VL can be positioned at the N-terminal end of VH, or vice versa.
  • C3 comprises an scFv having VL2 and VH2
  • the scFv can comprise, from N-terminus to C-terminus, VL2, a second linker, and VH2.
  • the scFv comprises, from N-terminus to C-terminus, VH2, a second linker, and VL2.
  • bispecific antibodies comprising a VL1/VH1 pair that specifically binds to human RANKL and a VL2/VH2 pair that specifically binds to human sclerostin.
  • the bispecific antibodies take the KIH configuration with an scFv on the second heavy chain.
  • the scFv comprises the VL2/VH2 pair that specifically binds to human sclerostin.
  • the scFv comprises the VL1/VH1 pair that specifically binds to human RANKL.
  • 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.
  • C1 first peptide chain
  • VH2 light chain constant region
  • CH1 heavy chain constant domain 1
  • Knob-Fc region a Knob-Fc region
  • 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.
  • C1 first peptide chain
  • VH2 light chain constant region
  • CH1 heavy chain constant domain 1
  • C3 a third peptide chain
  • 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.
  • the bispecific antibodies provided herein in KIH configuration comprise 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, the CL region, and the Fc region of the bispecific antibodies disclosed herein can be derived from any appropriate source, e.g., a constant region of an antibody such as an IgG1, IgG2, IgG3, or IgG4.
  • Antibody heavy and light chain constant regions amino acid sequences are well known in the art, e.g., those provided in the IMGT database (www. imgt. org) or at www. vbase2. org/vbstat. php., both of which are incorporated by reference herein.
  • 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 IgG1. 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.
  • the amino acid sequences of the CH1, the CL region, and the Fc region (hinge, CH2 and CH3) of the bispecific antibodies disclosed herein can comprise one or more amino acid substitutions that differ from the wild type immunoglobulin.
  • substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US 8323962, US6737056, and US7416727) .
  • the CH1 domain of the bispecific antibodies provided herein can be selected from the group consisting of a human IgG1 CH1 domain (SEQ ID NO: 35) , a human IgG2 CH1 domain (SEQ ID NO: 36) , a human IgG3 CH1 domain (SEQ ID NO: 37) , and a human IgG4 CH1 domain (SEQ ID NO: 38) .
  • the CH1 domain can be a human IgG1 CH1 domain (SEQ ID NO:35) or a variant thereof having up to ten amino acids substitutions.
  • the CH1 domain can be a human IgG2 CH1 domain (SEQ ID NO: 36) or a variant thereof having up to ten amino acids substitutions.
  • the CH1 domain can be a human IgG3 CH1 domain (SEQ ID NO: 37) or a variant thereof having up to ten amino acids substitutions.
  • the CH1 domain can be a human IgG4 CH1 domain (SEQ ID NO: 38) or a variant thereof having up to ten amino acids substitutions.
  • the CL region of the bispecific antibodies provided herein can be kappa CL (C ⁇ ; SEQ ID NO: 19) . In some embodiments, the CL region of the bispecific antibodies provided herein can be lambda CL (C ⁇ ; SEQ ID NO: 20) .
  • the Fc regions of the bispecific antibodies provided herein can be variants of the Fc region of human IgG2.
  • the Knob-Fc region is a human IgG2 Fc region variant having up to ten amino acids substitutions, including a T366W substitution; and the Hole-Fc region is a human IgG2 Fc region variant having up to ten amino acids substitutions, including a Y407V substitution.
  • the Hole-Fc region can further have T366S and L368A substitutions.
  • the Knob-Fc and Hole-Fc regions can further include S354C and Y349C substitutions, respectively.
  • the Knob-Fc and Hole-Fc regions can further include Y349C and S354C substitutions, respectively. All amino acid residues are numbered according to the EU Index.
  • the Knob-Fc region and the Hole-Fc region can have the amino acid sequences of (1) SEQ ID NOs: 29 and 32, respectively; (2) SEQ ID NOs: 30 and 33, respectively; or (3) SEQ ID NOs: 31 and 34, respectively.
  • the Knob-Fc region and the Hole-Fc region can have the amino acid sequences of SEQ ID NOs: 29 and 32, respectively.
  • the Knob-Fc region and the Hole-Fc region can have the amino acid sequences of SEQ ID NOs: 30 and 33, respectively.
  • the Knob-Fc region and the Hole-Fc region can have the amino acid sequences of SEQ ID NOs: 31 and 34, respectively.
  • the bispecific antibodies provided herein KIH format also comprise a linker connecting the scFv and the Knob-Fc, specifically, the C-terminus of the scFv and the N-terminus of the Knob-Fc.
  • the linker can be any suitable linker disclosed herein or otherwise known in the art.
  • the linker can be selected from those identified in Table 2.
  • the linker is a GS linker having the amino acid sequence of SEQ ID NO: 41.
  • the GS linker has the amino acid sequence of SEQ ID NO: 42.
  • the GS linker has the amino acid sequence of SEQ ID NO: 43.
  • the GS linker has the amino acid sequence of SEQ ID NO: 44.
  • the linker has the amino acid sequence of SEQ ID NO: 45.
  • the linker can be any suitable linker disclosed herein or otherwise known in the art.
  • the second linker can be selected from those identified in Table 2.
  • the linker is a GS linker having the amino acid sequence of SEQ ID NO: 41.
  • the GS linker has the amino acid sequence of SEQ ID NO: 42.
  • the GS linker has the amino acid sequence of SEQ ID NO: 43.
  • the GS linker has the amino acid sequence of SEQ ID NO: 44.
  • the linker has the amino acid sequence of SEQ ID NO: 45.
  • the CL region is kappa CL (C ⁇ ; SEQ ID NO: 19) or lambda CL (C ⁇ ; SEQ ID NO: 20) , or a variant thereof having up to ten amino acids substitutions;
  • the CH1 domain is human IgG2 CH1 domain (SEQ ID NO: 36) or a variant thereof having up to ten amino acids substitutions; and/or
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of (1) SEQ ID NOs: 29 and 32, respectively, (2) SEQ ID NOs: 30 and 33, respectively, or (3) SEQ ID NOs: 31 and 34, respectively, or a variant thereof having up to ten amino acids substitutions.
  • the CL region, CH1 domain, the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 19, 36, 29 and 32, respectively.
  • the CL region, CH1 domain, the Knob-Fc region and the Hole-Fc region have the amino acid sequences SEQ ID NOs: 19, 36, 30 and 33, respectively.
  • the CL region, CH1 domain, the Knob-Fc region and the Hole-Fc region have the amino acid sequences SEQ ID NOs: 19, 36, 31 and 34, respectively.
  • the CL region is C ⁇ (SEQ ID NO: 19)
  • the CH1 domain is human IgG2 CH1 domain (SEQ ID NO: 36)
  • the Knob-Fc and Hole-Fc have the amino acid sequences of SEQ ID NOs: 29 and 32, respectively.
  • the CL region is C ⁇ (SEQ ID NO: 19)
  • the CH1 domain is human IgG2 CH1 domain (SEQ ID NO: 36)
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 30 and 33, respectively.
  • the CL region is C ⁇ (SEQ ID NO: 19)
  • the CH1 domain is human IgG2 CH1 domain (SEQ ID NO: 36)
  • the Knob-Fc region and the Hole-Fc region have the amino acid sequences of SEQ ID NOs: 31 and 34, respectively.
  • Table 6A Provided in Table 6A are diagrams of the three peptide chains of exemplary bispecific antibodies in KIH format.
  • VL1/VH1 pair specifically binds to RANKL (e.g., SEQ ID NOs: 13 and 14) ; the VL2/VH2 specifically binds to sclerostin (e.g., SEQ ID NOs: 15 and 16) ; C ⁇ refers to the kappa CL.
  • L Linker
  • bispecific antibody that specifically bind to human RANKL and human sclerostin, wherein the bispecific antibody has a first peptide chain (C1) , a second peptide chain (C2) , and a third peptide chain (C3) , wherein C1, 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 NOs: 51, 53, and 54, respectively; (2) SEQ ID NOs: 51, 55, and 56, respectively; (3) SEQ ID NOs: 51, 57, and 58, respectively; (4) SEQ ID NOs: 51, 59, and 60, respectively.
  • C1, 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 NOs: 51, 53, and 54, respectively. In some embodiments, C1, 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 NOs: 51, 55, and 56, respectively. In some embodiments, C1, 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 NOs: 51, 57, and 58, respectively. In some embodiments, C1, 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 NOs: 51, 59, and 60, respectively.
  • the bispecific antibodies provided herein have the “IgG-scFv” structure depicted in FIG. 1B.
  • bispecific antibodies 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 (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.
  • the bispecific antibodies in IgG-scFv format comprises 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.
  • the bispecific antibodies in IgG-scFv format comprises 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.
  • the bispecific antibodies provided herein in 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 derived from any appropriate source, e.g., a constant region of an antibody such as an IgG1, IgG2, IgG3, or IgG4.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG1.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG2.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG3.
  • the constant regions of the bispecific antibodies provided herein are derived from human IgG4.
  • 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 that differ from the wildtype immunoglobulin. Such substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US 8323962, US6737056, and US7416727) .
  • the CL region of the bispecific antibodies provided herein can be kappa CL (C ⁇ ; SEQ ID NO: 19) , or a variant thereof having up to ten amino acids substitutions. In some embodiments, the CL region of the bispecific antibodies provided herein can be lambda CL (C ⁇ ; SEQ ID NO: 20) , or a variant thereof having up to ten amino acids substitutions.
  • the CH region of the bispecific antibodies provided herein can be selected from the group consisting of a human IgG1 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 ten amino acid substitutions.
  • the CH region is human IgG2 CH region (SEQ ID NO: 22) or a variant thereof having up to ten amino acid substitutions.
  • the CL region is C ⁇ (SEQ ID NO: 19) or C ⁇ (SEQ ID NO: 20) , or a variant thereof having up to ten amino acids substitutions; or (2) the CH region is 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 ten amino acids substitutions; or both (1) and (2) .
  • the CL region is C ⁇ (SEQ ID NO: 19)
  • the CH region is human IgG2 CH region (SEQ ID NO: 22) .
  • the bispecific antibodies provided herein in IgG-scFv format also comprise a linker connecting the heavy chain constant region (CH) and the scFv, specifically, the C-terminus of the CH and the N-terminus of the scFv.
  • the linker can be any suitable linker disclosed herein or otherwise known in the art.
  • the linker can be selected from those identified in Table 2.
  • the linker is a GS linker having the amino acid sequence of SEQ ID NO: 41.
  • the GS linker has the amino acid sequence of SEQ ID NO: 42.
  • the GS linker has the amino acid sequence of SEQ ID NO: 43.
  • the GS linker has the amino acid sequence of SEQ ID NO: 44.
  • the linker has the amino acid sequence of SEQ ID NO: 45.
  • the VL2 and VH2 are connected by a second linker.
  • 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.
  • the second linker can be selected from those identified in Table 2.
  • the linker is a GS linker having the amino acid sequence of SEQ ID NO: 41.
  • the GS linker has the amino acid sequence of SEQ ID NO: 42.
  • the GS linker has the amino acid sequence of SEQ ID NO:43.
  • the GS linker has the amino acid sequence of SEQ ID NO: 44.
  • the linker has the amino acid sequence of SEQ ID NO: 45.
  • Table 7A Provided in Table 7A are diagrams of the two peptide chains of exemplary bispecific antibodies in IgG-scFv format.
  • Table 7A Exemplary peptide chains of bispecific antibodies (IgG-scFv)
  • VL1/VH1 pair specifically binds to RANKL (e.g., SEQ ID NOs: 13 and 14) ; the VL2/VH2 specifically binds to sclerostin (e.g., SEQ ID NOs: 15 and 16) ; C ⁇ refers to the kappa CL.
  • L Linker
  • bispecific antibody that specifically bind to human RANKL and human sclerostin, wherein the bispecific antibody has 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 to the amino acid sequences of (1) SEQ ID NOs: 51 and 61 respectively; (2) SEQ ID NOs: 51 and 62 respectively; (3) SEQ ID NOs: 52 and 63 respectively; or SEQ ID NOs: 52 and 64 respectively.
  • C1 and C2 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 NOs: 51 and 61 respectively; (2) SEQ ID NOs: 51 and 62 respectively; (3) SEQ ID NOs: 52 and 63 respectively; or SEQ ID NOs: 52 and 64 respectively.
  • C1 and C2 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 NOs: 51 and 61 respectively. In some embodiments, C1 and C2 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 NOs: 51 and 62 respectively. In some embodiments, C1 and C2 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 NOs: 52 and 63, respectively. In some embodiments, C1 and C2 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 NOs: 52 and 64, respectively.
  • the present disclosure further contemplates additional variants and equivalents that are substantially homologous to the bispecific antibodies described herein.
  • it is desirable to modulate biological properties of the antibody including but not limited to, specificity, thermostability, expression level, effector function (s) , glycosylation, immunogenicity, and/or solubility.
  • amino acid changes may alter post-translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.
  • Antibodies comprising functional variants of the heavy chain, light chains, VL regions, VH regions, or one or more CDRs of the antibodies of the examples as also provided herein.
  • a functional variant of a heavy chain, a light chain, VL, VH, or CDRs used in the context of an antibody still allows the antibody to retain at least a substantial proportion (at least about 90%, 95%or more) of functional features of the “reference” and/or “parent” antibody, including affinity and/or the 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 length of heavy and light chains.
  • Exemplary variants include those which differ from heavy and/or light chains, VH and/or VL, and/or CDR regions of the parent antibody sequences mainly by conservative substitutions, e.g., 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant may be conservative amino acid residue replacements.
  • a variant of a bispecific antibody disclosed herein can retain its ability to bind RANKL and sclerostin to a similar extent, the same extent, or to a higher extent, as the parent bispecific antibody.
  • the variant can be at least 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.
  • a variant of a bispecific antibody disclosed herein comprises the amino acid sequence of the parent a bispecific antibody disclosed herein with one or more conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.
  • a variant of a bispecific antibody disclosed herein comprises the amino acid sequence of the parent antibody with one or more non-conservative amino acid substitutions. In some embodiments, a variant of a bispecific antibody disclosed herein comprises the amino acid sequence of the parent binding antibody with one or more non-conservative amino acid substitution, 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 a biological activity of the variant, such that the biological activity of the functional variant is increased as compared to the parent antibody.
  • the variant can have 1, 2, 3, 4, or 5 amino acid substitutions in the CDRs (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) of the binding moiety.
  • VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 amino acid substitutions in the CDRs (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) of the binding moiety.
  • the bispecific antibodies provided herein include modification in their Fc regions.
  • the modified antibodies e.g., modified Fc region
  • the deletion or inactivation (through point mutations or other means) of a constant region reduces Fc receptor binding of the modified antibody as it circulates.
  • the constant region modifications reduce the immunogenicity of the antibody.
  • the constant region modifications increase the serum half-life of the antibody.
  • the constant region modifications reduce the serum half-life of the antibody.
  • the constant region modifications decrease or remove ADCC and/or complement dependent cytotoxicity (CDC) of the antibody.
  • an antibody does not have one or more effector functions (e.g., “effectorless” antibodies) .
  • the antibody has no ADCC activity and/or no CDC activity.
  • the antibody does not bind an Fc receptor and/or complement factors.
  • the antibody has no effector function (s) .
  • the constant region modifications increase or enhance ADCC and/or CDC of the antibody.
  • the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. In some embodiments, the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.
  • the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor. In some embodiments of the bispecific antibodies provided herein, the Fc domain comprises one or more amino acid substitution that reduces the 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.
  • variants can include addition of amino acid residues at the amino-and/or carboxyl-terminal end of the antibody.
  • the length of additional amino acids residues can range from one residue to a hundred or more residues.
  • a variant comprises an N-terminal methionyl residue.
  • a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., a fluorescent tag or an enzyme) .
  • variant antibodies described herein can be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.
  • bispecific antibodies disclosed herein can be chemically modified naturally or by intervention.
  • 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 numerous chemical modifications can be carried out by known techniques.
  • the bispecific antibodies provided herein can comprise one or more analogs of an amino acid (including, for example, unnatural amino acids) , as well as other modifications known in the art.
  • bispecific antibodies of the present disclosure can be analyzed for their physical, chemical and/or biological properties by various methods known in the art.
  • a bispecific antibody provided herein is tested for its ability to bind human RANKL and sclerostin.
  • Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore) , ELISA, and FACS.
  • antibodies can be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.
  • bispecific antibodies disclosed herein can be conjugated to a detectable substance or molecule that allows the agent to be used for detection.
  • a detectable substance can include, but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine (s) ; fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC) , rhodamine, tetramethylrhodamine isothiocyanate (TRITC) , dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3) , and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials; positron emitting metals; and magnetic metal ions positron emitting metals; and magnetic metal ions.
  • the anti-RANKL/sclerostin bispecific antibodies disclosed herein can be attached to a solid support.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • an immobilized bispecific antibody is used in an immunoassay.
  • an immobilized bispecific antibody is used in purification.
  • polynucleotides encoding at least one light chain or one heavy chain of the anti-RANKL/sclerostin bispecific antibodies disclosed herein.
  • the polynucleotides provided herein encode one polypeptide, such as a light chain or a heavy chain of a bispecific antibody.
  • the polynucleotides provided herein encode more than one polypeptide.
  • the polynucleotides provided herein can encode, for example, the light chain and heavy chain of a bispecific antibody provided herein, respectively.
  • Cistrons can be separated by, for example, an internal ribosomal entry site (IRES) or 2A element.
  • An IRES refers to nucleotide sequences in an expression cassette which when transcribed into mRNA, can recruit ribosomes directly, without a previous scanning of untranslated region of mRNA by the ribosomes.
  • a 2A element as understood in the art, encoding self-cleaving short 2A peptides (about 20 amino acids) that provide a mechanism for subsequent separation of equimolarly produced polypeptides of interest.
  • the family of self-cleaving 2A peptides has been described in the art (see for example, Kim, J.H. et al. (2011) PLoS ONE 6: el8556) .
  • linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure) .
  • linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure) .
  • polycistronic constructs can be suitable for use as provided herein.
  • provided herein are polynucleotides encoding the C1, C2, C3, or any combination thereof, of the bispecific antibody designated as DR-P7. In some embodiments, provided herein are a plurality of polynucleotides that collectively encode the C1, C2, and C3 of the bispecific antibody designated as DR-P7. In some embodiments, provided herein are polynucleotides encoding the C1, C2, C3, or any combination thereof, of the bispecific antibody designated as DR-P8.In some embodiments, provided herein are a plurality of polynucleotides that collectively encode the C1, C2, and C3 of the bispecific antibody designated as DR-P8.
  • provided herein are polynucleotides encoding the C1, C2, C3, or any combination thereof, of the bispecific antibody designated as DR-P15. In some embodiments, provided herein are a plurality of polynucleotides that collectively encode the C1, C2, and C3 of the bispecific antibody designated as DR-P15. In some embodiments, provided herein are polynucleotides encoding the C1, C2, C3, or any combination thereof, of the bispecific antibody designated as DR-P16. In some embodiments, provided herein are a plurality of polynucleotides that collectively encode the C1, C2, and C3 of the bispecific antibody designated as DR-P16.
  • provided herein are polynucleotides encoding the LC, the HC, or both, of the bispecific antibody designated as DR-P25. In some embodiments, provided herein are a first and a second polynucleotides that encode the LC and HC of the bispecific antibody designated as DR-P25, respectively. In some embodiments, provided herein are polynucleotides encoding the LC, the HC, or both, of the bispecific antibody designated as DR-P26. In some embodiments, provided herein are a first and a second polynucleotides that encode the LC and HC of the bispecific antibody designated as DR-P26, respectively.
  • provided herein are polynucleotides encoding the LC, the HC, or both, of the bispecific antibody designated as DR-P27. In some embodiments, provided herein are a first and a second polynucleotides that encode the LC and HC of the bispecific antibody designated as DR-P27, respectively. In some embodiments, provided herein are polynucleotides encoding the LC, the HC, or both, of the bispecific antibody designated as DR-P28. In some embodiments, provided herein are a first and a second polynucleotides that encode the LC and HC of the bispecific antibody designated as DR-P28, respectively.
  • polynucleotide that encode a polypeptide encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.
  • the polynucleotides of the disclosure can be in the form of RNA or in the form of DNA.
  • 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 non-coding (anti-sense) strand.
  • the polynucleotides of the disclosure can be mRNA.
  • the present disclosure also provides variants of the polynucleotides described herein, wherein the variants have a nucleotide sequence 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/sclerostin bispecific antibody described herein.
  • a polynucleotide having a nucleotide sequence at least about 95%identical to a polynucleotide sequence means that the nucleotide sequence of the polynucleotide is identical to a reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence 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 another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted into the reference sequence.
  • These mutations of the reference sequence can occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both.
  • a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide.
  • a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code) .
  • Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) .
  • a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.
  • a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • a polynucleotide comprises the coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a polynucleotide which aids in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide) .
  • the polypeptide can have the leader sequence cleaved by the host cell to form a “mature” form of the polypeptide.
  • a polynucleotide comprises the coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a marker or tag sequence.
  • a marker sequence is a hexa-histidine tag (HIS-tag) that allows for efficient purification of the polypeptide fused to the marker.
  • a marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.
  • the marker sequence is a FLAG TM tag.
  • a marker can be used in conjunction with other markers or tags.
  • a polynucleotide is isolated. In some embodiments, a polynucleotide is substantially pure.
  • vectors comprising a polynucleotide disclosed herein.
  • vehicle and its grammatical equivalents as used herein refer to a vehicle that is used to carry genetic material (e.g., a polynucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed.
  • Vectors applicable for use 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 a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selection control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • both polynucleotides can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding polynucleotides can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • polynucleotides into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotides are expressed in a sufficient amount to produce a desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • vectors provided herein can be expression vectors.
  • vectors provided herein comprise a polynucleotide encoding at least one polypeptide chain of the anti-RANKL/sclerostin bispecific antibodies described herein.
  • recombinant expression vectors which can be used to amplify and express a polynucleotide encoding at least one polypeptide chain of the anti-RANKL/sclerostin bispecific antibodies described herein.
  • a recombinant expression vector can be a replicable DNA construct that includes synthetic or cDNA-derived DNA fragments encoding at least one polypeptide chain of the anti-RANKL/sclerostin bispecific antibodies described herein, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes.
  • a viral vector is used.
  • DNA regions are “operatively linked” when they are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • structural elements intended for use in certain expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • a polypeptide in situations where recombinant protein is expressed without a leader or transport sequence, can include an N-terminal methionine residue.
  • vectors are plasmid, autonomously replicating sequences, and transposable elements.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.
  • Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC)
  • bacteriophages such as lambda phage or M13 phage
  • animal viruses include, without limitation, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) .
  • expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST TM , pLenti6/V5-DEST TM , 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 such as Sleeping Beauty and PiggyBac can be used, which can be stably integrated into the genome (e.g., Ivics et al., Cell, 91 (4) : 501–510 (1997) ; et al., (2007) Nucleic Acids Research. 35 (12) : e87) .
  • the vector is an episomal vector or a vector that is maintained extrachromosomally.
  • episomal vector refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
  • the vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV.
  • the lymphotrophic herpes virus may be Epstein Barr virus (EBV) , Kaposi's sarcoma herpes virus (KSHV) , Herpes virus saimiri (HS) , or Marek's disease virus (MDV) .
  • Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.
  • the host cell comprises the viral replication transactivator protein that activates the replication.
  • “Expression control sequences, ” “control elements, ” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3'untranslated regions-which interact with host cellular proteins to carry out transcription and translation.
  • Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including ubiquitous promoters and inducible promoters can be used.
  • Illustrative ubiquitous expression control sequences that can be used in present disclosure include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) promoter (e.g., early or late) , a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an 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 70kDa protein 5 (H
  • inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone) , metallothionine promoter (inducible by treatment with various heavy metals) , MX-1 promoter (inducible by interferon) , the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323: 67) , the cumate inducible gene switch (WO 2002/088346) , tetracycline-dependent regulatory systems, etc.
  • steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone)
  • metallothionine promoter inducible by treatment with various heavy metals
  • MX-1 promoter inducible by interfer
  • the bispecific antibodies described herein can be produced by any method known in the art, including chemical synthesis and recombinant expression techniques.
  • the practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic 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. (1982) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons (1987 and annual updates) ; CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley &Sons (1987 and annual updates) Gait (ed. ) (1984) OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press; Eckstein (ed.
  • cells comprising the polynucleotides disclosed herein that encode at least one polypeptide chain of the anti-RANKL/sclerostin bispecific antibodies described herein.
  • cells provided herein comprise a polynucleotide that encodes the C1, C2, and C3 of the anti-RANKL/sclerostin bispecific antibodies disclosed herein having the KIH structure.
  • cells provided herein comprise a plurality of the polynucleotides that collectively encode the C1, C2, and C3 of the anti-RANKL/sclerostin bispecific antibodies disclosed herein having the KIH structure.
  • cells provided herein comprise a polynucleotide that encodes both the LC and the HC of the anti-RANKL/sclerostin bispecific antibodies disclosed herein having the IgG-scFv structure. In some embodiments, cells provided herein comprise a first polynucleotide that encodes the LC and a second polynucleotide that encodes HC of the anti-RANKL/sclerostin bispecific antibodies disclosed herein having the IgG-scFv structure.
  • host cells comprising vectors disclosed herein are also contemplated.
  • host cells comprising a vector comprising a polynucleotide disclosed herein.
  • host cells provided herein comprise a vector or multiple vectors that collectively comprise the polynucleotides encoding the polypeptide chains of the anti-RANKL/sclerostin bispecific antibodies described herein.
  • host cells provided herein produce the anti-RANKL/sclerostin bispecific antibodies described herein.
  • 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 cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • COS-7 monkey kidney-derived
  • L-929 murine fibroblast-derived
  • C127 murine mammary tumor-derived
  • 3T3 murine fibroblast-derived
  • CHO Choinese hamster ovary-derived
  • HeLa human cervical cancer-derived
  • BHK hamster kidney fibroblast-derived
  • HEK-293 human embryonic kidney-derived
  • Mammalian expression vectors can comprise 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, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • expression of recombinant proteins in insect cell culture systems e.g., baculovirus
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the bispecific antibodies disclosed herein are comprised of more than one polypeptide chain, which can be produced separately or together. In some embodiments, methods provided herein produce at least one polypeptide chain of the bispecific antibodies disclosed herein. In some embodiments, methods provided herein produce all polypeptide chains of the bispecific antibodies disclosed herein.
  • bispecific antibodies or polypeptides described herein can be produced and isolated using methods known in the art.
  • Polyeptides can be synthesized, in whole or in part, using chemical methods (see, e.g., 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 various solid phase techniques (see, e.g., Roberge, Science 269: 202 (1995) ; Merrifield, Methods. Enzymol.
  • host-expression vector systems can be utilized to recombinantly express the bispecific antibodies described herein or one or more of their polypeptide chains.
  • Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well-known in the art.
  • Such host-expression systems represent vehicles by which the coding sequences of the bispecific antibodies described herein can be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate polynucleotide coding sequences, express the bispecific antibodies described herein in situ.
  • These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for the compounds described herein; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the compounds described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baclovirus) 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 sequences encoding the molecules compounds described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S.
  • mammalian cell systems e.
  • Per C. 6 cells human retinal cells developed by Crucell harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • 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 that are readily purified can be desirable.
  • 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.
  • pGEX vectors can also be used to express polypeptides as fusion proteins with glutathione S-transferase (GST) .
  • GST glutathione S-transferase
  • proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising 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.
  • 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 cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • COS-7 monkey kidney-derived
  • L-929 murine fibroblast-derived
  • C127 murine mammary tumor-derived
  • 3T3 murine fibroblast-derived
  • CHO Choinese hamster ovary-derived
  • HeLa human cervical cancer-derived
  • BHK hamster kidney fibroblast-derived
  • HEK-293 human embryonic kidney-derived
  • Mammalian expression vectors can comprise 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, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Expression of recombinant proteins in insect cell culture systems e.g., baculovirus
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • modifications e.g., glycosylation
  • processing e.g., cleavage
  • 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 native or recombinant cellular mechanisms to form separate polypeptides of the bispecific antibodies described herein.
  • the disclosure thus encompasses engineering a nucleic acid sequence to encode a polyprotein precursor molecule comprising the polypeptides of the bispecific antibodies described herein, which includes coding sequences capable of directing post translational cleavage of said polyprotein precursor.
  • Post-translational cleavage of the polyprotein precursor results in the polypeptides of the bispecific antibodies described herein.
  • the post translational cleavage of the precursor molecule comprising the polypeptides of the compounds described herein can occur in vivo (i.e., within the host cell by native or recombinant cell systems/mechanisms, e.g. furin cleavage at an appropriate site) or can occur in vitro (e.g.
  • 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.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
  • cell lines which stably express compounds described herein can be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc. ) , and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • 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 confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method can advantageously be used to engineer cell lines which express the compounds described herein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the compounds described herein.
  • a number of selection systems may 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.
  • antimetabolite 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.
  • bispecific antibodies described herein or their polypeptide chains can be increased by vector amplification (for a review, see 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) .
  • vector amplification for a review, see 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) .
  • a marker in the vector system described herein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of a protein of interest, production of the protein of interest will also increase (Crouse et al. (1983) Mol. Cell. Biol.
  • the host cell can be co-transfected with more than one expression vectors, each encoding a polypeptide chain of a bispecific antibody described herein.
  • the vectors can contain identical selectable markers which enable equal expression of all polypeptides.
  • a single vector can be used which encodes two or more polypeptides.
  • the coding sequences for the polypeptides of compounds described herein can comprise cDNA or genomic DNA.
  • a bispecific antibody described herein or polypeptide described herein can be purified by any method known in the art for purification of polypeptides, polyproteins or antibodies (e.g., analogous to antibody purification schemes based on antigen selectivity) for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the compound comprises an Fc domain (or portion thereof) ) , and sizing column chromatography) , centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the compound comprises an Fc domain (or portion thereof)
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies.
  • kits for producing an anti-RANKL/sclerostin bispecific antibody described herein or a polypeptide chain of a bispecific antibody described herein comprising obtaining a cell described herein and expressing the polynucleotide described herein in said cell.
  • methods provided herein comprising culturing the cells under conditions that allow expression of the bispecific antibody.
  • the method further comprises isolating and purifying a bispecific antibody or polypeptide chain described herein.
  • the bispecific antibodies described herein can be tested for binding to human RANKL and/or sclerostin by, for example, standard ELISA. Briefly, microtiter plates are coated with purified antigen, and then blocked with bovine serum albumin. Dilutions of antibody are added to each well and incubated. The plates are washed and incubated with secondary reagent (e.g., for human antibodies, a goat-anti-human IgG Fc-specific polyclonal reagent) conjugated to horseradish peroxidase (HRP) . After washing, the plates can be developed and analyzed by a spectrophotometer.
  • secondary reagent e.g., for human antibodies, a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase (HRP)
  • Antibodies can be further tested by flow cytometry for binding to a cell line expressing human RANKL and/or sclerostin, but not to a control cell line that does not express the target antigen. Briefly, the binding of antibodies can be assessed by incubating RANKL and/or sclerostin expressing CHO cells with the bispecific antibody provided herein. The cells can be washed, and binding can be detected with an anti-human IgG Ab. Flow cytometric analyses can be performed using a FACS can flow cytometry (Becton Dickinson, San Jose, CA) .
  • the anti-RANKL/sclerostin bispecific antibodies provided herein can be further tested for reactivity with the target antigen (s) by Western blotting, and other methods known in the art for analyzing binding affinity, cross-reactivity, and binding kinetics of various anti-RANKL/sclerostin bispecific antibodies described herein include, for example, biolayer interferometry (BLI) using, for example, Gator system (Probe Life) or the Octet-96 system (Sartorius AG) , or BIACORE TM surface plasmon resonance (SPR) analysis using a BIACORE TM 2000 SPR instrument (Biacore AB, Uppsala, Sweden) .
  • BLI biolayer interferometry
  • Gator system Probe Life
  • Octet-96 system Sesartorius AG
  • SPR BIACORE TM surface plasmon resonance
  • compositions comprising the anti-RANKL/sclerostin bispecific antibodies disclosed herein.
  • the pharmaceutical composition comprises a therapeutically effective amount of the bispecific antibodies disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions are useful in treating an inflammatory disease or an autoimmune disease.
  • the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to a material that is suitable for drug administration to an individual along with an active agent without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.
  • the pharmaceutical compositions disclosed herein can comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof.
  • a buffer system a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof.
  • preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 19 th edition, 1995.
  • the pharmaceutical compositions provided herein comprise the anti-RANKL/sclerostin bispecific antibodies provided herein.
  • the anti-RANKL/sclerostin bispecific antibodies can be present at various concentrations.
  • the pharmaceutical compositions provided herein comprise the anti-RANKL/sclerostin bispecific antibodies provided herein at 1-1000 mg/mL.
  • the pharmaceutical compositions comprise the anti-RANKL/sclerostin bispecific antibodies provided herein at 10-500 mg/mL, 10-400 mg/mL, 10-300 mg/mL, 10-200 mg/mL, 10-100 mg/mL, 20-100 mg/mL, or 50-100 mg/mL.
  • the pharmaceutical compositions provided herein comprise the anti-RANKL/sclerostin bispecific antibodies provided herein at 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. Dosages can be readily adjusted by those skilled in the art; for example, a decrease in purity may require an increase in dosage.
  • 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.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) .
  • the active ingredient i.e., the anti-RANKL/sclerostin bispecific antibodies
  • the active ingredient 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.
  • the pharmaceutical composition or formulation disclosed herein comprises: (a) the anti-RANKL/sclerostin bispecific antibodies disclosed herein; (b) a buffering agent; (c) a stabilizing agent; (d) a salt; (e) a bulking agent; and/or (f) a surfactant.
  • 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.
  • the pharmaceutical composition or formulation is stable when stored at 4°C, 25°C, or 40°C.
  • Buffering agents useful in the pharmaceutical compositions or formulations disclosed herein can be a weak acid or base used to maintain the acidity (pH) of a solution near a chosen value after the addition of another acid or base.
  • Suitable buffering agents can maximize the stability of the pharmaceutical formulations by maintaining pH control of the formulation. Suitable buffering agents can also ensure physiological compatibility or optimize solubility. Rheology, viscosity and other properties can also depend on the pH of the formulation.
  • Common buffering agents include, but are not limited to, histidine, citrate, succinate, acetate and phosphate.
  • a buffering agent comprises histidine (e.g., L-histidine) with isotonicity agents and potentially pH adjustment with an acid or a base known in the art.
  • the buffering agent is L-histidine.
  • the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.
  • Stabilizing agents are added to a pharmaceutical product to stabilize that product. Such agents can stabilize proteins in different ways. Common stabilizing agents include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine, carbohydrates such as glucose, sucrose, trehalose, rafftnose, or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrins or destrans of any kind and molecular weight, or PEG. In some embodiments, the stabilizing agent is chosen to maximize the stability of antibodies in lyophilized preparations. In certain embodiments, the stabilizing agent is sucrose and/or arginine.
  • Bulking agents can be added to a pharmaceutical composition or formulation to add volume and mass to the product, thereby facilitating precise metering and handling thereof.
  • Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.
  • Surfactants are amphipathic substances with lyophilic and lyophobic groups.
  • a surfactant can be anionic, cationic, zwitterionic, or nonionic.
  • nonionic surfactants include, but are not limited to, alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethylamine oxide.
  • the surfactant is polysorbate 20 or polysorbate 80.
  • the pharmaceutical composition is an aqueous formulation.
  • aqueous formulation is typically a solution or a suspension, but can also include colloids, dispersions, emulsions, and multi-phase materials.
  • aqueous formulation is defined as a formulation comprising at least 50%w/w water.
  • aqueous solution is defined as a solution comprising at least 50 %w/w water
  • aqueous suspension is defined as a suspension comprising at least 50 %w/w water.
  • the pharmaceutical compositions disclosed herein are freeze-dried, to which the physician or the patient adds solvents and/or diluents prior to use.
  • compositions disclosed herein can also include a pharmaceutically acceptable antioxidant.
  • 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.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butyl
  • aqueous and nonaqueous carriers examples 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.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • 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 dispersions, and by the use of surfactants.
  • 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, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as 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 such as aluminum monostearate and gelatin.
  • compositions comprising the anti-RANKL/sclerostin bispecific antibodies or cells provided herein wherein the composition is suitable for local administration.
  • compositions or formulations typically 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, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • compositions can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • some methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the amount of active ingredient which can be combined with a carrier material in the pharmaceutical compositions or formulations disclosed herein can vary.
  • the amount of active ingredient which can be combined with a carrier material is the amount that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
  • 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.
  • 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 poly lactic acid.
  • Many methods for the preparation of such formulations are patented or 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.
  • kits for preparation of pharmaceutical compositions having the anti-RANKL/sclerostin bispecific antibodies disclosed herein comprising the anti-RANKL/sclerostin bispecific antibodies disclosed herein and a pharmaceutically acceptable carrier in one or more containers.
  • the kits can comprise the anti-RANKL/sclerostin bispecific antibodies disclosed herein for administration to a subject.
  • the kits comprise instructions regarding the preparation and/or administration of the anti-RANKL/sclerostin bispecific antibodies.
  • the anti-RANKL/sclerostin bispecific antibodies provided herein can be used in medical treatment.
  • Provided herein are also methods of increasing bone mass comprising administering to the subject in a subject in need thereof a therapeutically effective amount of the anti- RANKL/sclerostin bispecific antibodies disclosed herein.
  • the subject is a human.
  • the subject has primary osteoporosis.
  • the subject has secondary osteoporosis.
  • the subject is at risk of developing osteoporosis.
  • treat and its grammatical equivalents as used herein in connection with a disease or a condition, or a subject having a disease or a condition refer to an action, intervention and/or measure that suppresses, eliminates, reduces, and/or ameliorates a symptom, the severity of the symptom, and/or the frequency of the symptom associated with the disease or disorder being treated.
  • Treatment of osteoporosis can suppress, eliminate, reduce, or ameliorate the symptoms associated with osteoporosis. These symptoms primarily include bone loss, decreased bone density, and an increased risk of fractures, and the treatment aims to slow down the rate of bone loss, strengthen the bones, and reduce the likelihood of fractures.
  • prevent and its grammatical equivalents as used in connection with a disease or a condition, or a subject at risk of developing a disease or a condition, refer to actions taken to hinder, impede, or mitigate the occurrence, development, or progression of the disease or disorder.
  • the goal of prevention is to reduce the risk, delay the onset, or completely avoid the manifestation of the disease or condition in susceptible individuals.
  • administer and its grammatical equivalents as used herein refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition to the body of a subject by a method described herein or otherwise known in the art.
  • the therapeutic can be a compound, a polypeptide, an antibody, a cell, or a population of cells.
  • Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject.
  • Exemplary forms of administration 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, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.
  • 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, jellies, powders, or patches
  • buccal dosage forms inhalation powders, sprays, suspensions, and rectal suppositories.
  • an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject.
  • the therapeutically effective amount can be ascertained by measuring relevant physiological effects. The exact amount required varies from subject to subject, depending on the age, weight, 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.
  • subject refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be 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 who needs the treatment may be a human subject having, at risk for, or suspected of having a disease.
  • a subject having a disease can be identified by routine medical examination, e.g., a physical examination, a laboratory test, an organ functional test, a CT scan, or an ultrasound.
  • a subject suspected of having any of such a disease can show one or more symptoms of the disease.
  • a subject at risk for the disease can be a subject having one or more of the risk factors for that disease.
  • a subject can be a human.
  • a subject can have a particular disease or condition.
  • the bispecific antibodies and pharmaceutical compositions provided herein can be used for treating or preventing osteoporosis. In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein can be used for treating or preventing primary osteoporosis. In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein can be used for treating postmenopausal osteoporosis (Type I) . In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein can be used for treating senile osteoporosis (Type II) . In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein can be used for treating idiopathic osteoporosis.
  • the bispecific antibodies and pharmaceutical compositions provided herein can be used for preventing postmenopausal osteoporosis (Type I) . In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein can be used for preventing senile osteoporosis (Type II) . In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein can be used for preventing idiopathic osteoporosis.
  • the bispecific antibodies and pharmaceutical compositions provided herein can be used for treating secondary osteoporosis. In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein can be used for preventing secondary osteoporosis.
  • a person of ordinary skill in the art would understand that the secondary osteoporosis that can be treated or prevented with the bispecific antibodies and pharmaceutical compositions provided herein is not limited to any specific causes. For example, secondary osteoporosis with endocrine or metabolic causes, or associated with collagen/genetic disorders, medications, and/or nutritional deficiencies all can be treated with the bispecific antibodies and pharmaceutical compositions provided herein.
  • subjects that can be treated with the bispecific antibodies and pharmaceutical compositions provided herein can have osteoporosis or are at risk of having osteoporosis.
  • the subject has been diagnosed with osteoporosis.
  • the subject has been diagnosed with Type I osteoporosis.
  • the subject has been diagnosed with Type II osteoporosis.
  • the subject has been diagnosed with secondary osteoporosis.
  • the subject is at risk of having osteoporosis.
  • the subject at risk of having osteoporosis is a postmenopausal woman.
  • the subject at risk of having osteoporosis is age 50 or older.
  • the subject at risk of having osteoporosis has a family history of osteoporosis or fractures.
  • the subject at risk of having osteoporosis has hormonal imbalances, who can have conditions like hypogonadism, hyperthyroidism, and/or adrenal disorders.
  • the subject at risk of having osteoporosis have certain medical conditions, such as rheumatoid arthritis, celiac disease, kidney disease, and hormone-related disorders.
  • the subject at risk of having osteoporosis has long-term use of medications like corticosteroids (prednisone) , anticonvulsants, and some cancer treatments that weaken the bones.
  • the subject at risk of having osteoporosis has poor nutrition, low calcium and vitamin D intake, sedentary lifestyle, excessive alcohol consumption, smoking, and/or lack of weight-bearing exercise.
  • methods provided herein prevent or treat osteoporosis in a subject. In some embodiments, methods provided herein prevent fractures in the subject. In some embodiments, methods provided herein improve bone density. In some embodiments, methods provided herein increase bone mass. In some embodiments, methods provided herein slow or halt bone loss. In some embodiments, methods provided herein prevent further deterioration of bone density. In some embodiments, methods provided herein relieve symptoms associated with osteoporosis, including chronic pain. In some embodiments, methods provided herein enhance mobility and functionality of the subject. In some embodiments, methods provided herein minimize the risk of complications of osteoporosis, such as spinal deformities and loss of height.
  • the active ingredients i.e., the anti-RANKL/sclerostin bispecific antibodies disclosed herein
  • the selected dosage level will depend upon 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.
  • the anti-RANKL/sclerostin bispecific antibodies disclosed herein can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the anti-RANKL/sclerostin bispecific antibodies in the patient. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease.
  • the anti-RANKL/sclerostin bispecific antibodies or pharmaceutical compositions provided herein can be administered to a subject by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, for example by injection or infusion, or direct administration to the thymus.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
  • subcutaneous administration is adopted.
  • intravenous administration is adopted.
  • oral administration is adopted.
  • the antibodies or antigen-binding fragments provided herein can be delivered locally.
  • the antibodies or antigen-binding fragments provided herein can be administered systemically.
  • Anti-RANKL/sclerostin bispecific antibodies or pharmaceutical compositions provided herein can be administered with medical devices known in the art.
  • a needleless hypodermic injection device can be used, such as the devices 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.
  • Examples of well-known implants and modules for use 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.
  • the anti-RANKL/sclerostin bispecific antibodies or pharmaceutical compositions provided herein can be administered with an additional therapy.
  • the additional therapy can be administered prior to, concurrently with, or subsequent to administration of the bispecific antibodies or pharmaceutical compositions described herein.
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • a person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.
  • 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 VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH1 comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 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 VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH2 comprises VH CDR
  • Embodiment 2 The bispecific antibody of Embodiment 1, wherein VL1, VH1, VL2, and VH2 have the amino acid sequences of SEQ ID NOs: 13, 14, 15, and 16, respectively.
  • 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.
  • 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.
  • C1 first peptide chain
  • CL light chain constant region
  • 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.
  • 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.
  • 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.
  • C1 first peptide chain
  • C2 comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1)
  • CH1 heavy chain constant domain 1
  • Knob-Fc region a Knob-Fc region
  • 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.
  • C1 first peptide chain
  • CL light chain constant region
  • C2 comprising, from N-terminus to C-terminus, VH2, a heavy chain constant domain 1 (CH1) , and a Hole-Fc region
  • C3 a single chain variable fragment (scFv) comprising VL1 and VH1,
  • 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.
  • 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.
  • 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 ten amino acids substitutions, including a T366W substitution; and the Hole-Fc region is a human IgG2 Fc region variant having up to ten amino acids substitutions, including T366S, L368A, Y407V substitutions.
  • the Knob-Fc region is a human IgG2 Fc region variant having up to ten amino acids substitutions, including a T366W substitution
  • the Hole-Fc region is a human IgG2 Fc region variant having up to ten amino acids substitutions, including T366S, L368A, Y407V substitutions.
  • Embodiment 12 The bispecific antibody of Embodiment 11, wherein the Knob-Fc region further comprises S354C substitution, and the Hole-Fc region further comprises Y349C substitution.
  • Embodiment 13 The bispecific antibody of Embodiment 11, wherein the Knob-Fc region further comprises Y349C substitution, and the Hole-Fc region further comprises S354C substitution.
  • Embodiment 14 The bispecific antibody of Embodiment 11, wherein (i) the CL region is kappa CL (C ⁇ ; SEQ ID NO: 19) or lambda CL (C ⁇ ; SEQ ID NO: 20) , or a variant thereof having up to ten amino acids substitutions; (ii) the CH1 domain is human IgG2 CH1 domain (SEQ ID NO: 36) or a variant thereof having up to ten amino acids substitutions; and/or (iii) the Knob-Fc region and the Hole-Fc region have the amino acid sequences of (1) SEQ ID NOs: 29 and 32, respectively, (2) SEQ ID NOs: 30 and 33, respectively, or (3) SEQ ID NOs: 31 and 34, respectively, or a variant thereof having up to ten amino acids substitutions.
  • Embodiment 15 The bispecific antibody of Embodiment 14, wherein the CL region, CH1 domain, the Knob-Fc region and the Hole-Fc region have the amino acid sequences of (1) SEQ ID NOs: 19, 36, 29 and 32, respectively, (2) SEQ ID NOs: 19, 36, 30 and 33, respectively, or (3) SEQ ID NOs: 19, 36, 31 and 34, respectively.
  • Embodiment 16 The bispecific antibody of Embodiment 3, wherein C1, C2, and C3 have amino acid sequences of (1) SEQ ID NOs: 51, 53, and 54, respectively, (2) SEQ ID NOs: 51, 55, and 56, respectively, (3) SEQ ID NOs: 51, 57, and 58, respectively; or (4) SEQ ID NOs: 51, 59 and 60, respectively.
  • Embodiment 17 A 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.
  • C1 first peptide chain
  • CL light chain constant region
  • C2 a second peptide chain 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.
  • Embodiment 18 The bispecific antibody of Embodiment 17, wherein the scFv comprises, from N-terminus to C-terminus, VL2, a second linker, and VH2.
  • Embodiment 19 The bispecific antibody of Embodiment 17, wherein the scFv comprises, from N-terminus to C-terminus, VH2, a second linker, and VL2.
  • Embodiment 20 A 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.
  • C1 first peptide chain
  • CL light chain constant region
  • Embodiment 21 The bispecific antibody of Embodiment 20, wherein the scFv comprises, from N-terminus to C-terminus, VL1, a second linker, and VH1.
  • Embodiment 22 The bispecific antibody of Embodiment 20, wherein the scFv comprises, from N-terminus to C-terminus, VH1, a second linker, and VL1.
  • Embodiment 23 The bispecific antibody of any one of Embodiments 17 to 22, wherein (1) the CL region is kappa CL (C ⁇ ; SEQ ID NO: 19) or lambda CL (C ⁇ ; SEQ ID NO: 20) , or a variant thereof having up to ten amino acids substitutions; or (2) the CH region is 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 ten amino acids substitutions; or both (1) and (2) .
  • the CL region is kappa CL (C ⁇ ; SEQ ID NO: 19) or lambda CL (C ⁇ ; SEQ ID NO: 20) , or a variant thereof having up to ten amino acids substitutions
  • the CH region is human IgG1 CH region (SEQ ID NO: 21) , IgG2 CH region (
  • Embodiment 24 The bispecific antibody of Embodiment 23, wherein (1) the CL region is kappa CL (C ⁇ ; SEQ ID NO: 19) ; and (2) the CH region is human IgG2 CH region (SEQ ID NO: 22) .
  • Embodiment 25 The bispecific antibody of Embodiment 17, wherein the C1 and C2 have amino acid sequences of (1) SEQ ID NOs: 51 and 61, respectively, (2) SEQ ID NOs: 51 and 62, respectively.
  • Embodiment 26 The bispecific antibody of Embodiment 20, wherein the C1 and C2 have amino acid sequences of (1) SEQ ID NOs: 52 and 63, respectively, (2) SEQ ID NOs: 52 and 64, respectively.
  • 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.
  • Embodiment 28 A polynucleotide encoding a peptide chain of the bispecific antibody of any one of Embodiments 3 to 26.
  • Embodiment 29 The polynucleotide of Embodiment 28 encoding all peptide chains of the bispecific antibody.
  • Embodiment 30 A plurality of the polynucleotide of Embodiment 28 that collectively encode all peptide chains of the bispecific antibody.
  • Embodiment 31 A vector comprising the polynucleotide of Embodiment 28 or 29.
  • 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.
  • 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.
  • 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.
  • Embodiment 35 The method of Embodiment 34, wherein the subject has primary osteoporosis.
  • Embodiment 36 The method of Embodiment 35, wherein the subject has postmenopausal osteoporosis (Type I) .
  • Embodiment 37 The method of Embodiment 35, wherein the subject has senile osteoporosis (Type II) .
  • Embodiment 38 The method of Embodiment 34, wherein the subject has secondary osteoporosis.
  • Embodiment 39 The method of Embodiment 38, wherein the osteoporosis is secondary to an endocrine or metabolic cause, a collagen or genetic disorder, a medication cause, or a nutritional cause.
  • Embodiment 40 The method of any one of Embodiments 34 to 39, wherein the subject is a human.
  • Embodiment 41 Use of the bispecific antibody of any one of Embodiments 1 to 26 as a medicament.
  • Embodiment 42 Use of the bispecific antibody of any one of Embodiments 1 to 26 in preventing or treating osteoporosis.
  • Embodiment 43 Use of the bispecific antibody of any one of Embodiments 1 to 26 for the preparation of a medicament for preventing or treating osteoporosis.
  • the bispecific IgG2 antibodies that specifically bound to both human RANKL and human sclerostin having the knobs-in-the-holes configuration were generated (FIGs. 1A-1B; “DRAb” ) , including DR-P7, DR-P8, DR-P15, and DR-P16 (Table 6B) .
  • Point mutations were introduced in the CH3 domain of heavy chains. Specifically, in the “knobs” chain, S354C and T366W were introduced; in the “holes” chain, Y349C, T366S, L368A and Y407V were introduced.
  • the “knobs” chain included the antibody binding fragment (Fab) targeting RANKL, whereas the “holes” chain included a single chain variable fragment (scFv) targeting sclerostin. As such, mispairing of light chain should be eliminated.
  • ELISA was used to examine the binding affinities of some candidate bispecific antibodies against human RANKL protein and sclerostin. Briefly, ELISA plate was coated overnight at 4 °C with his-tagged human RANKL at a concentration of 5 ⁇ g/mL. The coated-plate was washed with PBST buffer for 3 times and blocked with PBS in the presence of 1 %BSA for 2 hours at room temperature.
  • the bispecific antibodies (50 ⁇ L) were added into the wells of his-tagged-human RANKL protein coated-ELISA plates at a series of concentrations (10 ⁇ g/mL being the highest concentration, 5-fold dilution, 7-point concentration) and incubated for 1 hour at room temperature.
  • ELISA stripes were washed with PBST for 3 times and HRP-conjugated goat anti-human Fc specific antibody (Abcam, ab97225) at a dilution of 1: 5000 in 1 %BSA in PBS was used to reveal the binding of the bispecific antibodies to the his-tagged RANKL.
  • DR-P7 For the binding to sclerostin, his-tagged human sclerostin protein was coated at a concentration of 5 ⁇ g/mL and incubated overnight at 4 °C. ELISA was performed as described above. An exemplary result is provided in FIG. 2. As shown, all three DRAbs: DR-P7, DR-P15, and DR-P16 specifically bound to human RANKL and human sclerostin. Denosumab and Romosozumab were used as reference antibodies.
  • RAW24.7 a murine macrophage precursor cell line
  • human RANKL was activated with human RANKL for osteoclast differentiation. Briefly, 100 ng/mL human RANKL was incubated with RAW264.7 for 7 days. Human RANKL-containing medium was changed every 2-3 days. Thereafter, the cells were fixed with 4%paraformaldehyde for 5 mins at room temperature and washed once with PBS. Cells were stained according to the TRAcP kit procedure (Wako, Fujifilm) , and photographed using a standard inverted microscope (Olympus, Japan) . Mature osteoclasts were counted when more than three nuclei were present in the stained multinucleated cells. Denosumab was used as a reference antibody. An exemplary result is provided in FIG. 3. As shown, bispecific antibodies disclosed herein, both DR-P7 and DR-P8 effectively inhibited osteoclast differentiation.
  • RAW24.7 a murine macrophage precursor cell line
  • RAW264.7 a murine macrophage precursor cell line
  • Human RANKL-containing medium was changed every 2-3 days.
  • RNA was collected with 500 ⁇ L RNAzol (MRC Inc, Ohio) and RNA was extracted according to manufacturer’s protocol.
  • 0.5 ⁇ g RNA was reverse transcribed to cDNA using PrimeScript RT Reagent Kit (Takara, Shiga) .
  • Quantitative PCR was performed using qPCR Master Mix and with the following cycling conditions: polymerase activation, 95 °C, 2 minutes; denaturation, 95 °C, 15 second; annealing and extension, 60 °C, 1 minute. 40 cycles of denaturation, annealing and extension were performed. Selected genes essential for osteoclast differentiation were examined. Denosumab was used as a reference antibody. An exemplary result is provided in FIG. 4. As shown, both DRAbs DR-P7 and DR-P8 effectively inhibited expression of osteoclast marker genes.
  • Example 5 DRAbs effectively blocked sclerostin-mediated inhibition on WNT signaling in HEK293 cells.
  • HEK293 cells were used to assess the activities of DRAbs in blocking sclerostin-mediated inhibition of WNT signaling.
  • the cells were plated at a density of 6, 000 cells/cm2 in 24-well culture plates, which were pre-coated with 50 ⁇ g/mL poly-D-lysine (Merck, A-003-E) for 1 hour.
  • the cells were maintained in complete growth medium (Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) containing 10%fetal bovine serum) at 37°C with 5%CO2.
  • the HEK293 cells were transfected with the pNL3.3 minP nanoluc TCE-LEF reporter plasmid (Promega) and a human WNT1-expressing plasmid (Genscript) .
  • the TCE-LEF reporter could monitor WNT signaling pathway activity.
  • a nanoluciferase assay (Promega) was performed to measure nanoluciferase levels in the culture medium, which indicated the activation level of the canonical WNT signaling pathway.
  • FIG. 5 An exemplary result is provided in FIG. 5. As shown, Romosozumab inhibited the sclerostin-mediated suppression of the WNT signaling pathway with an IC50 of 18.94 nM. DRAbs (DR-P7 and DR-P8) also inhibited sclerostin activity with IC50 values of 26.88 nM and 24.92 nM, respectively. These results demonstrate that DRAbs could effectively block sclerostin-mediated inhibition of the WNT signaling pathway in HEK293 cells.
  • mice expressing human RANKL were obtained from GemPharmatech Co., Ltd. Experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at GemPharmatech. Female mice were housed in cages and maintained under a strict 12-h light: 12-h dark cycle at 22°C with standard food pellets and free access to tap water. After a 1-week adaptation, 8-week-old female mice were randomly divided into following groups: Control group (ovary intact + vehicle (normal saline) i. v.
  • ovariectomy (OVX) group + IgG2 isotype 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 in a surgery room that had been exposed to ultraviolet radiation overnight. After anesthetization, the bilateral ovaries were exposed and removed to prepare the OVX animals; in the Control groups, the ovaries were exposed but left intact. Antibodies were administered intravenously weekly for 4 times starting from Day 0.
  • mice Twenty-eight days after ovariectomy, mice were sacrificed, and their femurs were collected and fixed with 4%paraformaldehyde for 2 days. Samples were scanned using a Scanco micro-CT 40 system (Scanco Medical, Brütisellen, Switzerland) with settings of 70 kVP for power, 113 ⁇ A for current, and an integration time of 200 ms.
  • the distal metaphysis was scanned at a nominal resolution of 10 ⁇ m/voxel.
  • the analyzed region of interest began 0.03 mm proximal to the growth plate and extended 0.9 mm proximally.
  • OVX treatment reduced trabecular number (Tb. N) and increased trabecular separation (Tb. Sp) .
  • Treatment with either Denosumab or Romosozumab partially restored Tb. N and Tb. Sp.
  • the group treated with DRAb showed a significantly greater increase in Tb. N and a more substantial reduction in Tb. Sp, demonstrating a synergistic effect in promoting bone regrowth by targeting both RANKL and sclerostin compared to targeting either one alone.

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Abstract

L'invention concerne des anticorps bispécifiques ciblant à la fois les protéines RANKL et sclérostine humaines. L'invention concerne également des polynucléotides codant pour les anticorps bispécifiques, des compositions pharmaceutiques comprenant les anticorps bispécifiques, et des procédés de production des anticorps bispécifiques. L'invention concerne également des utilisations médicales des anticorps bispécifiques décrits ici.
PCT/CN2024/112831 2023-08-17 2024-08-16 Anticorps ciblant les protéines rankl et sclérostine et leurs utilisations Pending WO2025036493A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008133722A2 (fr) * 2006-11-10 2008-11-06 Ucb Pharma S.A. Anticorps et diagnostics
WO2012118903A2 (fr) * 2011-03-01 2012-09-07 Amgen Inc. Agents liants bispécifiques
WO2016092101A1 (fr) * 2014-12-12 2016-06-16 Amgen Inc. Anticorps anti-sclérostine et utilisation de ceux-ci pour traiter des affections osseuses en tant qu'élements du protocole de traitement
WO2018115879A1 (fr) * 2016-12-21 2018-06-28 Mereo Biopharma 3 Limited Utilisation d'anticorps anti-sclérostine dans le traitement de l'ostéogenèse imparfaite
WO2018139623A1 (fr) * 2017-01-30 2018-08-02 Chugai Seiyaku Kabushiki Kaisha Anticorps anti-sclérostine et procédés d'utilisation
WO2018200918A1 (fr) * 2017-04-28 2018-11-01 Amgen Inc. Formulations d'anticorps anti-rankl humains, et leurs méthodes d'utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008133722A2 (fr) * 2006-11-10 2008-11-06 Ucb Pharma S.A. Anticorps et diagnostics
WO2012118903A2 (fr) * 2011-03-01 2012-09-07 Amgen Inc. Agents liants bispécifiques
WO2016092101A1 (fr) * 2014-12-12 2016-06-16 Amgen Inc. Anticorps anti-sclérostine et utilisation de ceux-ci pour traiter des affections osseuses en tant qu'élements du protocole de traitement
WO2018115879A1 (fr) * 2016-12-21 2018-06-28 Mereo Biopharma 3 Limited Utilisation d'anticorps anti-sclérostine dans le traitement de l'ostéogenèse imparfaite
WO2018139623A1 (fr) * 2017-01-30 2018-08-02 Chugai Seiyaku Kabushiki Kaisha Anticorps anti-sclérostine et procédés d'utilisation
WO2018200918A1 (fr) * 2017-04-28 2018-11-01 Amgen Inc. Formulations d'anticorps anti-rankl humains, et leurs méthodes d'utilisation

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