Anti-sclerostin constructs and uses thereof

Abstract

The present application provides anti-sclerostin molecules that bind to sclerostin (e.g., anti-sclerostin antibodies, e.g., bispecific anti-sclerostin antibodies), nucleic acid molecules encoding anti-sclerostin amino acid sequences, vectors containing the nucleic acid molecules, host cells containing the vectors, methods for preparing anti-sclerostin constructs, pharmaceutical compositions containing anti-sclerostin constructs, and methods for using anti-sclerostin constructs or compositions.

Description

Technical Field

[0001] <Cross-reference> This application claims priority based on an international patent application with application number PCT / CN2020 / 118387 filed on September 28, 2020, and the entire disclosure of that application is incorporated herein by reference.

[0002] This disclosure relates to anti-sclerostin constructs (e.g., anti-sclerostin antibodies) and their use. <Submission of Sequence Listing in ASCII Text File>

[0003] The content of the ASCII text file submitted below is incorporated herein by reference in its entirety: a sequence listing in computer-readable format (CRF) (file name: 210912000241SEQLIST.TXT, recording date: September 20, 2021, size: 386,061 KB).

Background Art

[0004] The function of sclerostin, a SOST gene product as a human bone formation inhibitor, was discovered by gene localization studies, in which a loss-of-function mutation of the SOST gene was identified as a pathogenic factor for high bone mass (HBM) osteopetrosis (Balemans et al., 2001 Hum. Mol. Genet. 10, 537 - 543; Brunkow et al., 2001 Am. J. Hum. Genet. 68, 577 - 589). In mice, deletion of the SOST gene increases bone mass and bone strength for increased bone formation, while overexpression of human sclerostin transgenic leads to decreased bone mass and reduced bone strength (Ke et al., 2012 Endocr. Rev. 33, 747 - 783.).

[0005] All disclosures of all disclosure explanations, patents, patent applications, and published patent applications cited in this specification are incorporated herein by reference.

Summary of the Invention

[0006] The following is for illustrative purposes only and is not intended to be limiting in any way. That is, the following is intended to introduce the advantages, beneficial effects, and uses of the novel molecular anti-sclerostin constructs of the present invention. Therefore, the following is not intended to limit the essential technical features of the subject matter sought to be protected, nor is it intended to determine the scope of the subject matter sought to be protected.

[0007] In one embodiment, the present application provides an anti-sclerostin construct comprising an antibody portion that specifically recognizes sclerostin (e.g., human sclerostin), wherein the antibody portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), for example, any of the anti-sclerostin constructs described herein. In some embodiments, the antibody portion is an antibody or antigen-binding fragment thereof selected from a full-length antibody, a bispecific antibody, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a dsscFv, a (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody. In some embodiments, the construct is a full-length antibody comprising an Fc fragment. In some embodiments, the antibody portion is an scFv fragment.

[0008] In another aspect, the present application provides anti-sclerostin constructs comprising an antibody portion that specifically recognizes sclerostin (e.g., human sclerostin) and a second portion. In some embodiments, the second portion comprises a half-life extending portion (e.g., an Fc fragment). In some embodiments, the second portion comprises a drug selected from parathyroid hormone (PTH), a selective estrogen receptor modulator (SERM), a bisphosphonate, a prostaglandin E (PGE) receptor agonist, a vascular endothelial growth factor (VEGF), a transforming growth factor-β (TGFβ), a growth factor (myostatin), and calcitonin.

[0009] In some embodiments, the second portion comprises a second antibody portion that specifically recognizes an antigen. In some embodiments, an anti-sclerostin construct is provided that comprises a first antibody portion that specifically recognizes sclerostin (including, but not limited to, the anti-sclerostin antibody portions described herein) and a second antibody portion that specifically recognizes Dickkopf-1 (DKK1) or Receptor Activator of Nuclear Factor Kappa beta Ligand (RANKL). The antibody portion that recognizes DKK1 or RANKL can be any anti-DKK1 or anti-RANKL antibody portion (including, but not limited to, the antibody portions described herein). In some embodiments, the second antibody portion is a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv) fragment, scFv-scFv, nanobody, diabody, or single-domain antibody (sdAb). In some embodiments, the second antibody portion is a full-length antibody comprising an Fc fragment, and the anti-sclerostin antibody portion is a single-chain Fv (scFv) fragment. In some embodiments, the second antibody portion is an scFv fragment, and the anti-sclerostin antibody portion is a full-length antibody comprising an Fc fragment. In some embodiments, the scFv fragment is fused (with or without a linker) to a heavy chain and / or a light chain (e.g., at the N-terminus and / or C-terminus) of a full-length antibody. In some embodiments, the construct comprises: a) a first polypeptide having a first light chain, the first light chain consisting of, from N-terminus to C-terminus: i) V L ii) a first polypeptide comprising a first light chain constant domain (“first CH1 domain”); b) a second polypeptide having a first heavy chain, comprising from N-terminus to C-terminus i) V H ii) a second polypeptide comprising a first heavy chain constant domain ("first CH1 domain") and iii) a first Fc domain; and c) a third polypeptide having a second heavy chain, said second heavy chain comprising from N-terminus to C-terminus i) V H-2ii) a third polypeptide comprising a second heavy chain constant domain (a "second CH1 domain") and iii) a second Fc domain; and d) a fourth polypeptide having a second light chain, the second light chain comprising from N-terminus to C-terminus: i) V L-2 ii) a fourth polypeptide comprising a second light chain constant domain ("second CL domain"), wherein the first Fc domain and the second Fc domain form an Fc fragment. The first CH1 or second CH1 and / or the first Fc domain or the second Fc domain can be modified or improved in some ways without departing from the concept of the present invention.

[0010] In another embodiment, the present application provides anti-sclerostin constructs that specifically bind to sclerostin in competition with any of the anti-sclerostin constructs described herein.

[0011] In another aspect, the present application provides pharmaceutical compositions comprising any of the anti-sclerostin constructs described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an agent selected from parathyroid hormone (PTH), a selective estrogen receptor modulator (SERM), VEGF, TGFβ, a growth factor (myostatin), and calcitonin.

[0012] In another aspect, the present application provides an isolated nucleic acid molecule encoding any of the anti-sclerostin constructs described herein or a portion of the anti-sclerostin constructs (e.g., one or more polypeptides thereof).

[0013] In another aspect, the present application provides a vector of any of the isolated nucleic acid molecules described herein.

[0014] In another aspect, the present application provides an isolated host cell comprising any of the isolated nucleic acid molecules and / or vectors described herein.

[0015] In another aspect, the present application provides a method for producing an anti-sclerostin construct, comprising: a) culturing any of the isolated host cells described herein under conditions that effectively express the anti-sclerostin construct or a portion thereof (e.g., one or more polypeptides thereof); and b) obtaining the expressed anti-sclerostin construct or a portion thereof from the host cell.

[0016] In another aspect, the present application provides methods for treating a disease or condition in an individual, comprising administering to the individual an effective amount of an anti-sclerostin construct, such as any of the anti-sclerostin constructs described herein or any of the pharmaceutical compositions described herein. In some embodiments, the disease or condition refers to a bone-related condition. In some embodiments, the bone-related condition includes osteogenesis imperfecta, osteosclerosis, osteoporosis (male and / or female), senile osteoporosis, delayed bone union, delayed or non-union of fractures, Paget's disease, bone loss due to immobility, glucocorticoid-induced osteoporosis, inflammation-induced bone loss including arthritis-induced bone loss, or other diseases or conditions associated with a) loss of bone mass or bone quality, or both, and / or b) abnormalities in bone structure and quality. In some embodiments, the anti-sclerostin construct or pharmaceutical composition is parenterally administered to the individual. In some embodiments, the method includes the use of a second drug or therapy (e.g., an anti-DKK1 antibody or an anti-RANKL antibody). In some embodiments, the second drug or therapy comprises a drug that is one selected from the group consisting of parathyroid hormone (PTH), selective estrogen receptor modulators (SERMs), bisphosphonates, prostaglandin E (PGE) receptor agonists, VEGF, TGFβ, growth factors (myostatin), and calcitonin. In some embodiments, the individual refers to a human. [Brief explanation of the drawings]

[0017] [Figure 1] The sequence and conformation of the "IGRGKWWR" motif in the second loop of sclerostin are shown to illustrate the interaction of sclerostin with LDL receptor-related protein 6 (LRP6). [Figure 2] 1 shows mapping of monoclonal antibody (mAb) 93B1B7 to a sclerostin epitope using a synthetic peptide fragment in the second loop of sclerostin. [Figure 3] 1 shows that romosozumab does not recognize the peptide derived from the second loop of human sclerostin that is bound by monoclonal antibody 93B1B7. [Figure 4] 1 shows that monoclonal antibodies 93B1B7 and romosozumab bind to different sclerostin epitopes and do not cross-block each other's binding to sclerostin. [Figure 5] 1 shows a schematic diagram of an exemplary bispecific antibody structure. [Figure 6] 1 shows results from Bio-Layer Interferometry (BLI) sensorgrams demonstrating increased binding of human sclerostin and DKK1 to the bispecific antibody. [Figure 7] 1 shows serum concentrations of bispecific antibodies in cynomolgus monkeys administered subcutaneously at 30 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present application provides novel anti-sclerostin constructs (e.g., anti-sclerostin monoclonal or multispecific antibodies) that specifically bind to sclerostin, methods for preparing the anti-sclerostin constructs, and methods for using the constructs (e.g., methods for treating diseases or conditions). The exemplary anti-sclerostin constructs described herein have been shown to have beneficial effects. For example, compared to romosozumab, the exemplary anti-sclerostin molecules exhibit higher binding affinity for sclerostin. See Examples 3, 5, and 6 (Tables 5, 7, and 8).

[0019] I. Definition

[0020] The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0021] A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for binding to an antigen. The variable regions of the heavy and light chains are referred to as "V H " and "V L The variable regions in the two chains typically contain three hypervariable loops called complementarity-determining regions (CDRs) (light chain (LC) CDRs contain LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs contain HC-CDR1, HC-CDR2, and HC-CDR3). The CDR segments of the antibodies and antigen-binding fragments disclosed herein can be defined or identified using the numbering systems of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are interspersed with flanking segments called framework regions (FR), which are more conserved than the CDRs and form brackets that support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit multiple effector functions. Antibodies are classified according to the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by having α, δ, ε, γ, and μ heavy chains, respectively. Some major antibody classes are further divided into subclasses, e.g., IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgG1 (α1 heavy chain), or IgGA2 (α2 heavy chain).

[0022] As used herein, the term "antigen-binding fragment" refers to antibody fragments, including, for example, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain Fvs (scFv), scFv dimers, multispecific antibodies consisting of one or more CDR-containing antibody portions, camelid single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. An antigen-binding fragment can bind to the same antigen as that bound by the parent antibody or parent antibody fragment (e.g., the parent scFv). In some embodiments, an antigen-binding fragment can comprise one or more CDRs from a particular human antibody grafted onto framework regions derived from one or more different human antibodies.

[0023] The variable region (Fv) is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain tightly non-covalently bound. The folding of these two domains forms six hypervariable regions (three regions in the heavy chain and three regions in the light chain) that contain the amino acid residues required for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although with lower affinity than a complete variable domain.

[0024] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a V linked to a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide is an antibody fragment comprising a V H Domains and V LThe scFv further comprises a polypeptide linker between the domains, which enables the scFv to form the structure necessary for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0025] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found in the variable regions of heavy and light chain polypeptides. These specific regions have been identified in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Immunologically Significant Protein Sequences" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), and overlapping or subsets of amino acid residues when compared with each other are included in the definitions. However, the CDRs of antibodies, grafted antibodies, or variants thereof to which either definition is applied are within the scope of the term as defined and used herein. For comparison, the amino acid residues comprising the CDRs defined in each of the aforementioned references are listed in Table 1 below. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein in their entirety as used in this application and as may be included in one or more claims of this application. In some embodiments, the CDR sequences provided herein are those defined based on IMGT numbering.For example, CDR sequences can be identified using the VBASE2 tool (http: / / www.vbase2.org / vbase2.php, or Retter I, Althaus HH, Munch R, Muller W: VBASE2, an integrated V gene database. Nucleic Acids Research 2005 Jan. 1, 33 (Database Release):D671-4, the entire contents of which are incorporated herein by reference).

[0026] As used herein, the term "osteoporosis" refers to premenopausal idiopathic osteoporosis, postmenopausal osteoporosis, menopausal osteoporosis, postoophorectomy osteoporosis, osteoporosis of disuse, drug-induced osteoporosis, osteoporosis due to malabsorption, post-surgical malabsorption osteoporosis, and / or senile osteoporosis.

[0027] As used herein, the term "osteopenia" refers to premenopausal idiopathic osteopenia, postmenopausal osteopenia, senile osteopenia, drug-induced osteopenia, osteopenia of disuse, neonatal osteopenia, and / or spaceflight osteopenia caused by reduced gravity.

[0028] The term "metabolic bone disease" as used herein includes, but is not limited to, renal osteodystrophy, primary and secondary hyperparathyroidism, familial hyperparathyroidism syndromes, parathyroid disorders, osteodystrophy, osteochondrosis, and hyperphosphatasia.

[0029] As used herein, the term "osteonecrosis" refers to avascular necrosis of bone, avascular necrosis secondary to diving, and osteonecrosis of the jaw.

[0030] As used herein, the term "bone loss" includes but is not limited to postmenopausal bone loss, immobilization-induced bone loss, weightlessness-induced bone loss, disease-associated facial bone loss, disease-associated cranial bone loss, disease-associated bone loss of the jaw, disease-associated bone loss of the skull, bone loss associated with space travel, glucocorticoid-induced bone loss, drug-induced bone loss, organ transplant-related bone loss, kidney transplant-related bone loss, HIV-associated bone loss, and bone loss associated with loss of growth hormone. hormone), bone loss associated with cystic fibrosis, chemotherapy-associated bone loss, tumor-induced bone loss, cancer-related bone loss, hormone ablative bone loss, oral bone loss, heparin-induced bone loss, arthritis-induced bone lossInflammation-induced bone loss, including bone loss due to: a) bone loss of quantity or quality, or both; and / or b) other diseases or conditions associated with abnormalities in bone structure and quality, including bone loss due to reduced gravity.

[0031] As used herein, the term "bone nonunion" or "delay bone healing" refers to delayed bone fractures or non-union bone fractures, hip fracture, pseudoarthritis after fusion or arthrodesis, osteolysis, postsurgical osteolysis, nonunion after spinal arthrodesis, enhancement / acceleration of spinal fusion, and chronic pain after arthroplasty.

[0032] As used herein, the term "osteomalacia" refers to vitamin D-resistant osteomalacia, calcium deficiency, sarcopenia, cancer-related sarcopenia, and tumor-induced osteomalacia.

[0033] The term "fracture" as used herein includes, but is not limited to, a compression fracture, a fragility fracture, a pathologic fracture, a stress fracture, a hip fracture, a fracture of the femoral neck, an atypical hip fracture, a femoral intertrochanter fracture, and a fracture of bone in neoplastic disease.

[0034] The term "hypercalcemia" as used herein includes hypercalcemia of malignancy, myopathy due to hypercalcemia, and hypercalcemia in chronic kidney disease.

[0035] As used herein, the term "multiple myeloma related bone disorders" refers to multiple myeloma bone disease and osteoporosis in multiple myelomatosis.

[0036] As used herein, the term "primary bone tumor" includes any of the following tumors: osteosarcoma, osteochondroma, osteoblastoma, osteochondromyxoma, osteoclastoma, osteoma, osteoid osteoma, chondrosarcoma, chondroblastoma, chondromyxoid fibroma, myxoid chondrosarcoma, sarcoma, Ewing sarcoma, Kaposi sarcoma, periosteal sarcoma, glomangiosarcoma, giant cell tumor, giant cell sarcoma, glioma, giant cell tumor, giant cell sarcoma, ... These include sarcoma, giant cell angiofibroma, hemangioendothelial sarcoma, undifferentiated sarcoma, fibrosarcoma, bone cyst, aneurysmal bone cyst, and multiple endocrine neoplasia.

[0037] The term "malignant tumor" used in "malignant tumor bone metastasis" includes breast cancer, lung cancer, liver cancer, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, thyroid cancer, and thymic cancer.

[0038] As used herein, the term "inflammatory or infectious bone disease" refers to osteomyelitis, pyogenic osteomyelitis, and ankylosing spondylitis.

[0039] As used herein, the term "bone marrow or hematological disorder" refers to leukemia, malignant lymphoma, hematological malignancies, blood disorders, and bone marrow disorders.

[0040] As used herein, the term "musculoskeletal rare disease" includes osteogenesis imperfecta, Albers-Schonberg disease, congenital pseudoarthrosis of the tibia, enchondromatosis, fibrous dysplasia, Gaucher's disease, Marfan's syndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteoporosis, osteopoikilosis, sclerotic lesions, pseudoarthrosis, melorheostosis, juvenile arthritis, and the like. arthritides, thalassemia, mucopolysaccharidoses, Turner syndrome, Pown syndrome, Klinefelter syndrome, leprosy, Perthes' disease, adolescent idiopathic scoliosis, Winchester syndrome, Menkes disease, ischemic bone disease (e.g., Legg-Calve-Perthes disease, regional migratory osteoporosis), idiopathic infantile hypercalcemiaHypercalcemia, Acromegaly, Hypogonadism, Albright-McCune-Sternberg syndrome, Aluminum bone disease, Camurati-Engelmann disease, Osteopetrosis and infantile neuroaxonal dystrophy, Dysosteosclerosis, Pycnodysostosis, Gorham-Stout syndrome, Cystic angiomatosis, Paget's disease, Juvenile Paget's disease, Osteoporosis-oculocutaneous-hypopigmentation syndrome, Osteoporosis in classical or atypical cystic fibrosis, Bowed tibiae-radial anomalies-osteopenia-fractures, X-linked hypophosphatemic osteomalacia, Familial expansile osteolysis, Osteopoikilosis, Melorheostosis, Craniometaphyseal dysplasia, Osteoporosis-pseudoglioma syndrome, Cleidocranial dysplasia, Hajdu-Cheney syndrome, Winchester-Torg syndromeSyndrome, Cole-Carpenter syndrome, Hypophosphatasia, Hereditary hyperphosphatasia, Fibrodysplasia ossificans progressive, Familial hypocalciuric hypercalcemia, Pseudohypoparathyroidism, Acrodysostosis, Eiken syndrome, Multiple enchondromatosis, Vitamin D hydroxylation-deficient rickets, and Hypophosphatemic rickets.

[0041] As used herein, the term "cartilage-related disorder" includes, but is not limited to, chondromatosis, chondrodysplasia, chondrodystrophic myotonia, juxtacortical chondroma, tear of cartilage of the knee, osteoarthritis, and osteochondrodystrophy.

[0042] As used herein, the term "muscle-related disorder" includes sarcopenia and cancer-related sarcopenia.

[0043] The term "surgeries" as used in "facilitation of heal after bone or joint surgeries" refers to orthopedic procedures, dental procedures, implant surgery, joint replacement, joint-preserving surgery, distraction osteogenesis, bone lengthening, bone grafting, bone cosmetic surgery and bone repair, such as fracture healing, nonunion healing, delayed union healing, and facial reconstruction.

[0044] [Table 1]

[0045] "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and variations thereof refer to the heavy chain variable domain or light chain variable domain numbering system used in antibody assembly by Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the framework regions (FRs) or hypervariable regions (HVRs) of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to the Kabat numbering system). The Kabat residue numbering of a given antibody can be determined by aligning the "standard" Kabat numbering sequence with regions of homology in the antibody sequence.

[0046] Unless otherwise specified herein, with reference to the preceding sentence, the numbering of residues in an immunoglobulin heavy chain is that of the EU index of Kabat et al. "Kabat EU index" refers to the numbering of residues in a human IgG1 EU antibody.

[0047] "Framework" or "FR" residues refer to variable domain residues excluding the CDR residues as herein defined.

[0048] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Often, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a recipient hypervariable region (HVR) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that has the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not present in the recipient or donor antibody. These modifications are intended to further improve antibody performance. Generally, humanized antibodies contain substantially all of the variable domains, at least one, and typically two, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are human immunoglobulin sequences. Optionally, the humanized antibody can further comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region (Fc). For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0049] A "human antibody" is an antibody having an amino acid sequence corresponding to that of a human-produced antibody and / or prepared using any of the techniques disclosed herein for producing human antibodies. This definition of human antibody expressly excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be prepared using a variety of techniques known in the art, including phage display libraries. See, for example, Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Human monoclonal antibodies can be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); and van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies may also be prepared by administering antigen to transgenic animals. Transgenic animals are engineered to produce such antibodies in response to antigenic challenge, but their endogenous sites have been disabled, e.g., immunized xenomouse. TM (For techniques, see, e.g., U.S. Patents US 6,075,181 and US 6,150,584. For human antibodies produced by human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0050] "Percent (%) amino acid sequence identity" or "homology" for the polypeptide and antibody sequences defined herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the compared polypeptide sequence, after aligning the sequences and accounting for any conservative substitutions as part of the sequence identity. To determine percent amino acid sequence identity, alignment can be performed in a variety of ways within the skill of the art using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared. However, the sequence alignment computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004) is used herein to generate percent amino acid sequence identity values.

[0051] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If the same position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if the same position in two DNA molecules is occupied by adenine, the two molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences, divided by the number of compared positions, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, two sequences are compared after being aligned to maximize homology.

[0052] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than another portion of the immunoglobulin (the variable domain) that contains the antigen-binding site. The constant domain is the C H 1. C H 2. C H 3 Domains (C H ) and light chain CHL (or C L ) domain.

[0053] The "light chains" of antibodies (immunoglobulins) of any mammalian species can be assigned to one of two clearly distinct types, called "κ" and "λ", depending on the amino acid sequence of their constant domain.

[0054] "C H The "H1 domain" (also called the "C1" of the "H1" domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0055] The "hinge region" is generally defined as the region of an IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). By locating the first and last cysteine ​​residues that form inter-heavy chain S—S bonds in the same positions, the hinge region of another IgG homolog can be aligned with the IgG1 sequence.

[0056] The "C" domain of the human IgG Fc region (also called the "C2" domain) H The "C2 domain" typically extends from about amino acid 231 to about amino acid 340. H The C2 domain is unique in that it pairs non-tightly with another C1 domain. In contrast, the two C2 domains of an intact native IgG molecule H Two N-linked branched carbohydrate chains are inserted between the two domains. The carbohydrates provide an alternative to domain-domain pairing, and C HIt has been speculated that this may contribute to stabilization of the two domains. Burton, Molec Immunol. 22:161-206 (1985).

[0057] "C H The C3 domain (also called the C3 domain) is the C domain in the Fc region. H The two domains comprise an extension to the C-terminal residue (ie, from about amino acid residue 341 to the C-terminus of the antibody sequence, usually at amino acid residue 446 or 447 for IgG).

[0058] The terms "Fc region" or "fragment crystallizable region" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is usually defined as stretching from the amino acid residue at Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a complete antibody composition can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies containing and not containing the K447 residue. Suitable native-sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0059] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. The FcR is most preferably a native-sequence human FcR. Furthermore, the FcR is preferably an FcR that binds to IgG antibodies (gamma receptors), and includes receptors of subclasses such as FcγRI, FcγRII, and FcγRIII, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains a single immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains a single immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Dae(umlaut e)ron, Annu. Rev. Immunol. 15:203-234 (1997). For reviews of FcRs, see Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including FcRs to be determined in the future, are also encompassed by the term "FcR" herein.)

[0060] As used herein, the term "epitope" refers to a specific atom or group of amino acids of an antigen that binds to an antibody or antibody portion. Two antibodies or antibody portions may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0061] As used herein, a first antibody or fragment thereof "competitively" binds to a target antigen if, when present at equimolar concentrations, the first antibody or fragment thereof inhibits target antigen binding of the second antibody to which it is a fragment by at least about 50% (e.g., at least any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), or vice versa. PCT Publication WO 03 / 48731 describes a high-throughput process for "binding" antibodies based on their cross-competition.

[0062] As used herein, the terms "specifically bind," "specifically recognize," and "to" refer to a measurable and reproducible interaction, such as the binding of a target to an antibody or antibody portion, that determines whether the target is present in the presence of a heterogeneous population, including biomolecules. For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to that target with greater affinity, greater ease, and / or longer duration than binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is about 10% less than the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target is about 10% less than the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 In some embodiments, the antibody specifically binds to a conserved protein epitope among proteins of different species. In some embodiments, specific binding can include, but does not require, exclusive binding. The binding specificity of an antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include Western blotting, ELISA, RIA, ECL, IRMA, EIA, BLI, BIACORE. TMThese include, but are not limited to, assays and peptide scans.

[0063] An "isolated" antibody (or construct) is an antibody (e.g., a natural antibody or a recombinant antibody) that has been identified, isolated, and / or recovered from a component of the environment in which it is produced. Preferably, the isolated polypeptide is free from binding to all other components from the environment in which it is produced.

[0064] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its environment of production. Preferably, an isolated nucleic acid is free from all components associated with its environment of production. The form of the isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is different from the form or environment in which they are found in nature. Thus, an isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies naturally present in a cell described herein. Isolated nucleic acid includes nucleic acid molecules contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0065] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a prepreprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Usually, "operably linked" means that the DNA sequences being linked are contiguous, and, if a secretory leader sequence is present, contiguous and in reading frame; however, enhancers are not necessarily linked. Linkage can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used, in accordance with conventional engineering protocols.

[0066] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0067] As used herein, the terms "transfection," "transformation," or "transduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected," "transformed," or "transduced" cell is one that has been transfected or transformed or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0068] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and progeny derived from that cell regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. As used herein, mutant progeny having similar function or biological activity, screened or selected from the initially transformed cell, are also included.

[0069] As used herein, "treatment" or "treatment" refers to a method of obtaining a beneficial or ideal result (including a clinical result). In this application, a beneficial or ideal clinical result includes, but is not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, reduction in the extent of the disease, stabilization of the disease, prevention or delay of disease spread, prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of the disease state, palliation of the disease (partially or completely), reduction in the dose of one or more other drugs required to treat the disease, delay of disease progression, improvement or improvement in quality of life, weight gain, and / or prolongation of survival. The methods of this application contemplate any one or more of these therapeutic concepts.

[0070] The term "inhibit" refers to the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the incidence, degree, or likelihood of that characteristic. "Reduce" or "inhibit" refers to a decrease, decrease, or cessation of activity, function, and / or amount compared to a reference. In some embodiments, "reduce" or "inhibit" refers to the ability to cause a 20% or greater overall reduction; in other embodiments, "reduce" or "inhibit" refers to the ability to cause a 50% or greater overall reduction. In yet other embodiments, "reduce" or "inhibit" refers to the ability to cause a 75%, 85%, 90%, 95%, or greater overall reduction.

[0071] As used herein, "reference" refers to any sample, control, or level used for comparison. A reference can be obtained from a healthy and / or non-diseased sample. In some examples, a reference can be obtained from a sample that has not received a treatment. In some examples, a reference is a non-diseased sample or a sample that has not received a treatment from an individual. In some examples, a reference is obtained from one or more healthy individuals that are not individuals or patients.

[0072] As used herein, "delaying disease progression" means to postpone, hinder, slow, retard, stabilize, inhibit, and / or slow the progression of the disease. This delay may have varying lengths of time depending on the history of the disease and / or condition of the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually include prevention, since the individual will not develop the disease.

[0073] "Prevention" as used herein includes providing prevention of the occurrence or recurrence of a disease in individuals who may be susceptible to the disease but have not yet been diagnosed with the disease.

[0074] As used herein, "inhibiting" a function or activity means decreasing the function or activity when all other conditions are the same, or when compared to another condition, other than the condition or parameter of interest. For example, an antibody that inhibits tumor growth decreases the growth rate of a tumor compared to the growth rate of the tumor in the absence of the antibody.

[0075] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, an individual refers to a human.

[0076] An "effective amount" of a drug refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The specific dose may vary depending on one or more factors selected from the particular drug selected, the administration regimen followed, whether it is used in combination with other compounds, the time of administration, the tissue being imaged, and the physical delivery system used to deliver it.

[0077] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a formulation in which the active ingredient is present in a form that effectively activates the biological activity of the active ingredient and does not contain additional ingredients that are unacceptably toxic to the individual using the formulation. Such formulations may be sterile.

[0078] The term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier commonly used in the art with therapeutic drugs, including a "drug composition" used in an individual with the drug. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation in which it is used. A pharmaceutically acceptable carrier or adjuvant preferably meets the standards required for toxicology and manufacturing testing and / or is included in the guidelines for inactive ingredients compiled by the U.S. Food and Drug Administration.

[0079] A "sterile" formulation is sterile or substantially free of living microorganisms and their spores.

[0080] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0081] The term "concurrently" is used herein to refer to the use of two or more therapeutic agents, where the administration at least partially overlaps in time, or the use of one therapeutic agent is shortly spaced relative to the use of the other therapeutic agent, e.g., the two or more therapeutic agents are administered about 60 minutes or less apart, e.g., about 30 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 1 minute or less apart.

[0082] The term "sequence" is used herein to refer to the use of two or more therapeutic drugs, where one or more drugs are used consecutively after the administration of one or more other drugs has ceased. For example, the interval between the use of the two or more therapeutic drugs is greater than about 15 minutes, e.g., about 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, or longer.

[0083] As used herein, "in conjunction" refers to the use of another therapeutic method in addition to another therapeutic modality. Thus, "in conjunction" refers to administering one therapeutic method to an individual before, during, or after administering the other therapeutic method to the individual.

[0084] The term "pharmaceutical label" refers to instructions typically included in commercial packaging of a therapeutic product, including information regarding indications, uses, dosage, administration, general medical treatment, contraindications, and / or warnings regarding the use of such therapeutic product.

[0085] An "article of manufacture" is any product (e.g., packaging or container) or kit that includes at least one reagent, such as a drug for treating a disease or condition, or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is advertised, distributed, or sold as a unit for performing the methods described herein.

[0086] It should be understood that the applied embodiments described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0087] As used herein, references to (and statements about) a value or parameter include modifications to that value or parameter itself. For example, reference to "about X" includes reference to "X."

[0088] As used herein, "about XY" has the same meaning as "about X to about Y."

[0089] As used in this specification and the appended claims, the singular forms "a," "an," "or," and "said" include plural references unless the context clearly dictates otherwise.

[0090] II. Anti-Sclerostin Constructs

[0091] The present application provides anti-sclerostin constructs comprising an anti-sclerostin antibody portion, which specifically binds to sclerostin (the protein product of the SOST gene expression) as described herein.

[0092] Sclerostin is a secreted glycoprotein with a C-terminal cysteine ​​knot-like (CTCK) domain and shares sequence similarity with the DAN (differential screening-selected gene aberrative in neuroblastoma) family of bone morphogenetic protein (BMP) antagonists. Disruptive mutations in this gene are associated with sclerosis, a possible autosomal recessive disorder that causes progressive bone overgrowth. Deletions downstream of this gene lead to decreased sclerostin expression, which is associated with a milder form of the disease called van Buchem disease.

[0093] In some embodiments, an anti-sclerostin construct is provided that comprises an antibody portion that specifically recognizes sclerostin, wherein the antibody portion binds to an epitope of sclerostin, and the epitope comprises the amino acid sequence set forth in SEQ ID NO:186.

[0094] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing VH-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0095] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0096] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0097] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0098] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:16, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0099] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 3, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0100] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:16, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0101] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 8, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 11, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:17, an LC-CDR2 having the amino acid sequence of SEQ ID NO:19, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:21.

[0102] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0103] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:85, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0104] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:86, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20.

[0105] In some embodiments, the V H i) HC-CDR1 having an amino acid sequence of any one of SEQ ID NOs: 1 to 4 and 12, ii) HC-CDR2 having an amino acid sequence of any one of SEQ ID NOs: 5 to 8 and 13, and iii) HC-CDR3 having an amino acid sequence of any one of SEQ ID NOs: 9 to 11 and 14, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these HC-CDRs, L comprises i) an LC-CDR1 having the amino acid sequence of any one of SEQ ID NOs: 15 to 17, ii) an LC-CDR2 having the amino acid sequence of any one of SEQ ID NOs: 18 and 19, and iii) an LC-CDR3 having the amino acid sequence of any one of SEQ ID NOs: 20 and 21, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs.

[0106] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 14, L-2comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15 or 16, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having the amino acid sequence of any one of SEQ ID NO: 20.

[0107] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs, and V L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, the V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20. Hcomprises the amino acid sequence of SEQ ID NO: 87 or a variant of the amino acid sequence having at least about 80% (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and L comprises the amino acid sequence of SEQ ID NO:88 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). H comprises the amino acid sequence of SEQ ID NO:22 or a variant of the amino acid sequence having at least about 80% (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and L comprises the amino acid sequence of SEQ ID NO:23 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). H comprises the amino acid sequence of SEQ ID NO:26 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:27 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0108] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V Hcomprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs; L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 24 or a variant of the amino acid sequence having 80% (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and L comprises the amino acid sequence of SEQ ID NO:25 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). H comprises the amino acid sequence of SEQ ID NO: 36 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L includes the amino acid sequence of SEQ ID NO:37 or a variant having an amino acid sequence with at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0109] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 2, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs; L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO:28 or a variant of the amino acid sequence having at least about 80% (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and L comprises the amino acid sequence of SEQ ID NO:29 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0110] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V Hcomprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 2, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs, L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having an amino acid sequence identity of at least about 80% (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:31 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0111] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs;L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:16, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 32 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L includes the amino acid sequence of SEQ ID NO:33 or a variant having an amino acid sequence with at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0112] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:3, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:7, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:10, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs; Lcomprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 34 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L includes the amino acid sequence of SEQ ID NO:35 or a variant having an amino acid sequence with at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0113] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs; Lcomprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:16, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 38 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:39 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0114] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:4, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:8, iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:11, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs, Lcomprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:17, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:19, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:21, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 40 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:41 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0115] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs, and V Lcomprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:85, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:9, L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:85, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20. H comprises the amino acid sequence of SEQ ID NO: 87 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:89 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0116] In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), including V Hcomprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs, and V L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:86, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein. In some embodiments, V H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:9, L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:86, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20. H comprises the amino acid sequence of SEQ ID NO: 87 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:90 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0117] In some embodiments, an anti-sclerostin construct comprises an antibody portion that specifically recognizes sclerostin, said antibody portion comprising a heavy chain variable region (V H ) and the light chain variable region (V L ), a) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:22 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 23, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in b) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:24 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 25, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in c) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:26 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 27, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in d) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:28 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 29, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in e) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO: 30 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 31, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in f) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO: 32 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 33, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in g) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:34 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 35, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in h) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO: 36 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 37, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in i) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:38 Hand LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 39, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in j) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:40 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 41, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in k) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO: 87 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 88, respectively. L and the amino acid sequences of CDR1, CDR2, and CDR3 in l) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:87 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 89, respectively. L or comprising the amino acid sequences of CDR1, CDR2 and CDR3 in m) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO: 87 H and LC-CDR1, LC-CDR2 and LC-CDR3 have the amino acid sequences shown in SEQ ID NO: 90, respectively. LThe present invention provides an anti-sclerostin construct comprising the amino acid sequences of CDR1, CDR2, and CDR3 of:

[0118] In some embodiments, the construct comprises a full-length antibody, a bispecific antibody, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide bond stabilized Fv fragment (dsFv), a dsscFv, a (dsFv)2, a V H The antibody or antigen-binding fragment thereof may be selected from H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, and tetrabody.

[0119] In some embodiments, the anti-sclerostin antibody portion is a full-length antibody.

[0120] In some embodiments, the anti-sclerostin antibody portion is an scFv or dsscFv.

[0121] In some embodiments, the anti-sclerostin antibody portion comprises an Fc fragment of an immunoglobulin selected from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the anti-sclerostin antibody portion or full-length antibody comprises an Fc fragment of an immunoglobulin selected from IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof. In some embodiments, the Fc fragment has reduced effector function compared to the corresponding wild-type Fc fragment. In some embodiments, the Fc fragment has enhanced effector function compared to the corresponding wild-type Fc fragment.

[0122] In some embodiments, the antibody portion comprises a humanized antibody of any of the antibody portions described herein.

[0123] In some embodiments, the anti-sclerostin construct comprises or is an anti-sclerostin fusion protein.

[0124] In some embodiments, the anti-sclerostin construct comprises or is a multispecific anti-sclerostin construct (eg, a bispecific antibody).

[0125] In some embodiments, the sclerostin is human sclerostin.

[0126] a) Antibody affinity

[0127] The binding specificity of an antibody moiety can be tested and determined by methods known in the art, such as Western blotting, ELISA, RIA, ECL, IRMA, EIA, BLI, BIACORE. TM These include, but are not limited to, assays and peptide scans.

[0128] In some embodiments, the K of binding between the antibody moiety and sclerostin D is about 10 -7 M~about 10 -12 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, about 10 -11 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -7 M~about 10 -11 M, about 10-8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -7 M~about 10 -10 M, about 10 -8 M~about 10 -10 M, or about 10 -7 M~about 10 -9 In some embodiments, the K of binding between the antibody moiety and sclerostin is D is about 10 -7 M, about 10 -8 M, about 10 -9 M, about 10 -10 M, about 10 -11 M, or about 10 -12 M. In some embodiments, the sclerostin is human sclerostin. In some embodiments, the sclerostin is cynomolgus monkey sclerostin.

[0129] In some embodiments, the K of binding between the antibody moiety and sclerostin on is about 10 3 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 3 M -1 s -1 ~about 10 4 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 7 M -1 s-1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and sclerostin is on is about 10 3 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 5 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and sclerostin is on is 10 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s -1 , 10 6 M -1 s -1 , 10 7 M -1 s -1 Or 10 8 M -1 s -1In some embodiments, the sclerostin is human sclerostin. In some embodiments, the sclerostin is cynomolgus monkey sclerostin.

[0130] In some embodiments, the K of binding between the antibody moiety and sclerostin off is about 1 s -1 ~about 10 -6 s -1 , about 1 s -1 ~about 10 -2 s -1 , about 10 -2 s -1 ~about 10 -3 s -1 , about 10 -3 s -1 ~about 10 -4 s -1 , about 10 -4 s -1 ~about 10 -5 s -1 , about 10 -5 s -1 ~about 10 -6 s -1 , about 1 s -1 ~about 10 -5 s -1 , about 10 -2 s -1 ~about 10 -6 s -1 , about 10 -3 s -1 ~about 10 -6 s -1 , about 10 -4 s -1 ~about 10 -6 s -1 , about 10 -2 s -1 ~about 10 -5 s -1 , or about 10 -3 s -1 ~about 10 -5 s -1 In some embodiments, the K of binding between the antibody moiety and sclerostin is off is about 1 s -1 , about 10 -2 s -1 , about 10 -3 s-1 , about 10 -4 s -1 , about 10 -5 s -1 or about 10 -6 s -1 In some embodiments, the sclerostin is human sclerostin. In some embodiments, the sclerostin is cynomolgus monkey sclerostin.

[0131] In some embodiments, the binding affinity of the anti-sclerostin antibody portion or anti-sclerostin construct is low (e.g., low K D values) higher than conventional anti-sclerostin antibodies (e.g., romosozumab).

[0132] b) Chimeric or humanized antibodies

[0133] In some embodiments, the anti-sclerostin construct (e.g., anti-sclerostin antibody portion) is a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region of murine origin) and a human constant region. In some embodiments, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed compared to the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0134] In some embodiments, an anti-sclerostin construct (e.g., an anti-sclerostin antibody portion) is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity in humans, while preserving the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains, where the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, a humanized antibody further comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., antibody from which HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0135] For a review of humanized antibodies and methods for their preparation, see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and for further description, see, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents US 5,821,337, US 7,527,791, US 6,982,321, and US 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (description of SDR (a-CDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (description of "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (description of "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (description of the "guided selection" method of FR shuffling).

[0136] The human framework regions used for humanization may be framework regions selected using the "best fitting" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)), framework regions of consensus sequences of human antibodies derived from particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions, or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0137] It should be understood that humanization of murine-derived antibodies is a well-known and commonly used technique. Accordingly, it should be understood that any and all humanized forms of the anti-sclerostin antibodies disclosed in the Sequence Listing can be used in preclinical or clinical settings. If any humanized form of the referenced anti-sclerostin antibodies or their antigen-binding regions is used in such preclinical or clinical settings, the humanized form at that time is expected to have the same or similar biological activity and characteristics as the original, non-humanized form.

[0138] c) Human antibodies

[0139] In some embodiments, the anti-sclerostin construct (e.g., the anti-sclerostin antibody portion) is a human antibody (also called a human domain antibody or human dAb). Human antibodies can be generated using a variety of techniques known in the art. For a description of human antibodies, see generally van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, MolImmunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single domain antibodies (or sdAbs) are known in the art. See, for example, US20090307787A1, US Patent No. US8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0140] Human antibodies (e.g., human dAbs) can be prepared by administering immunogens to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. These animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci, are present extrachromosomally, or are randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., XENOMOUSE. TM(See U.S. Patents US 6,075,181 and US 6,150,584, which describe the HuMab® technology, US 5,770,429, which describes the HuMab® technology, US 7,041,870, which describes the KM MOUSE® technology, and US Patent Application Publication US 2007 / 0061900, which describes the VelociMouse® technology.) The human variable regions derived from intact antibodies produced in such animals can be further modified, for example, by attaching them to different human constant regions.

[0141] Human antibodies (e.g., human dAbs) can be prepared by hybridoma methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). See also Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for a description of human antibodies produced by human B cell hybridoma technology. For other methods, see, for example, U.S. Patent No. 7,189,826 (which describes the production of human monoclonal IgM antibodies using hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006), which describes human-human hybridomas. For a description of human hybridoma technology (trioma technology), see Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005), and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0142] Human antibodies (e.g., human dAbs) can also be generated by isolating Fv clone variable domain sequences selected from human phage display libraries. These variable domain sequences can then be combined with suitable human constant domains. Techniques for selecting human antibodies from antibody libraries are as follows.

[0143] d) Library-derived antibodies

[0144] The anti-sclerostin antibody portions described herein can be used to screen combinatorial libraries to isolate antibodies with the desired activity or activities. For example, phage display libraries can be generated to obtain antibodies with ideal binding characteristics, and various methods for screening such libraries are known in the art. For a review of such methods, see, e.g., Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further descriptions can be found in, e.g., McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003; Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004). Methods for constructing single-domain antibody libraries have been previously described; see, for example, U.S. Patent No. US7,371,849.

[0145] In some phage display methods, V is isolated by polymerase chain reaction (PCR), as described, for example, in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). H and V LGene libraries are individually cloned and randomly recombined in a phage library, followed by screening for antigen-binding phages. Phages typically display antibody fragments in the form of scFv or Fab fragments. Immune-derived libraries provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, as described, for example, in Griffiths et al., EMBO J, 12:725-734 (1993), naive libraries (e.g., human) can be cloned without any immunization to provide single-origin antibodies against a wide range of non-self and self antigens. Finally, as described, for example, in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992), naive libraries can be synthesized by cloning unrearranged V gene fragments from stem cells, using PCR primers containing random sequences to encode hypervariable CDR3 regions, and completing rearrangements in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications US2005 / 0079574, US2005 / 0119455, US2005 / 0266000, US2007 / 0117126, US2007 / 0160598, US2007 / 0237764, US2007 / 0292936, and US2009 / 0002360.

[0146] Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

[0147] e) Substitutions, insertions, deletions and variants

[0148] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutations include HVRs (or CDRs) and FRs. Conservative substitutions are found in the "Preferred Substitutions" section of Table 2. More substantial changes are provided in the "Exemplary Substitutions" section of Table 2 and are described in detail below with reference to types of amino acid side chains. Amino acid substitutions can be introduced into a target antibody, and the product can be screened for the desired activity, e.g., retaining / improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

[0149] [Table 2]

[0150] Amino acids are divided into the following groups according to their general side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect main-chain conformation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0151] Non-conservative substitutions involve replacing one member of one class with another class.

[0152] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further study have altered (e.g., improved) biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or substantially retain certain biological properties of the parent antibody. Exemplary substitutional variants are affinity matured antibodies, which are amenable to generation, for example, using the phage display-based affinity maturation techniques described herein. Briefly, these are variant antibodies in which one or more HVR residues are mutated, displayed on phage, and selected for a particular biological activity (e.g., binding affinity).

[0153] Modifications (e.g., substitutions) may be made in HVRs to, for example, improve antibody affinity. Such modifications may be made in HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting mutant V H or V L The binding affinity of the antibody is then detected. For a description of affinity maturation by constructing a secondary library and reselecting from this library, see, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. The secondary library is then screened to identify any antibody variants with ideal affinities. Another method for introducing diversity involves HVR blending, in which several HVR residues (e.g., every 4-6 residues) are randomly grouped. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

[0154] In some embodiments, substitutions, insertions, or deletions may be made in one or more HVRs as long as such modifications do not significantly reduce the antigen-binding ability of the antibody. For example, conservative modifications (e.g., conservative substitutions provided herein) can be made in HVRs without significantly reducing binding affinity. Such modifications may be outside of HVR "hot spots" or CDRs.

[0155] One useful method for identifying potential antibody residues or regions to target for mutagenesis is called "alanine scanning mutagenesis" and is described, for example, in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether this affects the antibody-antigen interaction. Further substitutions are introduced at the amino acid position to demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex can be used to determine contact points between the antibody and antigen. These contact residues and neighboring residues can be targeted as candidates for substitution or removed. Multiple mutants can be screened to determine whether they have the desired properties.

[0156] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, and intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion of the antibody to the N- or C-terminus of an enzyme (e.g., to ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0157] f) Glycosylation variants

[0158] In some embodiments, an anti-sclerostin construct (e.g., an anti-sclerostin antibody portion) is altered to increase or decrease the degree of structural glycosylation. The addition or deletion of antibody glycosylation sites can be facilitated by altering the amino acid sequence to create or delete one or more glycosylation sites.

[0159] If the antibody moiety comprises an Fc region, the carbohydrate attached thereto can be varied. Natural antibodies produced by mammalian cells typically contain one branched, biantennary oligosaccharide, which is typically N-linked to the Fc region C. H The oligosaccharide is attached to Asn297 of the 2 domain. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and the fucose of GlcNAc attached to the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides on the antibody moiety can be modified to generate antibody variants with improved properties.

[0160] In some embodiments, an anti-sclerostin construct (e.g., an anti-sclerostin antibody portion) has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is measured by calculating the average amount of fucose in the Asn297 glycan relative to the sum of all glycostructures attached to Asn297 (e.g., complex, heteroconjugate, and high mannose structures) as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of residues in the Fc region); however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor antibody sequence variations. Such fucosylation variants may have improved ADCC function. See U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0 109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 0357778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1; Presta, L. and WO 2004 / 056312 A1; Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng, 94(4):680-688 (2006) and WO 2003 / 085107).

[0161] In some embodiments, the anti-sclerostin construct (e.g., anti-sclerostin antibody portion) has a bisected oligosaccharide, e.g., a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. For exemplary descriptions of such antibody variants, see, e.g., WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Pat. No. 2005 / 0123546 (Umana et al.). Further provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. For a description of such antibody variants, see, for example, WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0162] g) Fc region variants and light chain constant region variants In some embodiments, the anti-sclerostin construct (eg, the anti-sclerostin antibody portion) comprises an Fc fragment.

[0163] The terms "Fc region," "Fc domain," "Fc fragment," or "Fc" refer to the C-terminal non-antigen-binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine of the Fc region (Lys447) does not affect the structure or stability of the Fc region and may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in an IgG or Fc region is based on the EU numbering system for antibodies, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0164] In some embodiments, the Fc fragment is derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

[0165] In some embodiments, the Fc fragment has reduced effector function compared to a corresponding wild-type Fc fragment (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% reduced effector function as measured by antibody-dependent cellular cytotoxicity (ADCC) levels).

[0166] In some embodiments, the Fc fragment refers to an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises an L234A mutation and / or an L235A mutation. In some embodiments, the Fc fragment refers to an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment refers to an IgG4 Fc fragment comprising an S228P, F234A, and / or an L235A mutation. In some embodiments, the Fc fragment comprises an N297A mutation. In some embodiments, the Fc fragment comprises an N297G mutation.

[0167] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody portion to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions. In some embodiments, the Fc fragment is derived from a rat Fc region sequence (e.g., a rat IgG2 Fc) or a mouse Fc region sequence (e.g., a mouse IgG1 Fc).

[0168] In some embodiments, the Fc fragment comprises a human IgG2 Fc region.

[0169] In some embodiments, the Fc fragment comprises a human IgG4 Fc region. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising one or more (e.g., two, three, or four) substitutions selected from S228P, T366W, and optionally H435, and optionally Y436F. Unless otherwise specified, the numbering of modifications described herein is according to the EU index. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising S228P, T366W, and optionally H435R, and optionally Y436F. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising one or more (e.g., two, three, four, five, or six) substitutions selected from F126C, L128C, C131S, F170C, P161C, V173C, S228P, T366S, L368A, and Y407V, and optionally H435 and optionally Y436F. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising: a) C131S, S228P, T366S, L368A, and Y407V, b) one substitution selected from F126C, L128C, F170C, P161C, and V173C, and c) optionally H435R and optionally Y436F. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising one or more (e.g., two, three, four, five, or six) substitutions selected from F126C, C131S, S228P, T366S, L368A, and Y407V, and optionally H435R, and optionally Y436F. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising F126C, C131S, S228P, T366S, L368A, and Y407V, and optionally H435R, and optionally Y436F. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising L128C, C131S, S228P, T366S, L368A and Y407V, and optionally H435R, and optionally Y436F.In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising C131S, F170C, S228P, T366S, L368A, and Y407V, and optionally H435R, and optionally Y436F. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising C131S, V173C, S228P, T366S, L368A, Y407V, and optionally H435R, and optionally Y436F. In some embodiments, the Fc fragment comprises a modified human IgG4 heavy chain Fc region comprising C131S, P171C, S228P, T366S, L368A, and Y407V, and optionally H435R, and optionally Y436F.

[0170] In some embodiments, the anti-sclerostin construct comprises an Fc fragment, the Fc fragment comprising: a) a first modified human IgG4 heavy chain Fc region comprising S228P, T366W; and b) a second modified human IgG4 heavy chain Fc region comprising: a) C131S, S228P, T366S, L368A, Y407V, and optionally H435R, and optionally Y436F; and b) one substitution selected from F126C, L128C, F170C, P161C, and V173C.

[0171] In some embodiments, the anti-sclerostin construct comprises an Fc fragment, the Fc fragment comprising a) a first modified human IgG4 heavy chain Fc region, and b) a second modified human IgG4 heavy chain Fc region, wherein the first modified human IgG4 heavy chain Fc region comprises S228P, T366W, and the second modified human IgG4 heavy chain Fc region comprises F126C, C131S, S228P, T366S, L368A, Y407V, H435R, and Y436F.

[0172] In some embodiments, the anti-sclerostin construct comprises an Fc fragment, the Fc fragment comprising: a) a first modified human IgG4 heavy chain Fc region; and b) a second modified human IgG4 heavy chain Fc region, wherein the first modified human IgG4 heavy chain Fc region comprises S228P, T366W, and the second modified human IgG4 heavy chain Fc region comprises L128C, C131S, S228P, T366S, L368A, Y407V, H435R, and Y436F.

[0173] In some embodiments, the anti-sclerostin construct comprises an Fc fragment, the Fc fragment comprising: a) a first modified human IgG4 heavy chain Fc region; and b) a second modified human IgG4 heavy chain Fc region, wherein the first modified human IgG4 heavy chain Fc region comprises S228P, T366W, and the second modified human IgG4 heavy chain Fc region comprises C131S, F170C, S228P, T366S, L368A, Y407V, H435R, and Y436F.

[0174] In some embodiments, the anti-sclerostin construct comprises an Fc fragment, the Fc fragment comprising: a) a first modified human IgG4 heavy chain Fc region; and b) a second modified human IgG4 heavy chain Fc region, wherein the first modified human IgG4 heavy chain Fc region comprises S228P, T366W, and the second modified human IgG4 heavy chain Fc region comprises C131S, V173C, S228P, T366S, L368A, Y407V, H435R, and Y436F.

[0175] In some embodiments, the anti-sclerostin construct comprises an Fc fragment, the Fc fragment comprising: a) a first modified human IgG4 heavy chain Fc region; and b) a second modified human IgG4 heavy chain Fc region, wherein the first modified human IgG4 heavy chain Fc region comprises S228P, T366W, and the second modified human IgG4 heavy chain Fc region comprises C131S, F171C, S228P, T366S, L368A, Y407V, H435R, and Y436F.

[0176] In some embodiments, the anti-sclerostin construct comprises a human Igκ light chain constant region. In some embodiments, the anti-sclerostin construct comprises a modified human Igκ light chain constant region having F118C, S121C, Q160C, S162C, S176C, and / or C214S. In some embodiments, the anti-sclerostin construct comprises a modified human Igκ light chain constant region having S121C and C214S. In some embodiments, the anti-sclerostin construct comprises a modified human Igκ light chain constant region having F118C and C214S. In some embodiments, the anti-sclerostin construct comprises a modified human Igκ light chain constant region having S176C and C214S. In some embodiments, the anti-sclerostin construct comprises a modified human Igκ light chain constant region having Q160C and C214S. In some embodiments, the anti-sclerostin construct comprises a modified human Ig kappa light chain constant region having S162C and C214S.

[0177] In some embodiments, Fc fragments retain some, but not all, effector functions, making them desirable candidates for some applications. In these applications, the in vivo half-life of the antibody portion is important, but some effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 (Ravetch and Kinet, Annu Rev. Immunol. 9:457-492 (1991) p. 464). Examples of in vitro assays for assessing ADCC activity of target molecules are described, but are not limited to, in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods (e.g., flow cytometric ACTI) may be used. TMNon-radioactive cytotoxicity assays, such as those described in Cell Technology, Inc., Mountain View, CA, and CytoTox96® (Promega, Madison, WI), may be employed. Effector cells for use in such assays include peripheral blood monocytes (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule can be assayed in vivo, e.g., as described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Furthermore, C1q binding assays can be performed to confirm the inability of the antibody to bind C1q and thus the lack of CDC activity. See, for example, the ELISA binding of C1q and C3c in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Measurements of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0178] Antibodies with reduced effector function include antibodies with one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc fragment variants include Fc fragment variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc fragment variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). In some embodiments, the Fc fragment comprises an N297A mutation. In some embodiments, the Fc fragment comprises an N297G mutation.

[0179] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0180] In some embodiments, the Fc fragment refers to an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises an L234A mutation and / or an L235A mutation. In some embodiments, the Fc fragment refers to an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising an S228P, F234A, and / or an L235A mutation.

[0181] In some embodiments, the antibody portion comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 in the Fc region (EU numbering of residues).

[0182] In some embodiments, the Fc region has altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0183] In some embodiments, the antibody partial variant comprises a variant Fc region, the variant Fc region comprising amino acid substitutions that alter half-life and / or binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US 2005 / 0014934 A1 (Hinton et al.), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies comprise an Fc region with one or more substitutions that alter binding of the Fc region to FcRn. Such Fc variants include variants with substitutions at one or more of the Fc region residues at positions 250, 252, 254, 256, 307, 308, 428, and 434 (U.S. Patent No. 7,371,826), including the so-called "LS" Fc variant with M428L and N434S (WO 2009 / 086320), and the so-called "YTE" Fc variant with M252Y, S254T, and T256E (WO 2002 / 060919).

[0184] See also Duncan & Winter, Nature 322:738-40 (1988), US Pat. No. 5,648,260, US Pat. No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0185] h) Cysteine ​​Engineered Antibody Variants

[0186] In some embodiments, it is necessary to produce cysteine ​​engineered antibody moieties, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In specific embodiments, the substituted residues are located at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody, allowing the antibody to be coupled to other moieties (e.g., drug moieties or linker-drug moieties) to generate antibody-drug conjugates, as further described herein. In some embodiments, one or more of residues A118 (EU numbering) of the heavy chain and S400 (EU numbering) of the heavy chain Fc region are substituted with cysteine. Cysteine ​​engineered antibody moieties can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0187] i) Antibody derivative

[0188] In some embodiments, the antibody moieties described herein may be further modified to include other nonproteinaceous moieties known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly(N-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene glycol / propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. Polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when more than one type of polymer is attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymer used for derivatization can be determined based on criteria such as, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used in diagnosis in a defined setting, etc.

[0189] In some embodiments, the antibody portion may be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biological activity" or "biologically active" refers to the in vivo biological activity required to perform a specific function. For example, the term refers to combining with a specific biomolecule, such as a protein or DNA, and promoting or inhibiting the activity of such a biomolecule. In some embodiments, biologically active proteins or fragments thereof include proteins and polypeptides administered to patients as active pharmaceutical ingredients to prevent or treat a disease or condition, diagnostic proteins and polypeptides, e.g., enzymes used in diagnostic tests or in vitro detection, and proteins and polypeptides administered to patients to prevent a disease (e.g., vaccines).

[0190] Multispecific anti-sclerostin constructs

[0191] In some embodiments, the anti-sclerostin construct comprises a multispecific (e.g., bispecific) anti-sclerostin construct comprising any of the anti-sclerostin antibody moieties described herein and a second binding moiety (e.g., a second antibody moiety) that specifically recognizes a second antigen. In some embodiments, the multispecific anti-sclerostin molecule comprises an anti-sclerostin antibody moiety and a second antibody moiety that specifically recognizes a second antigen. In some embodiments, the second antigen is an immune checkpoint molecule. In some embodiments, the second antigen is DKK1 (Dickkopf Wnt signaling pathway inhibitor 1) or RANKL (NF-κB receptor activator ligand).

[0192] Multispecific constructs targeting sclerostin and RANKL

[0193] The present application provides multispecific constructs that target sclerostin and RANKL. In some embodiments, the multispecific constructs include a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL. In some embodiments, sclerostin refers to human sclerostin. In some embodiments, RANKL refers to human RANKL.

[0194] RANKL (receptor activator of NF-κB ligand), also known as tumor necrosis factor ligand superfamily member 11 (TNFSF11), is a ligand for osteoprotegerin and is a key factor in osteoclast differentiation and activation. This protein has been shown to be a dendritic cell survival factor involved in regulating T cell-dependent immune responses. T cell activation has been reported to induce expression of this gene, leading to increased osteoclast production and bone loss. This protein activates the anti-apoptotic kinase AKT / PKB through a signaling complex involving SRC kinase and tumor necrosis factor receptor-associated factor (TRAF6), indicating that this protein plays a role in regulating apoptosis. Targeted disruption of the relevant gene in mice leads to severe osteosclerosis and osteoclast deficiency. Deficient mice exhibit early differentiation defects in T and B lymphocytes and fail to form lobulo-alveolar mammary structures throughout gestation.

[0195] Exemplary Anti-RANKL Antibody Moieties

[0196] In some embodiments, the anti-RANKL antibody portion (e.g., scFv) used in the multispecific anti-sclerostin constructs described herein comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), and the antibody portion comprises an antibody portion having a second heavy chain variable region (V) for a binding epitope of RANKL. H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0197] In some embodiments, the anti-RANKL antibody portion (e.g., scFv) used in the multispecific anti-sclerostin construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), and V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68, L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71. H comprises the amino acid sequence of SEQ ID NO: 72 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:73 or a variant of the amino acid sequence having at least about 80% (e.g., at least any one selected from about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0198] In some embodiments, the anti-RANKL moiety comprises HC-CDR1, HC-CDR2, and HC-CDR3, each of which is V HThe amino acid sequences of CDR1, CDR2 and CDR3 in the chain region are set forth in SEQ ID NO: 72, and each of V L The chain region comprises the amino acid sequences of CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:73.

[0199] In some embodiments, a multispecific construct comprising a first antibody portion that specifically recognizes RANKL and a second antibody portion that specifically recognizes sclerostin, wherein the first antibody portion comprises an anti-RANKL single domain antibody (sdAb) portion and the second antibody portion comprises a heavy chain variable region (V H ), the second light chain variable region (V L and a full-length antibody comprising an Fc fragment. In some embodiments, the anti-RANK LsdAb is fused to the two heavy chains of the full-length antibody comprising an Fc fragment. In some embodiments, the anti-RANK LsdAb is fused to the two light chains of the full-length antibody. In some embodiments, the anti-RANK LsdAb is fused to the N-terminus of the heavy or light chain of the full-length antibody. In some embodiments, the anti-RANK LsdAb is fused to the C-terminus of the heavy or light chain of the full-length antibody. In some embodiments, the anti-RANK LsdAb is fused to the full-length antibody via a linker (e.g., any of the linkers described herein). In some embodiments, the anti-RANK LsdAb is fused to the full-length antibody without a linker.

[0200] In some embodiments, a multispecific construct comprising a first antibody portion that specifically recognizes RANKL and a second antibody portion that specifically recognizes sclerostin, wherein the first antibody portion comprises an anti-RANKL single domain antibody (sdAb) portion and the second antibody portion comprises a heavy chain variable region (V H ) and a second light chain variable region (V L ), wherein the construct comprises: a) each chimeric heavy chain comprises, from N-terminus to C-terminus: i) V H ii) the first heavy chain constant domain ("C H1 domain"), iii) an anti-RANKLsdAb, and iv) two chimeric heavy chains comprising an Fc domain, wherein the two Fc domains form an Fc fragment; b) a V L and the light chain constant domain ("C L In some embodiments, the anti-RANK LsdAb is fused to the Fc domain via a first linker. In some embodiments, the anti-RANK LsdAb is fused to the Vc domain via a second linker. H to be merged into

[0201] In some embodiments, a multispecific construct comprising a first antibody portion that specifically recognizes RANKL and a second antibody portion that specifically recognizes sclerostin, wherein the first antibody portion comprises an anti-RANKL single domain antibody (sdAb) portion and the second antibody portion comprises a heavy chain variable region (V H ) and a second light chain variable region (V L ), comprising: a) a first heavy chain comprising, from N-terminus to C-terminus: i) an anti-RANKLsdAb; and ii) a first Fc domain; b) a first heavy chain comprising, from N-terminus to C-terminus: i) a V H ii) the first heavy chain constant domain ("C H 1 domain") and iii) a second heavy chain comprising a second Fc domain, and c) a V L and the light chain constant domain ("C L In one embodiment, the multispecific construct comprises a light chain comprising a first Fc domain and a second Fc domain, wherein the first Fc domain and the second Fc domain form an Fc fragment.

[0202] In some embodiments, one of the first and second Fc domains comprises a T366W mutation and optionally a S354C mutation, and the other Fc domain comprises a T366S mutation, a L368A mutation, a Y407V mutation, and optionally a Y349C mutation.

[0203] In some embodiments, the second antibody portion comprises a second heavy chain variable region (V) for a binding epitope of RANKL. H-2) and a second light chain variable region (V L-2 ) and competes with a third antibody portion comprising V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71. H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68, L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0204] In some embodiments, the anti-sclerostin construct is a multispecific (e.g., bispecific) anti-sclerostin construct comprising: a) an anti-sclerostin antibody moiety described in any of the anti-sclerostin antibody moieties herein; and b) a second antibody moiety (an anti-RANKL antibody moiety, e.g., any of the anti-RANKL antibody moieties described herein) that specifically recognizes RANKL. In some embodiments, the anti-sclerostin V construct is a multispecific (e.g., bispecific) anti-sclerostin V construct. H the anti-sclerostin V antibody comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these HC-CDRs; Lcomprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs.

[0205] In some embodiments, the anti-sclerostin V H the anti-sclerostin V antibody comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these HC-CDRs; L comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:85, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs.

[0206] In some embodiments, the amino acid substitutions are limited to the "exemplary substitutions" shown in Table 2 herein. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 2 herein.

[0207] In some embodiments, the anti-sclerostin construct comprises: a) an anti-sclerostin full-length antibody having two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region (V H ), and each light chain comprises a light chain variable region (V La) a full-length anti-sclerostin antibody comprising: a) an anti-RANKL antibody moiety (e.g., any of the antibody moieties described herein) fused to at least two heavy chains of the full-length anti-sclerostin antibody; and b) a multispecific (e.g., bispecific) anti-sclerostin construct comprising: a) an anti-RANKL antibody moiety (e.g., any of the antibody moieties described herein) fused to at least two heavy chains of the full-length anti-sclerostin antibody. In some embodiments, the anti-RANKL antibody moiety is fused to the N-terminus of the two heavy chains. In some embodiments, the anti-RANKL antibody moiety is fused to the C-terminus of the two heavy chains.

[0208] In some embodiments, the anti-sclerostin construct comprises: a) an anti-RANKL antibody portion comprising a full-length antibody having two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region (V H ), and each light chain comprises a light chain variable region (V L a) an anti-RANKL antibody moiety comprising: ... (e.g., any of the anti-sclerostin antibody moieties described herein) fused to at least one or two heavy chains of an anti-RANKL full-length antibody; and b) an anti-sclerostin antibody moiety (e.g., any of the anti-sclerostin antibody moieties described herein) fused to at least one or two heavy chains of an anti-RANKL full-length antibody. In some embodiments, the anti-sclerostin antibody moiety is fused to the N-terminus of the two heavy chains. In some embodiments, the anti-sclerostin antibody moiety is fused to the C-terminus of the two heavy chains.

[0209] In some embodiments, the anti-sclerostin construct comprises: a) an anti-sclerostin full-length antibody having two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region (V H ), and each light chain comprises a light chain variable region (V L a) a full-length anti-sclerostin antibody comprising: a) an anti-RANKL antibody moiety (e.g., any of the antibody moieties described herein) fused to at least two light chains of the full-length anti-sclerostin antibody; and b) a multispecific (e.g., bispecific) anti-sclerostin construct comprising: a) an anti-RANKL antibody moiety (e.g., any of the antibody moieties described herein) fused to at least two light chains of the full-length anti-sclerostin antibody. In some embodiments, the anti-RANKL antibody moiety is fused to the N-terminus of the two light chains. In some embodiments, the anti-RANKL antibody moiety is fused to the C-terminus of the two light chains.

[0210] In some embodiments, the anti-sclerostin construct comprises: a) an anti-sclerostin full-length antibody comprising two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region (V H ), and each light chain comprises a light chain variable region (V L a) a full-length anti-sclerostin antibody comprising: a) a full-length anti-sclerostin antibody comprising: a) an anti-sclerostin antibody moiety (e.g., any of the antibody moieties described herein) fused to at least two light chains of an anti-RANKL full-length antibody; and b) an anti-sclerostin antibody moiety (e.g., any of the antibody moieties described herein) fused to at least two light chains of an anti-RANKL full-length antibody. In some embodiments, the anti-sclerostin antibody moiety is fused to the N-terminus of the two light chains. In some embodiments, the anti-sclerostin antibody moiety is fused to the C-terminus of the two light chains.

[0211] In some embodiments, a multispecific construct comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, wherein the first antibody portion comprises a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 ), and the second antibody portion comprises a single-chain Fv fragment (scFv) comprising a second heavy chain variable region (V H-2 ), the second light chain variable region (V L-2 and an Fc fragment. In some embodiments, the first antibody portion is fused to one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to one or two light chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the N-terminus of one or two heavy or light chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two heavy or light chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the full-length antibody via a first linker (e.g., any of the linkers described herein). In some embodiments, the first antibody portion is fused to the full-length antibody without a linker. In some embodiments, the first antibody portion is fused to the full-length antibody via a first linker (e.g., any of the linkers described herein). H-1 is connected to V via a second linker (e.g., any linker described herein). L-1 to be merged into

[0212] In some embodiments, a multispecific construct comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, wherein the first antibody portion comprises a first heavy chain variable region (V H-1 ), the first light chain variable region (V L-1 ) and an Fc fragment, wherein the second antibody portion is a full-length antibody comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 In some embodiments, the second antibody portion is fused to the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the two light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the N-terminus of a heavy or light chain of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of one of the heavy or light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the full-length antibody via a first linker (e.g., any of the linkers described herein). In some embodiments, the second antibody portion is fused to the full-length antibody. In some embodiments, the V H-2 is connected to the V via a second linker (e.g., any of the linkers described herein) to allow for proper assembly of the scFv. L-2 In some embodiments, V H-2 is not connected via a linker, V L-2 to be merged into

[0213] In some embodiments, a multispecific construct that specifically recognizes clerostin and RANKL comprises a first antibody portion and a second antibody portion, wherein the first antibody portion comprises a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 ), wherein the second antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), said construct comprising: a) from N-terminus to C-terminus: i) V L-1ii) a first polypeptide having a first light chain comprising a first light chain constant domain (“first CH1 domain”); b) a polypeptide comprising, from N-terminus to C-terminus: i) V H-1 ii) a first heavy chain constant domain (“first CH1 domain”) and iii) a first Fc domain; and c) a second polypeptide having a first heavy chain comprising, from N-terminus to C-terminus: i) V H-2 ii) a second heavy chain comprising a second heavy chain constant domain (a "second CH1 domain") and iii) a second Fc domain; and d) a third polypeptide comprising, from N-terminus to C-terminus: i) V L-2 and ii) a fourth polypeptide having a second light chain comprising a second light chain constant domain ("second CL domain"), wherein the first Fc domain and the second Fc domain form an Fc fragment. In some embodiments, the first antibody portion specifically recognizes sclerostin and the second antibody portion specifically recognizes RANKL. In some embodiments, the first antibody portion specifically recognizes RANKL and the second antibody portion specifically recognizes sclerostin.

[0214] In some embodiments, one of the first and second Fc domains comprises a T366W mutation and optionally a S354C mutation, and the other Fc domain comprises a T366S mutation, a L368A mutation, a Y407V mutation and optionally a Y349C mutation, where numbering is based on the EU index.

[0215] In some embodiments, i) the first CH1 domain and the first CL domain, or ii) the second CH1 domain and the second CL domain each comprise: a) a CH1 domain in which the amino acid at position 141 has been substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or at position 220 (e.g., in IgG1) has been substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 116 has been substituted with a cysteine ​​and the cysteine ​​at position 214 has been substituted with a non-cysteine ​​amino acid; b) a CL domain in which the amino acid at position 168 has been substituted with a cysteine; a) a CH1 domain in which the amino acid at position 164 is substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g. in IgG2 or IgG4) or 220 (e.g. in IgG1) is substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 164 is substituted with a cysteine ​​and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid; b) a CH1 domain in which the amino acid at position 126 is substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g. in IgG2 or IgG4) or 220 (e.g. in IgG1) is substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 128 has been substituted with a cysteine ​​and the cysteine ​​at position 214 has been substituted with a non-cysteine ​​amino acid; d) a CH1 domain in which the amino acid at position 128 has been substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or 220 (e.g., in IgG1) has been substituted with a non-cysteine ​​amino acid; and a CL domain in which the amino acid at position 118 has been substituted with a cysteine ​​and the cysteine ​​at position 214 has been substituted with a non-cysteine ​​amino acid; e) a CH1 domain in which the amino acid at position 170 has been substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or 220 (e.g., in IgG1) has been substituted with a non-cysteine ​​amino acid. a CH1 domain in which the cysteine ​​at position 171 is substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g. in IgG2 or IgG4) or 220 (e.g. in IgG1) is substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 176 is substituted with a cysteine ​​and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid; f) a CH1 domain in which the amino acid at position 171 is substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g. in IgG2 or IgG4) or 220 (e.g. in IgG1) is substituted with a non-cysteine ​​amino acid, and a CL domain in which the cysteine ​​at position 162 is substituted with a cysteine;a) a CL domain in which the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid; b) a CH1 domain in which the amino acid at position 173 is substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or 220 (e.g., in IgG1) is substituted with a non-cysteine ​​amino acid; and c) a CL domain in which the amino acid at position 160 is substituted with a cysteine ​​and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid, wherein numbering is according to the EU index.

[0216] In some embodiments, a multispecific construct comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, wherein each heavy chain of the first antibody portion comprises a first heavy chain variable region (V H-1 ), each light chain comprising a first light chain variable region (V L-1 and a second antibody portion comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), wherein the second antibody portion is fused to the C-terminus of two heavy chains of an anti-sclerostin full-length antibody. H-1 comprises the amino acid sequence of SEQ ID NO:22 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L-1 comprises the amino acid sequence of SEQ ID NO:23 or a variant of the amino acid sequence having at least about 80% (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68,L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71. H-2 comprises the amino acid sequence of SEQ ID NO: 72 or a variant of the amino acid sequence having at least about 80% (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and L-2 comprises the amino acid sequence of SEQ ID NO:73 or a variant of the amino acid sequence having at least about 80% (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0217] In some embodiments, a multispecific construct that specifically recognizes sclerostin and RANKL comprises a first antibody portion and a second antibody portion, wherein the first antibody portion comprises a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 ), wherein the second antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), said construct comprising: a) from N-terminus to C-terminus: i) V L-1 ii) a first polypeptide having a first light chain comprising a first light chain constant domain (“first CH1 domain”); b) a polypeptide comprising, from N-terminus to C-terminus: i) V H-1 ii) a first heavy chain constant domain ("first CH1 domain"), and iii) a first Fc domain; and c) a second polypeptide having a first heavy chain comprising, from N-terminus to C-terminus: i) V H-2 ii) a second heavy chain constant domain ("second CH1 domain"), and iii) a second Fc domain; and d) a third polypeptide having a second heavy chain comprising, from N-terminus to C-terminus: i) V L-2and ii) a fourth polypeptide having a second light chain comprising a second light chain constant domain ("second CL domain"), wherein the first Fc domain and the second Fc domain form an Fc fragment, and wherein the first antibody portion specifically recognizes sclerostin and the second antibody portion specifically recognizes RANKL. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO:22 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L-1 comprises the amino acid sequence of SEQ ID NO:23 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71. H-2 comprises the amino acid sequence of SEQ ID NO: 72 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L-2 comprises the amino acid sequence of SEQ ID NO:73 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0218] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having an amino acid sequence of SEQ ID NO: 134; b) a first light chain variable region (V L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 156; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 157; H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes RANKL.

[0219] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 134; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 158; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 159. H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2and V L-2 provides a multispecific (e.g., bispecific) construct comprising a second antibody portion that specifically recognizes RANKL. In some embodiments, the V H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having any of the amino acid sequences of SEQ ID NO: 20.

[0220] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first light chain variable region (V) and a first heavy chain constant region, wherein V comprises the amino acid sequence of SEQ ID NO: 134; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 160; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 161; H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes RANKL.

[0221] In some embodiments, a) a first variable heavy chain variable region (V H-1a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 134; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain constant region, the second heavy chain comprising the amino acid sequence of SEQ ID NO: 162; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 163; H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes RANKL.

[0222] In some embodiments, a) a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 a first antibody portion that specifically recognizes sclerostin, comprising: H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1 a) a first antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 a second antibody portion that specifically recognizes RANKL, having the V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 68, L-2 provides a multispecific (e.g., bispecific) anti-sclerostin construct comprising a second antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 22, L-1 comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 88 or 169. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 72, L-2 comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the second antibody portion is a V H-2 and V L-2 In some embodiments, the V H-2 V L-2 In some embodiments, the V H-2 is arbitrarily V H-2 V with a single alanine amino acid attached to the target C-terminus L-2In some embodiments, the V H-2 and the V L-2 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 In some embodiments, the scFv is a dsscFv, and is fused without a linker. H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2 In some embodiments, the second antibody portion is fused to the N-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the N-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the first antibody portion is V H-1 and V L-1 In some embodiments, the V H-1 V L-1 In some embodiments, the V H-1 is arbitrarily V H-1 V with a single alanine amino acid attached to the C-terminus of L-1 In some embodiments, the V H-1 and the V L-1 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the scFv is a dsscFv, and is a fusion protein comprising: a) V according to the numbering of SEQ ID NO:60; H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2In some embodiments, the first antibody moiety is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody moiety and the second antibody moiety are fused via a linker (e.g., a GS linker, e.g., (GGGGS)4). In some embodiments, the first antibody moiety and the second antibody moiety are fused without a linker.

[0223] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:126, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:129.

[0224] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:127, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:129.

[0225] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:128, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:129.

[0226] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:133, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:130.

[0227] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:133, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:131.

[0228] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL, the multispecific construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:133, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:132.

[0229] In some embodiments, a) a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 a first antibody portion that specifically recognizes sclerostin, comprising: H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1a) a first antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:85, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 a second antibody portion that specifically recognizes RANKL, comprising: H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 68, L-2 provides a multispecific (e.g., bispecific) anti-sclerostin construct comprising a second antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 89 or 170. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 72, L-2 comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the second antibody portion is a V H-2 and V L-2 In some embodiments, the V H-2 VL-2 In some embodiments, the V H-2 is arbitrarily V H-2 V with a single alanine amino acid attached to the C-terminus of L-2 In some embodiments, the V H-2 and the V L-2 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 In some embodiments, the scFv is fused without a linker. H-2 G44C mutation in V L-2 The antibody fragment was converted to a dsscFv with a H44-L100 disulfide bond consisting of a G100C mutation at V. In some embodiments, the second antibody moiety is fused to the N-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody moiety is fused to the C-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody moiety is fused to the N-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody moiety is fused to the C-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody moiety is a full-length antibody comprising two heavy chains and two light chains, and the first antibody moiety is a full-length antibody comprising two heavy chains and two light chains. H-1 and V L-1 In some embodiments, the V H-1 V L-1 In some embodiments, the V H-1 is arbitrarily V H-1 V with a single alanine amino acid attached to the C-terminus of L-1 In some embodiments, the V H-1 and the V L-1 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1The scFv is a dsscFv, and a) V according to the numbering of SEQ ID NO:60. H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2 In some embodiments, the first antibody portion is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion and the second antibody portion are fused via a linker (e.g., a GS linker, e.g., (GGGGS)3). In some embodiments, the first antibody portion and the second antibody portion are fused without a linker.

[0230] In some embodiments, the anti-RANKL antibody moiety and the anti-sclerostin antibody moiety are fused to each other via a linker (e.g., any of the linkers described herein) in any operable form that can serve as a suitable binding moiety.

[0231] Multispecific construct targeting sclerostin and DKK1

[0232] In some embodiments, the anti-sclerostin construct is a multispecific (e.g., bispecific) anti-sclerostin construct comprising: a) an anti-sclerostin antibody moiety that is any of the anti-sclerostin antibody moieties described herein; and b) a second antibody moiety (anti-DKK1 antibody moiety) that specifically recognizes DKK1.

[0233] DKK1 (Dickkopf-related protein 1) is a member of the Dickkopf protein family. Members of this family are secreted proteins characterized by two cysteine-rich domains that mediate protein-protein interactions. DKK1 binds to the LRP6 coreceptor and inhibits β-catenin-dependent Wnt signaling. This gene plays a role in embryonic development and may play an important role in adult bone formation.

[0234] Exemplary Anti-DKK1 Antibody Moieties

[0235] In some embodiments, the anti-DKK1 antibody portion (e.g., scFv) used in the multispecific anti-sclerostin construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) for a binding epitope of DKK1, and the antibody portion comprises an antibody portion having a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:45, an LC-CDR2 having the amino acid sequence of SEQ ID NO:46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:47.

[0236] In some embodiments, the anti-DKK1 antibody portion (e.g., scFv) used in the multispecific anti-sclerostin construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) for a binding epitope of DKK1, and the antibody portion comprises an antibody portion having a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 50, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:51, an LC-CDR2 having the amino acid sequence of SEQ ID NO:52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:58.

[0237] In some embodiments, the anti-DKK1 antibody portion (e.g., scFv) used in the multispecific anti-sclerostin construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) for a binding epitope of DKK1, and the antibody portion comprises an antibody portion having a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ) competes with an antibody or antibody fragment containing V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:57, an LC-CDR2 having the amino acid sequence of SEQ ID NO:58, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:59.

[0238] In some embodiments, the anti-DKK1 antibody portion (e.g., scFv) used in the multispecific anti-sclerostin construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), and V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, Lcomprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 45, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 47. H comprises the amino acid sequence of SEQ ID NO:60 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:61 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0239] In some embodiments, the anti-DKK1 antibody portion (e.g., scFv) used in the multispecific anti-sclerostin construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), and V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 50, L comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 51, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 58. H comprises the amino acid sequence of SEQ ID NO:62 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), Lcomprises the amino acid sequence of SEQ ID NO:63 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0240] In some embodiments, the anti-DKK1 antibody portion (e.g., scFv) used in the multispecific anti-sclerostin construct comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), and V H comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56, L comprises a C-CDR1 having the amino acid sequence of SEQ ID NO: 57, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 59. H comprises the amino acid sequence of SEQ ID NO:64 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L comprises the amino acid sequence of SEQ ID NO:65 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0241] In some embodiments, each anti-DKK1 moiety is V H HC-CDR1, HC-CDR2 and HC-CDR3, each having the amino acid sequence of CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 60, 62 or 64 in the chain region; LThe chain region includes LC-CDR1, LC-CDR2, and LC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 61, 63, or 65.

[0242] In some embodiments, a) an anti-sclerostin full-length antibody comprising two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region (V H ), and each light chain comprises a light chain variable region (V L a) a full-length anti-sclerostin antibody comprising: ...

[0243] In some embodiments, the anti-DKK1 antibody portion comprises a) a full-length antibody having two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region (V H ), and each light chain comprises a light chain variable region (V L a) an anti-DKK1 antibody portion comprising: a) an anti-DKK1 antibody portion comprising: a) an anti-sclerostin antibody portion (e.g., any of the anti-sclerostin antibody portions described herein) fused to at least one or two heavy chains of an anti-DKK1 full-length antibody; and b) an anti-sclerostin antibody portion (e.g., any of the anti-sclerostin antibody portions described herein) fused to at least one or two heavy chains of an anti-DKK1 full-length antibody. In some embodiments, the anti-sclerostin antibody portion is fused to the N-terminus of the two heavy chains. In some embodiments, the anti-sclerostin antibody portion is fused to the C-terminus of the two heavy chains.

[0244] In some embodiments, a) an anti-sclerostin full-length antibody comprising two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region (V H ), and each light chain comprises a light chain variable region (V La) a full-length anti-sclerostin antibody comprising: a) a heavy chain of the full-length anti-sclerostin antibody; and b) an anti-DKK1 antibody moiety (e.g., any of the antibody moieties described herein) fused to at least two heavy and light chains of the full-length anti-sclerostin antibody. In some embodiments, the anti-DKK1 antibody moiety is fused to the N-terminus of the two light chains. In some embodiments, the anti-DKK1 antibody moiety is fused to the C-terminus of the two light chains.

[0245] In some embodiments, the anti-DKK1 antibody portion comprises a) a full-length antibody having two heavy chains and two light chains, each heavy chain comprising a heavy chain variable region (V H ), and each light chain comprises a light chain variable region (V L a) an anti-DKK1 antibody portion comprising a) an anti-DKK1 antibody portion comprising a) an anti-sclerostin antibody portion (e.g., any of the antibody portions described herein) fused to at least one or two light chains of an anti-DKK1 full-length antibody; and b) an anti-sclerostin antibody portion (e.g., any of the antibody portions described herein) fused to at least one or two light chains of an anti-DKK1 full-length antibody. In some embodiments, the anti-sclerostin antibody portion is fused to the N-terminus of the two light chains. In some embodiments, the anti-sclerostin antibody portion is fused to the C-terminus of the two light chains.

[0246] In some embodiments, the anti-sclerostin antibody portion and the anti-DKK1 antibody portion are fused to each other via a linker (e.g., any linker described herein) in any operable form that adequately serves as a binding moiety. In some embodiments, the anti-sclerostin antibody portion and the anti-DKK1 antibody portion are fused without a linker.

[0247] In some embodiments, a) a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 a first antibody portion that specifically recognizes sclerostin, comprising: H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9,L-1 a) a first antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 a second antibody portion that specifically recognizes DKK1, comprising: H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-2 provides a multispecific (e.g., bispecific) anti-sclerostin construct comprising a second antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:45, an LC-CDR2 having the amino acid sequence of SEQ ID NO:46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:47. In some embodiments, V H-1 comprises the amino acid sequence of SEQ ID NO:22, L-1 comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 88 or 169. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 60, 164 or 166, L-2comprises the amino acid sequence of SEQ ID NO: 61, 165 or 167. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 164, L-2 comprises the amino acid sequence of SEQ ID NO: 165. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 166, L-2 comprises the amino acid sequence of SEQ ID NO: 167. H-2 comprises the amino acid sequence of SEQ ID NO:61, L-2 comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, the first antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the second antibody portion is a full-length antibody comprising V H-2 and V L-2 In some embodiments, the V H-2 is arbitrarily V H-2 V with a single alanine amino acid attached to the C-terminus of L-2 In some embodiments, the V H-2 V L-2 In some embodiments, the V H-2 and the V L-2 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 In some embodiments, the scFv is a dsscFv, and is fused without a linker. H , V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2In some embodiments, the second antibody portion is fused to the N-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the N-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the first antibody portion is V H-1 and V L-1 In some embodiments, the V H-1 V L-1 In some embodiments, the V H-1 is arbitrarily V H-1 V with a single alanine amino acid attached to the C-terminus of L-1 In some embodiments, the V H-1 and the V L-1 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 The scFv is a dsscFv, and is fused without a linker, and a) V according to the numbering of SEQ ID NO:60 H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2In some embodiments, the first antibody portion is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion and the second antibody portion are fused via a linker (e.g., a GS linker, e.g., (GGGGS)3). In some embodiments, the first antibody portion and the second antibody portion are fused without a linker.

[0248] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:91, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:93.

[0249] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:92, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:93.

[0250] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:94, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:96.

[0251] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:95, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:96.

[0252] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:97, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:99.

[0253] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:98, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:99.

[0254] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:100, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:102.

[0255] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:101, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:102.

[0256] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:103, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:104.

[0257] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:103, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:105.

[0258] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:106, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:107.

[0259] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:106, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:108.

[0260] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:109, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:110.

[0261] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:109, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:111.

[0262] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:112, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:113.

[0263] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:112, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:114.

[0264] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:117, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:119.

[0265] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:118, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:119.

[0266] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:123, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:125.

[0267] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:124, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:125.

[0268] In some embodiments, a) a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 a first antibody portion that specifically recognizes sclerostin, comprising: H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1 a) a first antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:85, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 a second antibody portion that specifically recognizes DKK1, comprising: H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-2 provides a multispecific (e.g., bispecific) anti-sclerostin construct comprising a second antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:45, an LC-CDR2 having the amino acid sequence of SEQ ID NO:46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:47. In some embodiments, V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1comprises the amino acid sequence of SEQ ID NO: 89 or 170. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 60, 164 or 166, L-2 comprises the amino acid sequence of SEQ ID NO: 61, 165 or 167. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 164, L-2 comprises the amino acid sequence of SEQ ID NO: 165. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 166, L-2 comprises the amino acid sequence of SEQ ID NO: 167. H-2 comprises the amino acid sequence of SEQ ID NO:61, L-2 comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, the first antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the second antibody portion is a full-length antibody comprising V H-2 and V L-2 In some embodiments, the V H-2 V L-2 In some embodiments, the V H-2 is arbitrarily V H-2 V with a single alanine amino acid attached to the C-terminus of L-2 In some embodiments, the V H-2 and the V L-2 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1and the V L-1 In some embodiments, the scFv is a dsscFv, and is fused without a linker. H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2 In some embodiments, the second antibody portion is fused to the N-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the N-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the first antibody portion is V H-1 and V L-1 In some embodiments, the V H-1 V L-1 In some embodiments, the V H-1 is arbitrarily V H-1 V with a single alanine amino acid attached to the C-terminus of L-1 In some embodiments, the V H-1 and the V L-1 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 In some embodiments, the scFv is fused without a linker. H-1 G44C mutation in V L-1The antibody fragment was converted to a dsscFv with a H44-L100 disulfide bond consisting of a G100C mutation at the H44-L100 position. In some embodiments, the first antibody moiety is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody moiety and the second antibody moiety are fused via a linker (e.g., a GS linker, e.g., (GGGGS)3). In some embodiments, the first antibody moiety and the second antibody moiety are fused without a linker.

[0269] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:115, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:119.

[0270] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:116, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:119.

[0271] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:120, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:122.

[0272] In some embodiments, a multispecific (e.g., bispecific) construct is provided that comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, the multispecific (e.g., bispecific) construct comprising two heavy chains, each heavy chain comprising the amino acid sequence of SEQ ID NO:121, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:122.

[0273] In some embodiments, a) a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 a first antibody portion that specifically recognizes sclerostin, comprising: H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1 a) a first antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:85, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 a second antibody portion that specifically recognizes DKK1, comprising said V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 50, L-2provides a multispecific (e.g., bispecific) anti-sclerostin construct comprising the second antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:51, an LC-CDR2 having the amino acid sequence of SEQ ID NO:52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:58. In some embodiments, V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 89 or 170. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO:62, L-2 comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the second antibody portion is a full-length antibody comprising two heavy chains and two light chains. H-2 and V L-2 In some embodiments, the V H-2 is arbitrarily V H-2 V with a single alanine amino acid attached to the C-terminus of L-2 In some embodiments, the V H-2 V L-2 In some embodiments, the V H-2 and the V L-2 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 In some embodiments, the scFv is a dsscFv, and is fused without a linker.H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2 In some embodiments, the second antibody portion is fused to the N-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the N-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the first antibody portion is V H-1 and V L-1 In some embodiments, the V H-1 V L-1 In some embodiments, the V H-1 is arbitrarily V H-1 V with a single alanine amino acid attached to the C-terminus of L-1 In some embodiments, the V H-1 and V L-1 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 The scFv is a dsscFv, and a) V according to the numbering of SEQ ID NO:60. H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2In some embodiments, the first antibody portion is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion and the second antibody portion are fused via a linker (e.g., a GS linker, e.g., (GGGGS)3). In some embodiments, the first antibody portion and the second antibody portion are fused without a linker.

[0274] In some embodiments, a) a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 a first antibody portion that specifically recognizes sclerostin, comprising: H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1 a) a first antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:85, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 a second antibody portion that specifically recognizes DKK1, comprising said V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, L-2provides a multispecific (e.g., bispecific) anti-sclerostin construct comprising the second antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:57, an LC-CDR2 having the amino acid sequence of SEQ ID NO:58, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:59. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 89 or 170. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO:64, L-1 comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, the first antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the second antibody portion is a full-length antibody comprising V H-2 and V L-2 In some embodiments, the V H-2 V L-2 In some embodiments, the V H-2 is arbitrarily V H-2 V with a single alanine amino acid attached to the C-terminus of L-2 In some embodiments, the V H-2 and the V L-2 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 In some embodiments, the scFv is a dsscFv, and is fused without a linker.H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2 In some embodiments, the second antibody portion is fused to the N-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the N-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the first antibody portion is V H-1 and V L-1 In some embodiments, the V H-1 V L-1 In some embodiments, the V H-1 is arbitrarily V H-2 V with a single alanine amino acid attached to the C-terminus of L-1 In some embodiments, the V H-1 and the V L-1 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 The scFv is a dsscFv, and is fused without a linker, and a) V according to the numbering of SEQ ID NO:60 H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2In some embodiments, the first antibody moiety is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody moiety is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody moiety and the second antibody moiety are fused via a linker (e.g., a GS linker, e.g., (GGGGS)3). In some embodiments, the first antibody moiety and the second antibody moiety are fused without a linker.

[0275] In some embodiments, a) a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 a first antibody portion that specifically recognizes sclerostin, comprising: H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1 a) a first antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 20; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 a second antibody portion that specifically recognizes DKK1, comprising said V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 50, L-2provides a multispecific (e.g., bispecific) anti-sclerostin construct comprising the second antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:51, an LC-CDR2 having the amino acid sequence of SEQ ID NO:52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:58. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 22, L-1 comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 88 or 169. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO:62, L-2 comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the second antibody portion is a full-length antibody comprising two heavy chains and two light chains. H-2 and V L-2 In some embodiments, the V H-2 V L-2 In some embodiments, the V H-2 is arbitrarily V H-2 V with a single alanine amino acid attached to the C-terminus of L-2 In some embodiments, the V H-2 and the V L-2 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the VH-1 and the V L-1 In some embodiments, the scFv is a dsscFv, and is fused without a linker. H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2 In some embodiments, the second antibody portion is fused to the N-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the N-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the first antibody portion is V H-1 and V L-1 In some embodiments, the V H-1 V L-1 In some embodiments, the V H-1 is arbitrarily V H-1 V with a single alanine amino acid attached to the C-terminus of L-1 In some embodiments, the V H-1 and the V L-1 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 The scFv is a dsscFv, and is fused without a linker, and a) V according to the numbering of SEQ ID NO:60 H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2In some embodiments, the first antibody portion is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion and the second antibody portion are fused via a linker (e.g., a GS linker, e.g., (GGGGS)3). In some embodiments, the first antibody portion and the second antibody portion are fused without a linker.

[0276] In some embodiments, a) a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 a first antibody portion that specifically recognizes sclerostin, comprising: H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1 a) a first antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:15, an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:20; and b) a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 a second antibody portion that specifically recognizes DKK1, comprising said V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, L-2provides a multispecific (e.g., bispecific) anti-sclerostin construct comprising a second antibody portion comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:57, an LC-CDR2 having the amino acid sequence of SEQ ID NO:58, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:59. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 22, L-1 comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 88 or 169. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO:64, L-2 comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, the first antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the second antibody portion is a full-length antibody comprising V H-2 and V L-2 In some embodiments, the V H-2 V L-2 In some embodiments, the V H-2 is arbitrarily, V H-2 V with a single alanine amino acid attached to the C-terminus of L-2 In some embodiments, the V H-2 and the V L-2 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the VH-1 and the V L-1 In some embodiments, the scFv is a dsscFv, and is fused without a linker. H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2 In some embodiments, the second antibody portion is fused to the N-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two heavy chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the N-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is fused to the C-terminus of the two light chains of the full-length antibody. In some embodiments, the second antibody portion is a full-length antibody comprising two heavy chains and two light chains, and the first antibody portion is V H-1 and V L-1 In some embodiments, the V H-1 V L-1 In some embodiments, the V H-1 is arbitrarily V H-1 V with a single alanine amino acid attached to the C-terminus of L-1 In some embodiments, the V H-1 and the V L-1 is fused via a linker (e.g., a GS linker, e.g., (GGGGS)). In some embodiments, the V H-1 and the V L-1 The scFv is a dsscFv, and is fused without a linker, and a) V according to the numbering of SEQ ID NO:60 H or V H-2 and b) a G44C mutation in V according to SEQ ID NO:61 numbering. L or V L-2In some embodiments, the first antibody portion is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the first antibody portion and the second antibody portion are fused via a linker (e.g., a GS linker, e.g., (GGGGS)4). In some embodiments, the first antibody portion and the second antibody portion are fused without a linker.

[0277] In some embodiments, a multispecific construct that specifically recognizes sclerostin and DKK1 comprises a first antibody portion and a second antibody portion, wherein the first antibody portion comprises a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 ), wherein the second antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), the construct comprising: a) from N-terminus to C-terminus: i) V L-1 ii) a first polypeptide having a first light chain comprising a first light chain constant domain (“first CH1 domain”); b) a polypeptide comprising, from N-terminus to C-terminus: i) V H-1 ii) a first heavy chain constant domain ("first CH1 domain"), and iii) a first Fc domain; and c) a second polypeptide having a first heavy chain comprising, from N-terminus to C-terminus: i) V H-2 ii) a second heavy chain constant domain ("second CH1 domain"), and iii) a second Fc domain; and d) a third polypeptide having a second heavy chain comprising, from N-terminus to C-terminus: i) V L-2and ii) a fourth polypeptide having a second light chain comprising a second light chain constant domain ("second CL domain"), wherein the first Fc domain and the second Fc domain form an Fc fragment. In some embodiments, the first antibody portion specifically recognizes sclerostin and the second antibody portion specifically recognizes DKK1. In some embodiments, the first antibody portion specifically recognizes DKK1 and the second antibody portion specifically recognizes sclerostin.

[0278] In some embodiments, one of the first and second Fc domains comprises a T366W mutation and optionally a S354C mutation, and the other Fc domain comprises a T366S mutation, a L368A mutation, a Y407V mutation, and optionally a Y349C mutation, where numbering is based on the EU index.

[0279] In some embodiments, i) the first CH1 domain and the first CL domain, or ii) the second CH1 domain and the second CL domain each comprise: a) a CH1 domain in which the amino acid at position 141 has been substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or at position 220 (e.g., in IgG1) has been substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 116 has been substituted with a cysteine ​​and the cysteine ​​at position 214 has been substituted with a non-cysteine ​​amino acid; b) a CL domain in which the amino acid at position 168 has been substituted with a cysteine; a) a CH1 domain in which the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or at position 220 (e.g., in IgG1) is substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 164 is substituted with a cysteine ​​and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid; b) a CH1 domain in which the amino acid at position 126 is substituted with a cysteine ​​and the cysteine ​​at position 220 is substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 121 is substituted with a cysteine ​​and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid; d) CH1 domains in which the amino acid at position 128 has been substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or 220 (e.g., in IgG1) has been substituted with a non-cysteine ​​amino acid, and CL domains in which the amino acid at position 118 has been substituted with a cysteine ​​and the cysteine ​​at position 214 has been substituted with a non-cysteine ​​amino acid; e) CH1 domains in which the amino acid at position 170 has been substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or 220 (e.g., in IgG1) has been substituted with a non-cysteine ​​amino acid. a) a CH1 domain in which the amino acid at position 171 is substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or 220 (e.g., in IgG1) is substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 162 is substituted with a cysteine ​​and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid; b) a CH1 domain in which the amino acid at position 173 is substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or 220 (e.g., in IgG1) is substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 162 is substituted with a cysteine ​​and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid; c) a CH1 domain in which the amino acid at position 173 is substituted with a cysteine ​​and the cysteine ​​at position 131 (e.g., in IgG2 or IgG4) or 220 (e.g., in IgG1) is substituted with a non-cysteine ​​amino acid.a CH1 domain in which the cysteine ​​at position 160 (e.g., in IgG2 or IgG4) or at position 220 (e.g., in IgG1) has been substituted with a non-cysteine ​​amino acid, and a CL domain in which the amino acid at position 160 has been substituted with a cysteine ​​and the cysteine ​​at position 214 has been substituted with a non-cysteine ​​amino acid, numbering being according to the EU index.

[0280] In some embodiments, the first heavy chain comprises a modified human IgG4 heavy chain Fc region having S228P, T366W, H435R and Y436F, the second heavy chain comprises a modified human IgG4 heavy chain Fc region having F126C, C131S, S228P, T366S, L368A and Y407V, and the second light chain comprises a modified human Igκ light chain constant region having S121C and C214S.

[0281] In some embodiments, the first heavy chain comprises a modified human IgG4 heavy chain Fc region having S228P, T366S, L368A and Y407V, the second heavy chain comprises a modified human IgG4 heavy chain Fc region having F126C, C131S, S228P, T366W, H435R and Y436F, and the second light chain comprises a modified human Igκ light chain constant region having S121C and C214S.

[0282] In some embodiments, the first heavy chain comprises a modified human IgG4 heavy chain Fc region having S228P, T366W, H435R and Y436F, the second heavy chain comprises a modified human IgG4 heavy chain Fc region having L128C, C131S, S228P, T366S, L368A and Y407V, and the second light chain comprises a modified human Igκ light chain constant region having F118C and C214S.

[0283] In some embodiments, the first heavy chain comprises a modified human IgG4 heavy chain Fc region having S228P, T366S, L368A and Y407V, the second heavy chain comprises a modified human IgG4 heavy chain Fc region having L128C, C131S, S228P, T366W and H435R, and the second light chain comprises a modified human Igκ light chain constant region having F118C and C214S.

[0284] In some embodiments, the first heavy chain comprises a modified human IgG4 heavy chain Fc region having S228P, T366S, L368A and Y407V, the second heavy chain comprises a modified human IgG4 heavy chain Fc region having F170C, C131S, S228P, T366W, H435R and Y436F, and the second light chain comprises a modified human Igκ light chain constant region having S176C and C214S.

[0285] In some embodiments, the first heavy chain comprises a modified human IgG4 heavy chain Fc region having S228P, T366S, L368A and Y407V, the second heavy chain comprises a modified human IgG4 heavy chain Fc region having V173C, C131S, S228P, T366W, H435R and Y436F, and the second light chain comprises a modified human Igκ light chain constant region having Q160C and C214S.

[0286] In some embodiments, the first antibody portion specifically recognizes sclerostin and the second antibody portion specifically recognizes DKK1. H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-1 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15 or 85, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 20. H-1comprises the amino acid sequence of SEQ ID NO: 22, L-1 comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 88 or 169. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-1 comprises the amino acid sequence of SEQ ID NO: 89 or 160. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 87, L-1 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO: 168, L-1 comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 45, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 47; and b) said V H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 50, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 51, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or c) said V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:57, an LC-CDR2 having the amino acid sequence of SEQ ID NO:58, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:59.

[0287] In some embodiments, the first antibody portion specifically recognizes DKK1 and the second antibody portion specifically recognizes sclerostin. H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15 or 85, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 20. H-2 has the amino acid sequence of SEQ ID NO:22, L-2 has the amino acid sequence of SEQ ID NO: 23. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 87 or 168, L-2comprises the amino acid sequence of SEQ ID NO: 88 or 169. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 87, L-2 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 168, L-2 comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 87 or 169, L-2 comprises the amino acid sequence of SEQ ID NO: 89 or 170. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 87, L-2 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the V H-2 comprises the amino acid sequence of SEQ ID NO: 168, L-2 comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-1 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 45, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 47; and b) said V H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 50, L-1comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 51, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or c) said V H-1 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, L-1 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:57, an LC-CDR2 having the amino acid sequence of SEQ ID NO:58, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:59.

[0288] In some embodiments, a multispecific construct comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1, wherein each heavy chain of the first antibody portion comprises a first heavy chain variable region (V H-1 ), each light chain comprising a first light chain variable region (V L-1 and a second antibody portion comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), wherein the second antibody portion is fused to the C-terminus of the two heavy chains of the anti-sclerostin full-length antibody. H-1 comprises the amino acid sequence of SEQ ID NO:22 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L-1 comprises the amino acid sequence of SEQ ID NO:23 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). H-2comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 45, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 47. H-2 comprises the amino acid sequence of SEQ ID NO:60 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), or L-2 comprises the amino acid sequence of SEQ ID NO:61 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0289] In some embodiments, a multispecific construct that specifically recognizes sclerostin and DKK1 comprises a first antibody portion and a second antibody portion, wherein the first antibody portion comprises a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 ), wherein the second antibody portion comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 ), comprising: a) from the N-terminus to the C-terminus: i) V L-1 ii) a first polypeptide having a first light chain comprising a first light chain constant domain (“first CH1 domain”); b) a polypeptide comprising, from N-terminus to C-terminus: i) V H-1 ii) a first heavy chain constant domain ("first CH1 domain"), and iii) a first Fc domain; and c) a second polypeptide having a first heavy chain comprising, from N-terminus to C-terminus: i) V H-2ii) a second heavy chain constant domain ("second CH1 domain"), and iii) a second Fc domain; and d) a third polypeptide having a second heavy chain comprising, from N-terminus to C-terminus: i) V L-2 and ii) a fourth polypeptide having a second light chain comprising a second light chain constant domain ("second CL domain"), wherein the first Fc domain and the second Fc domain form an Fc fragment, and wherein the first antibody portion specifically recognizes sclerostin and the second antibody portion specifically recognizes DKK1. In some embodiments, the V H-1 comprises the amino acid sequence of SEQ ID NO:22 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L-1 comprises the amino acid sequence of SEQ ID NO:23 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 45, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 47. H-2 comprises the amino acid sequence of SEQ ID NO:60 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), L-2comprises the amino acid sequence of SEQ ID NO:61 or a variant of the amino acid sequence having at least about 80% sequence identity (e.g., at least any one selected from 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0290] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 134; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 136; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 137, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0291] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 134; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain comprising a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 138; and L-2) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 139, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provides multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0292] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 134; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 140; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 141. H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0293] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 134; L-1a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 142; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 143, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0294] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 134; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 135; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 144; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 145. H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0295] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 146; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 147; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 148; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 149, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0296] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 146; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 147; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 150; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 151. H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and VL-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0297] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 146; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 147; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 152; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 153, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0298] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 146; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 147; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 154; and L-2) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 155, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0299] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 146; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 147; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 156; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 157, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0300] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 146; L-1a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 147; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 158; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 159, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0301] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 146; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 147; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 160; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 161; H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0302] In some embodiments, a) a first variable heavy chain variable region (V H-1 a) a first heavy chain having a first heavy chain constant region (V) and a first heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 146; L-1 a) a first light chain having a first variable heavy chain constant region (V) and a first light chain constant region (V) comprising the amino acid sequence of SEQ ID NO: 147; H-2 d) a second heavy chain having a second heavy chain constant region (V) and a second heavy chain constant region, the second heavy chain comprising the amino acid sequence of SEQ ID NO: 162; and L-2 ) and a second light chain having a second light chain constant region, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 163, H-1 and V L-1 comprises a first antibody portion that specifically recognizes sclerostin, H-2 and V L-2 provide multispecific (eg, bispecific) constructs that include a second antibody portion that specifically recognizes DKK1.

[0303] Anti-sclerostin fusion protein or antibody-drug conjugate

[0304] In some embodiments, the anti-sclerostin construct is a fusion protein or antibody drug conjugate comprising an anti-sclerostin antibody portion (eg, an anti-sclerostin scFv) and a second portion.

[0305] In some embodiments, the second moiety comprises a half-life extending moiety. In some embodiments, the half-life extending moiety is an albumin binding moiety (e.g., an albumin binding antibody moiety).

[0306] In some embodiments, the second moiety comprises a drug selected from parathyroid hormone (PTH), a selective estrogen receptor modulator (SERM), a bisphosphonate, a prostaglandin E (PGE) receptor agonist, VEGF, TGFβ, a growth factor (myostatin), and calcitonin.

[0307] In some embodiments, the anti-sclerostin antibody portion and the second portion are fused via a linker (e.g., any of the linkers described in the "Linkers" section). In some embodiments, the anti-sclerostin antibody portion and the second portion are fused without a linker.

[0308] Linker

[0309] In some embodiments, the anti-sclerostin constructs described herein include one or more linkers between two moieties (e.g., the anti-sclerostin antibody moiety and half-life extending moiety, or the anti-sclerostin antibody moiety and second binding moiety in the multispecific constructs described above). The length, degree of flexibility, and / or other properties of the linker used in the anti-sclerostin construct will influence its properties to some extent, including, but not limited to, affinity, specificity, or affinity for one or more particular antigens or epitopes. For example, a long linker can be selected to ensure that two adjacent domains do not spatially interfere with each other. In some embodiments, the linker (e.g., a peptide linker) includes flexible residues (e.g., glycine and serine) to allow adjacent domains to move freely relative to each other. For example, a glycine-serine bisconjugate may be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.

[0310] Other factors to consider in the linker include its effect on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and expected degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, ability to be incorporated into micelles or liposomes, etc.

[0311] Peptide linker

[0312] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence, for example, a sequence derived solely from the hinge region of a heavy chain antibody can be used as a linker (see, for example, WO 1996 / 34103).

[0313] The peptide linker can have any suitable length. In some embodiments, the peptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more amino acids in length. In some embodiments, the peptide linker is 100 or less, 75 or less, 50 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or fewer amino acids in length. In some embodiments, the length of the peptide linker is selected from about 1 to about 10 amino acids, about 1 to about 20 amino acids, about 1 to about 30 amino acids, about 5 to about 15 amino acids, about 10 to about 25 amino acids, about 5 to about 30 amino acids, about 10 to about 30 amino acids, about 30 to about 50 amino acids, about 50 to about 100 amino acids, and about 1 to about 100 amino acids.

[0314] One of the basic technical features of such peptide linkers is that they do not contain any polymerization activity. As is known in the art and described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80), the characteristics of peptide linkers include not promoting secondary structures. A particularly preferred amino acid in "peptide linkers" is Gly. Also preferred are peptide linkers that do not promote any secondary structures. The interconnection of the domains can be provided, for example, by genetic engineering as described in the Examples. Methods for preparing fused, operably linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0315] The peptide linker is a stable linker that is not cleaved by proteases, particularly matrix metalloproteinases (MMPs).

[0316] The linker may be a flexible linker. An exemplary flexible linker is a glycine polymer (G) n (SEQ ID NO:78), glycine-serine polymers (e.g., (GS) n (SEQ ID NO:79), (GSGGS) n (SEQ ID NO:80), (GGGGS) n(SEQ ID NO: 81) and (GGGS) n (SEQ ID NO:82), where n in each sequence is an integer equal to or greater than 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively conformationally undefined and can therefore function as neutral tethers between components. Glycine can occupy more phi-psi space than alanine and is much less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). As known to those skilled in the art, the design of antibody fusion proteins can include a fully or partially flexible linker, such that the linker contains a flexible linker portion and one or more portions that confer a weakly flexible structure to provide the desired antibody fusion protein structure.

[0317] Exemplary linkers also include, for example, (GGGGS) n (SEQ ID NO:81), where n is an integer from 1 to 8, e.g., (GGGGS)3 (SEQ ID NO:76), (GGGGS)4 (SEQ ID NO:77), or (GGGGS)6 (SEQ ID NO:83). In some embodiments, the peptide linker comprises the amino acid sequence of (STSGSGKPGSGEGS) n (SEQ ID NO:84) (where n is an integer of 1 to 3).

[0318] Non-peptide linkers

[0319] Coupling of the two moieties can be achieved by any chemical reaction that bonds the two molecules, so long as the two moieties retain their respective activities, i.e., as long as the two moieties of the anti-sclerostin multispecific antibody can bind to sclerostin and a second antigen, respectively. The linker can be formed by many chemical mechanisms, including covalent bonding, affinity bonding, intercalation, coordinate bonding, and complex formation. In some embodiments, the term "bonding" refers to a covalent bond. Covalent bonding can be achieved either by traditional direct condensation of side chains or by incorporating an external crosslinking molecule. In this case, many bivalent or polyvalent linkers are used to couple protein molecules. For example, representative coupling agents include organic compounds such as thioesters, carbodiimides, succinimidyl esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be an exhaustive list of the various types of coupling agents known in the art, but rather represents the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0320] Linkers used herein are described in references (e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). In some embodiments, non-peptide linkers used herein include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxy-α-methyl-α-(2-pyrimidinyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat. (21558G)), (iv) PEG-100 (PEG-1000)), (v) PEG-1000 (PEG-1000)), (vi) PEG-1000 (PEG-1000)), (vii) PEG-1000 (PEG-1000)), (viii) PEG-1000 (PEG-1000)), (viii ... #21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co., Cat. #2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) coupled with EDC.

[0321] The linkers contain components with different properties, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, which can form antibody fusion proteins with increased stability. Disulfide bonds are less stable than other bonds because they are cleaved in vitro, resulting in less available antibody fusion protein. Sulfo-NHS, in particular, can enhance the stability of carbodiimide coupling. Carbodiimide coupling (such as EDC) when used with sulfo-NHS forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0322] III.Preparation method

[0323] In some embodiments, methods for preparing anti-sclerostin constructs or antibody portions that specifically bind sclerostin, and compositions produced in preparing the anti-sclerostin constructs or antibody portions, such as polynucleotides, nucleic acid constructs, vectors, host cells, or media, are provided. The anti-sclerostin constructs or antibody portions or compositions described herein can be prepared by a variety of methods, as generally described below and in more detail in the examples.

[0324] Antibody expression and production

[0325] The antibodies described herein (including anti-sclerostin monoclonal antibodies, anti-sclerostin bispecific antibodies, and anti-sclerostin antibody portions) can be prepared using any method known in the art, including those described below and in the examples.

[0326] Monoclonal antibodies

[0327] Monoclonal antibodies are obtained from a population of essentially homogeneous antibodies, i.e., the individual antibodies comprising the antibody population are identical except for minor, naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. For example, monoclonal antibodies may be prepared using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be prepared using recombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridoma method, as described above, a mouse or other suitable host animal, such as a hamster or camel, is immunized to induce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing protein. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). For immunization of camels, see Example 1.

[0328] The immunizing agent typically contains an antigen protein or a fusion variant thereof. The immunizing agent consists of purified full-length or truncated sclerostin polypeptide or a variant or fragment thereof (i.e., a peptide). Such peptides may be produced by proteolytic cleavage of a larger polypeptide, by recombinant molecular methods, or chemically synthesized using methods described herein and known in the art. Based on the crystal structure of the complex between human sclerostin and LRP6 (Kim, J. 2020, Nat Commun 11:5357-5357), the tip of the second loop, containing the "IGRGKWWR" motif (SEQ ID NO: 186), is found to be the primary binding determinant for sclerostin binding to the first propeller domain of LRP6. Peptides used as immunizing agents typically have an amino acid sequence consisting of at least 7 contiguous amino acids from the sclerostin amino acid sequence encompassing the "IGRGKWWR" motif in the second loop, as described herein, and preferably at least 8, 9, 10, 11, 12, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 contiguous amino acids, the contiguous amino acids comprising the "IGRGKWWR" motif. Other preferred peptide formulations include 8 or more but not more than 12 contiguous amino acids in the sclerostin sequence encompassing the "IGRGKWWR" motif, or more than 24 contiguous amino acids in the second loop sclerostin polypeptide. Other preferred peptide formulations include any integer number between 8 and 24 contiguous amino acids encompassing the "IGRGKWWR" motif in the second loop, or include polypeptides of the full-length sclerostin sequence. In one embodiment, the immunizing agent is full-length human sclerostin, or preferably a portion of the second loop thereof, e.g., a peptide covering amino acids 110-133, or preferably 119-130, or more preferably 116-126, or more preferably 118-125, in the second loop is synthesized to generate antibodies that specifically recognize the "IGRGKWWR" motif in the second loop of sclerostin, as described herein.Generally, if human-derived cells are required, peripheral blood lymphocytes ("PBLs") are used, and if non-human mammalian cells are required, spleen cells or lymph node cells are used. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent (e.g., polyethylene glycol) to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103.

[0329] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells derived from rodents, cows, and humans. Rat or mouse myeloma cell lines are typically used. The hybridoma cells thus prepared are seeded and grown in a suitable medium. Preferably, the medium contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells.

[0330] Immortalized myeloma cells are preferably cells that fuse efficiently, maintain stable high-level antibody production in selected antibody-producing cells, and are sensitive to culture media (e.g., HAT medium). Mouse myeloma cell lines, such as cell lines derived from MOPC-21 and MPC-11 mouse tumors, are available from the Salk Institute Cell Distribution Center (San Diego, Calif., USA), and SP-2 cells (and their derivatives, e.g., X63-Ag8-653) are available from the American Type Culture Collection (Manassas, Va., USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for use in producing human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0331] The culture medium in which the hybridoma cells are grown is analyzed to produce monoclonal antibodies directed against the antigen of interest. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

[0332] The culture medium in which the hybridoma cells are cultured may be analyzed for the presence of monoclonal antibodies against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody are determined by immunoprecipitation or an in vitro binding assay (e.g., radioimmunoassay (RIA) or enzyme-linked assay (ELISA)). Such techniques and tests are known in the art. For example, binding affinity is determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980). To select monoclonal antibodies that specifically neutralize the ability of sclerostin to inhibit Wnt activity, the resulting hybridomas can be screened by ELISA or BLI to determine the presence of antibodies that react with full-length human sclerostin and the polypeptide from the second loop containing the "IGRGKWWR" motif. Preferably, the hybridomas produce monoclonal antibodies that specifically bind to full-length sclerostin and its peptide containing the "IGRGKWWR" motif, as described in Examples 3 and 4. As described in Example 7, HEK293 / TCF / LEF / Wnt1 reporter gene analysis (Hannoush R N., 2008, PLoS ONE, 3:e3498) is used to select monoclonal antibodies that can block, impair, or inhibit the binding of full-length human sclerostin to low-density lipoprotein receptor-related protein family members, such as LRP5 or LRP6.

[0333] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones are subcloned by limiting dilution and expanded by standard methods (Goding, supra). For this purpose, suitable media include, for example, D-MEM or RPMI-1640 medium. The hybridoma cells are then grown as tumors in a mammal.

[0334] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-agarose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0335] Monoclonal antibodies, as noted above, can also be prepared by recombinant DNA techniques, e.g., those described in U.S. Pat. No. 4,816,567. DNA encoding a monoclonal antibody can be readily isolated by conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of a mouse antibody). Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA can be introduced into a vector and transfected into host cells, e.g., E. coli cells, COS monkey cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not otherwise produce immunoglobulins, thereby enabling the recombinant host cells to synthesize the monoclonal antibody. Reviews on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Rev. 130:151-188 (1992).

[0336] In another embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the use of phage libraries to isolate murine and human antibodies, respectively. Subsequent disclosures have described the generation of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0337] For example, the DNA may be altered by substituting the coding sequence for human heavy and light chain constant domains for the cognate murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such a non-immunoglobulin polypeptide replaces the constant domains of an antibody or replaces the variable domains of one antigen-binding site of an antibody to produce a chimeric bivalent antibody having one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

[0338] The monoclonal antibodies described herein can be monovalent, and methods for their preparation are known in the art. For example, methods involve recombinant expression of immunoglobulin light chains and altered heavy chains. The heavy chains are typically truncated at a site in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residues are substituted with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also applicable to the preparation of monovalent antibodies. Digestion to generate antibody fragments, particularly Fab fragments, can be performed using conventional techniques known in the art.

[0339] Chimeric or hybrid antibodies may be prepared in vitro by known methods in synthetic protein chemistry, including those using crosslinking agents; for example, immunotoxins can be constructed by a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate salts and 4-mercaptobutylpyrimidine methyl.

[0340] Nucleic acid molecules encoding antibody portions

[0341] In some embodiments, a polynucleotide encoding any one of the anti-sclerostin constructs or antibody moieties described herein is provided. In some embodiments, a polynucleotide prepared using any one of the methods described herein is provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an antibody moiety (e.g., an anti-sclerostin antibody moiety). In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an antibody moiety (e.g., an anti-sclerostin antibody moiety). In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding the heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding the light chain.

[0342] In some embodiments, the heavy and light chains are expressed into two separate polypeptides by one nucleic acid molecule or two separate nucleic acid molecules. In some embodiments, for example, when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising the linked heavy and light chains.

[0343] In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody moiety (e.g., an anti-sclerostin antibody moiety) includes a nucleotide sequence encoding a leader sequence, which, when translated, is located at the N-terminus of the heavy or light chain. As noted above, the leader sequence may be the native heavy or light chain leader sequence or may be another heterologous leader sequence.

[0344] In some embodiments, polynucleotide refers to DNA. In some embodiments, polynucleotide refers to RNA. In some embodiments, RNA refers to mRNA.

[0345] Nucleic acid molecules can be constructed by recombinant DNA techniques common in the art. In some embodiments, the nucleic acid molecule is in an expression vector suitable for expression in a selected host cell.

[0346] nucleic acid construct

[0347] In some embodiments, there is provided a nucleic acid construct comprising any one of the polynucleotides described herein. In some embodiments, there is provided a nucleic acid construct prepared by any of the methods described herein.

[0348] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the polynucleotide, hi some embodiments, the polynucleotide corresponds to a gene and the promoter is the wild-type promoter of the gene.

[0349] vector

[0350] In some embodiments, a vector is provided that includes any polynucleotide encoding the heavy and / or light chain of any of the antibody moieties (e.g., anti-sclerostin antibody moieties) described herein or the nucleic acid construct described herein. In some embodiments, a vector is provided that is prepared by any of the methods described herein. The vector includes a polynucleotide encoding any of the anti-sclerostin constructs, such as antibodies, scFvs, fusion proteins, or other forms of constructs described herein (e.g., anti-sclerostin scFv). Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector includes a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, for example, when the antibody is an scFv, the heavy and light chains are expressed as part of a single polypeptide.

[0351] In some embodiments, a first vector comprises a polynucleotide encoding a heavy chain, and a second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (e.g., similar molar amounts or similar masses). In some embodiments, the first vector and the second vector are transfected into host cells in a molar or mass ratio of between 5:1 and 1:5. In some embodiments, the vector encoding the heavy chain and the vector encoding the light chain are used in a mass ratio of 1:1 to 1:5. In some embodiments, the vector encoding the heavy chain and the vector encoding the light chain are used in a mass ratio of 1:2.

[0352] In some embodiments, a vector optimized for expression of a polypeptide in CHO or CHO-derived cells, or NS0 cells is selected. Exemplary descriptions of such vectors are provided, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0353] ●Host cell

[0354] In some embodiments, a host cell is provided comprising any of the polypeptides, nucleic acid constructs, and / or vectors described herein. In some embodiments, a host cell is provided prepared by any of the methods described herein. In some embodiments, the host cell is capable of producing any of the antibody portions described herein under fermentation conditions.

[0355] In some embodiments, antibody portions (e.g., anti-sclerostin antibody portions) described herein are expressed in prokaryotic cells (e.g., bacterial cells) or eukaryotic cells, such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out according to procedures known in the art. Exemplary eukaryotic cells for expressing polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NS0 cells. In some embodiments, antibody portions (e.g., anti-sclerostin antibody portions) described herein are expressed in yeast. See, e.g., U.S. Publication No. US 2006 / 0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy and / or light chains of the antibody portion. For example, in some embodiments, a polypeptide produced in CHO cells has a higher level of sialylation compared to a similar polypeptide produced in 293 cells.

[0356] The nucleic acid(s) can be introduced into the desired host cell by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary method descriptions can be found, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press, 2001. The nucleic acid is transiently or stably transfected into the desired host cells by any suitable method.

[0357] The present application further provides host cells comprising any one of the polynucleotides or vectors described herein. In some embodiments, the present invention provides host cells comprising an anti-sclerostin antibody. Any host cell capable of overexpressing heterologous DNA may be used to isolate genes encoding target antibodies, polypeptides, or proteins. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or B. subtilis) and yeast (e.g., S. cerevisae, S. pombe, or K. lactis).

[0358] In some embodiments, the antibody portion is produced in a cell-free system. For descriptions of non-limiting exemplary cell-free systems, see, e.g., Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0359] Culture medium

[0360] In some embodiments, a medium is provided comprising any of the antibody portions, polynucleotides, nucleic acid constructs, vectors, and / or host cells described herein. In some embodiments, a medium is provided prepared by any of the methods described herein.

[0361] In some embodiments, the medium contains hypoxanthine, aminopterin, and / or thymidine (e.g., HAT medium). In some embodiments, the medium does not contain serum. In some embodiments, the medium contains serum. In some embodiments, the medium refers to D-MEM or RPMI-1640 medium. Romo

[0362] Purification of antibody moieties

[0363] Anti-sclerostin constructs (e.g., anti-sclerostin monoclonal or multispecific antibodies) can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the ROR1 extracellular domain and antibody constant regions. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify anti-sclerostin constructs that bind to the constant region and contain an Fc fragment. Hydrophobic interaction chromatography, such as butyl or phenyl columns, is applicable to the purification of some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange and / or cation exchange chromatography) is also applicable to the purification of some polypeptides, such as antibodies. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) is also applicable to the purification of some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0364] IV. Treatment method

[0365] In some embodiments, provided are methods for treating a disease or condition in an individual, comprising administering to the individual an effective amount of an anti-sclerostin construct or pharmaceutical composition described herein, wherein the disease or condition is a bone-related or cartilage-related disease, a bone marrow or hematological disease, a rare musculoskeletal disease, a muscle-related disease, or cancer.

[0366] The methods described herein are applicable to any bone-related disease or condition. In some embodiments, the conditions include osteogenesis imperfecta, osteoporosis or osteopenia (male and female), osteonecrosis, delayed bone union, non-union of fractures, multiple myeloma, multiple myeloma-related bone disease, primary bone tumors, bone metastases from malignant tumors, inflammatory or infectious bone diseases, osteomalacia, hypercalcemia, Paget's disease, bone loss due to immobility, glucocorticoid-induced bone loss, inflammation-induced bone loss including arthritis-induced bone loss, osteoporosis / osteopenia in astronauts and bone loss due to weightlessness, or other diseases or conditions associated with a) loss of bone mass or quality, or both, and / or b) abnormalities in bone structure and quality. In some embodiments, the bone-related disease refers to osteoporosis or osteopenia. In some embodiments, the bone-related condition refers to osteogenesis imperfecta. In some embodiments, the bone-related condition refers to multiple myeloma and multiple myeloma-related bone disease.

[0367] In some embodiments, the disease or condition refers to a cartilage condition, which in some embodiments includes chondromatosis, chondrodysplasia, achondroplasia, epiphyseal dysplasia, chondrodystrophic myoankylosis, cortical parachondroma, knee cartilage laceration, osteofibrous dysplasia, osteoarthritis, osteogenesis imperfecta, hypophosphatemic rickets, or osteochondrodystrophy.

[0368] In some embodiments, the disease or condition refers to a muscle-related disease, hi some embodiments, the muscle-related disease refers to sarcopenia and cancer-related sarcopenia.

[0369] In some embodiments, the disease or condition refers to cancer (e.g., a hematological malignancy, e.g., multiple myeloma).

[0370] In some embodiments, methods are provided for promoting fusion following bone or joint surgery in an individual, comprising administering to the individual an effective amount of an anti-sclerostin construct (e.g., any of the anti-sclerostin constructs described herein).

[0371] In some embodiments, a method of treating a disease or condition (e.g., a bone-related disease) in an individual comprises administering to said individual an anti-sclerostin construct that effectively assembles, wherein the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL), H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO: 5, iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO: 9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs, L provides a method for detecting an LC-CDR comprising: i) an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15 or 85; ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18; or iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO: 20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs.

[0372] In some embodiments, methods are provided for treating a disease or condition (e.g., a bone-related disease) in an individual, comprising administering to the individual an effective amount of a multispecific construct, wherein the multispecific construct comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes RANKL. In some embodiments, the first antibody portion comprises a first heavy chain variable region (V H -1) and a first light chain variable region (VL-1), HVL-1 comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs; VL-1 comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15 or 85, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the second antibody portion comprises a second heavy chain variable region (V H -2) and a second light chain variable region (V L-2 ), including V H -2 comprises HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0373] In some embodiments, methods are provided for treating a disease or condition (e.g., a bone-related disease) in an individual, comprising administering to the individual an effective amount of a multispecific construct, wherein the multispecific construct comprises a first antibody portion that specifically recognizes sclerostin and a second antibody portion that specifically recognizes DKK1. In some embodiments, the first antibody portion comprises a first heavy chain variable region (V H -1) and a first light chain variable region (VL-1), HVL-1 comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs; VL-1 comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15 or 85, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the second antibody portion comprises a second heavy chain variable region (VH-2) and a second light chain variable region (VL-3). L-2 a) said V H -2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-2 a) the VH-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 45, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 47; b) the VH-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 50; L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 51, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 58, or c) said V H -2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, L-2comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:57, an LC-CDR2 having the amino acid sequence of SEQ ID NO:58, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:59.

[0374] In some embodiments, the subject is a mammal (eg, a human).

[0375] Dosage and Administration of Anti-Sclerostin Constructs

[0376] The dosing regimen (e.g., specific dose and frequency) for administering an anti-sclerostin construct to an individual to treat a disease or condition described herein will vary depending on the particular anti-sclerostin construct (e.g., an anti-sclerostin monoclonal antibody or multispecific antibody, e.g., an anti-sclerostin fusion protein), the mode of administration, and the type of disease or condition being treated.

[0377] In some embodiments of any of the foregoing, an effective amount of the anti-sclerostin construct (e.g., an anti-sclerostin monoclonal or multispecific antibody) is in the range of about 0.001 μg / kg to about 500 mg / kg of total body weight, e.g., about 0.005 μg / kg to about 100 mg / kg, about 0.01 μg / kg to about 50 mg / kg, or about 0.01 μg / kg to about 5 mg / kg.

[0378] In some embodiments, treatment involves multiple administrations of the anti-sclerostin construct (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations of the anti-sclerostin construct). In some embodiments, the anti-sclerostin construct is administered about daily, weekly, twice weekly, monthly, once every three months, once every six months, or once every year.

[0379] Anti-sclerostin constructs can be administered to an individual (e.g., a human) by a variety of routes, including, for example, intravenous, intra-articular, intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesical, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, the anti-sclerostin construct is administered to the individual in a pharmaceutical composition. In some embodiments, a sustained release formulation of the composition may be used. In some embodiments, the composition is injected intravenously. In some embodiments, the composition is injected intraperitoneally. In some embodiments, the composition is injected intravenously. In some embodiments, the composition is injected intraperitoneally. In some embodiments, the composition is injected intramuscularly. In some embodiments, the composition is injected subcutaneously. In some embodiments, the composition is injected intravenously. In some embodiments, the composition is administered orally.

[0380] Combination treatment

[0381] The present application further provides methods of administering an anti-sclerostin construct to an individual to treat a disease or condition (e.g., a bone-related disease), comprising administering a second drug or therapy. In some embodiments, the second drug or therapy is a standard or common drug or therapy for treating the disease or condition.

[0382] In some embodiments, methods are provided for treating a disease or condition (e.g., a bone-related disease) in an individual, comprising administering to the individual: a) an effective amount of an anti-sclerostin construct (e.g., any of the anti-sclerostin constructs described herein); and b) a second therapy or drug. In some embodiments, the second therapy or drug is an anti-DKK1 antibody or an anti-RANKL antibody. In some embodiments, the second drug or therapy comprises a drug selected from parathyroid hormone (PTH), a selective estrogen receptor modulator (SERM), a bisphosphonate, a prostaglandin E (PGE) receptor agonist, VEGF, TGFβ, a growth factor (myostatin), and calcitonin.

[0383] In some embodiments, methods are provided for treating a disease or condition (e.g., a bone-related disease) in an individual, comprising administering to the individual: a) an effective amount of an anti-sclerostin construct (e.g., any of the anti-sclerostin constructs described herein); and b) an anti-DKK1 antibody. In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL), H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs; and the VL comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15 or 85, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the anti-DKK1 antibody comprises a second heavy chain variable region (V H -2) and the second light chain variable region (V L-2 ), including V H -2 comprises HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 68, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:69, an LC-CDR2 having the amino acid sequence of SEQ ID NO:70, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:71.

[0384] In some embodiments, methods are provided for treating a disease or condition (e.g., a bone-related disease) in an individual, comprising administering to the individual: a) an effective amount of an anti-sclerostin construct (e.g., any of the anti-sclerostin constructs described herein); and b) an anti-RANKL antibody. In some embodiments, the anti-sclerostin construct comprises an antibody portion, the antibody portion comprising a heavy chain variable region (V H ) and a light chain variable region (VL), H comprises i) an HC-CDR1 having the amino acid sequence of SEQ ID NO:1, ii) an HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and iii) an HC-CDR3 having the amino acid sequence of SEQ ID NO:9, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs; and the VL comprises i) an LC-CDR1 having the amino acid sequence of SEQ ID NO:15 or 85, ii) an LC-CDR2 having the amino acid sequence of SEQ ID NO:18, and iii) an LC-CDR3 having the amino acid sequence of SEQ ID NO:20, or a variant having up to 5, 4, 3, 2, or 1 amino acid substitution in these LC-CDRs. In some embodiments, the anti-RANKL antibody comprises a second heavy chain variable region (VH-2) and a second light chain variable region (VH-3). L-2 a) said V H -2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 44, L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 45, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 47; and b) said V H -2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 50, L-2comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 51, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 58; or c) said V H V-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, or L-2 comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO:57, an LC-CDR2 having the amino acid sequence of SEQ ID NO:58, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:59.

[0385] In some embodiments, the anti-sclerostin construct is administered in conjunction with a second drug or therapy. In some embodiments, the anti-sclerostin construct is administered in conjunction with a second drug or therapy. In some embodiments, the anti-sclerostin construct is administered sequentially with the second drug or therapy.

[0386] V. INGREDIENTS, KITS AND PRODUCTS

[0387] The present application further provides compositions (e.g., formulations, e.g., pharmaceutical compositions) comprising the anti-sclerostin constructs or anti-sclerostin antibody portions described herein, nucleic acids encoding the antibody portions, vectors of nucleic acids encoding the antibody portions, or host cells containing the nucleic acids or vectors.

[0388] In some embodiments, a pharmaceutical composition is provided comprising an anti-sclerostin construct (e.g., any of the anti-sclerostin constructs described herein) and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a drug selected from parathyroid hormone (PTH), a selective estrogen receptor modulator (SERM), VEGF, TGFβ, a growth factor (myostatin), and calcitonin.

[0389] Suitable formulations of the anti-sclerostin constructs described herein can be obtained by mixing an anti-sclerostin construct or anti-sclerostin antibody portion having the desired purity with any selected pharmaceutically acceptable carrier, excipient, or stabilizer, and are present in the form of a lyophilized formulation or aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, adjuvants, or stabilizers are non-toxic to subjects at the dosages and concentrations used and include the following: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, sorbitol, and the like; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or, for example, TWEEN. TM , PLURONICS TM or non-ionic surfactants such as polyethylene glycol (PEG). Lyophilized formulations suitable for subcutaneous injection are described, for example, in WO 97 / 04801. Such lyophilized formulations can be reconstituted with an appropriate diluent to a high protein concentration, and the reconstituted formulation can be injected subcutaneously into the individual to be imaged, diagnosed, or treated.

[0390] Formulations for internal administration must be sterile, which is readil...

Claims

1. An anti-sclerostin construct comprising an antibody portion that specifically recognizes sclerostin, the antibody portion comprising a heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ), The V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, SEQ The VHC-CDR2 has the amino acid sequence of SEQ ID NO: 5, and the HC-CDR3 has the amino acid sequence of SEQ ID NO:

9. Ｌ comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having any one of the amino acid sequences of SEQ ID NO: 20; The V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, SEQ The VHC-CDR2 has the amino acid sequence of SEQ ID NO: 5, and the HC-CDR3 has the amino acid sequence of SEQ ID NO:

9. Ｌ comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 85, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having the amino acid sequence of any one of SEQ ID NO: 20, The V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, SEQ The VHC-CDR2 has the amino acid sequence of SEQ ID NO: 5, and the HC-CDR3 has the amino acid sequence of SEQ ID NO:

9. Ｌ comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 86, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having the amino acid sequence of any one of SEQ ID NO: 20, The V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, SEQ HC-CDR2 having the amino acid sequence of SEQ ID NO: HC-CDR3 having an amino acid sequence of 10, Ｌ comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having any one of the amino acid sequences of SEQ ID NO: 20; The V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO:2, SEQ HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, Ｌ comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having any one of the amino acid sequences of SEQ ID NO: 20; The V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO: 3, SEQ HC-CDR2 having the amino acid sequence of SEQ ID NO: 6, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, Ｌ comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having any one of the amino acid sequences of SEQ ID NO: 20; The V Ｈ comprises an HC-CDR1 having an amino acid sequence of SEQ ID NO: 199, an HC-CDR2 having an amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 having an amino acid sequence of SEQ ID NO: 10; Ｌ comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having any one of the amino acid sequences of SEQ ID NO: 20; The V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO: 3, SEQ HC-CDR2 having the amino acid sequence of SEQ ID NO: 200, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 10, Ｌ comprises an LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and an LC-CDR3 having the amino acid sequence of any one of SEQ ID NO: 20, or The V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO: 4, SEQ The VHC-CDR2 has an amino acid sequence of SEQ ID NO: 8, and the HC-CDR3 has an amino acid sequence of SEQ ID NO:

11. Ｌ an anti-sclerostin construct comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:17, an LC-CDR2 having the amino acid sequence of SEQ ID NO:19, and an LC-CDR3 having the amino acid sequence of any one of SEQ ID NO:

21.

2. An anti-sclerostin construct comprising an antibody portion that specifically recognizes sclerostin, the antibody portion comprising a heavy chain variable region (V Ｈ ) and the light chain variable region (V Ｌ ), a) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

22. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

23. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: b) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

24. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

25. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: c) HC-CDR1, HC-CDR2 and HC-CDR3 are each represented by SEQ ID NO: V having the sequence shown in NO:26 Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

27. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: d) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

28. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

29. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: e) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

30. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

31. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: f) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

32. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

33. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: g) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

34. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

35. Ｌ LC-CDR1, LC-CDR2 and LC-CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 in h) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

36. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

37. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: i) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

38. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

39. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: j) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

40. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

41. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: k) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

87. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

88. Ｌ and the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: l) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

87. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

89. Ｌ or m) HC-CDR1, HC-CDR2 and HC-CDR3 each have the sequence shown in SEQ ID NO:

87. Ｈ and LC-CDR1, LC-CDR2 and LC-CDR3 each have the sequence shown in SEQ ID NO:

90. Ｌ An anti-sclerostin construct having the amino acid sequences of CDR1, CDR2 and CDR3 in:

3. a) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 22, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 23, b) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 24, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 25, c) Said V Ｈ comprises the amino acid sequence of SEQ ID NO: 26, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 27, d) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 28, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 29, e) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 30, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 31, f) Said V Ｈ comprises the amino acid sequence of SEQ ID NO: 32, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 33, g) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 34, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 35, h) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 36, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 37, i) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 38, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 39, j) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 40, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 41, k) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 87, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 88, l) Said V Ｈ comprises the amino acid sequence of SEQ ID NO: 87, The V Ｌ comprises the amino acid sequence of SEQ ID NO: 89; or m) Said V Ｈ comprises the amino acid sequence of SEQ ID NO: 87, The V Ｌ The anti-sclerostin construct of claim 1 or claim 2, comprising the amino acid sequence of SEQ ID NO:

90.

4. The antibody portion comprises a heavy chain variable region (V Ｈ ) and a second light chain variable region (V Ｌ ), a) the V Ｈ HC-CDR1 having the amino acid sequence of SEQ ID NO: 1; HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO:9; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 having an amino acid sequence of any one of SEQ ID NO: 20; b) the V Ｈ teeth, HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO: 10; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 having an amino acid sequence of any one of SEQ ID NO: 20; c) Said V Ｈ teeth, HC-CDR1 having the amino acid sequence of SEQ ID NO: 2, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO: 10; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 having an amino acid sequence of any one of SEQ ID NO: 20; d) the V Ｈ teeth, HC-CDR1 having the amino acid sequence of SEQ ID NO: 3, HC-CDR2 having the amino acid sequence of SEQ ID NO:6, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO: 10; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 having an amino acid sequence of any one of SEQ ID NO: 20; e) the V Ｈ teeth, HC-CDR1 having the amino acid sequence of SEQ ID NO: 199, HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO: 10; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 16, LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 having an amino acid sequence of any one of SEQ ID NO: 20; f) Said V Ｈ teeth, HC-CDR1 having the amino acid sequence of SEQ ID NO: 3, HC-CDR2 having the amino acid sequence of SEQ ID NO: 200, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO: 10; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 15, LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and LC-CDR3 having an amino acid sequence of any one of SEQ ID NO: 20; g) the V Ｈ teeth, HC-CDR1 having the amino acid sequence of SEQ ID NO: 4, HC-CDR2 having the amino acid sequence of SEQ ID NO: 8, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO: 11; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 17, LC-CDR2 having the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 having an amino acid sequence of any one of SEQ ID NO: 21, h) the V Ｈ teeth, HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO:9; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 85, LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and or an LC-CDR3 having the amino acid sequence of any one of SEQ ID NO: 20; i) the V Ｈ teeth, HC-CDR1 having the amino acid sequence of SEQ ID NO: 1, HC-CDR2 having the amino acid sequence of SEQ ID NO:5, and comprising a HC-CDR3 having the amino acid sequence of SEQ ID NO:9; The V Ｌ teeth, LC-CDR1 having the amino acid sequence of SEQ ID NO: 86, LC-CDR2 having the amino acid sequence of SEQ ID NO: 18, and 3. The anti-sclerostin construct of claim 1 or claim 2, comprising an LC-CDR3 having an amino acid sequence of any one of SEQ ID NO:

20.

5. The antibody portion may be a full-length antibody, a bispecific antibody, a single chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, an F(ab') fragment, or a combination thereof. ２ , Fv fragment, disulfide bond stabilized Fv fragment (dsFv), disulfide bond stabilized scFv (dsscFv), (dsFv) ２ 5. The anti-sclerostin construct of any one of claims 1 to 4, which is an antibody or antigen-binding fragment thereof selected from the group consisting of an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody and a tetrabody.

6. The anti-sclerostin construct of any one of claims 1 to 4, wherein the construct is a full-length antibody comprising an Fc fragment.

7. The anti-sclerostin construct of claim 5 , wherein the antibody portion is an scFv fragment.

8. The anti-sclerostin construct of any one of claims 1 to 7, wherein the sclerostin is human sclerostin.

9. The anti-sclerostin construct of any one of claims 1 to 5, 7 and 8, further comprising a second moiety.

10. 10. The anti-sclerostin construct of claim 9, wherein the second portion comprises a second antibody portion that specifically recognizes an antigen.

11. The anti-sclerostin construct of claim 10, wherein the second antibody portion comprises a second heavy chain variable region (VH-2) and a light chain variable region (VL-2).

12. The anti-sclerostin construct of claim 10 or 11, wherein the antigen is DKK1 or RANKL.

13. The anti-sclerostin construct of claim 12, wherein the DKK1 is human DKK1 or the RANKL is human RANKL.

14. The V Ｈ－２ has an HC-CDR1 having the amino acid sequence of SEQ ID NO: 42, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 43, and and a HC-CDR3 having the amino acid sequence of SEQ ID NO:44, Ｌ－２ LC-CDR1 having the amino acid sequence of SEQ ID NO: 45, LC-CDR2 having the amino acid sequence of SEQ ID NO: 46, and or comprising an LC-CDR3 having the amino acid sequence of SEQ ID NO:47; The V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:

50. or said V L-2 comprises an HC-CDR3 having the amino acid sequence of SEQ ID NO:50, said V L-2 comprising an LC-CDR1 having the amino acid sequence of SEQ ID NO:51, an LC-CDR2 having the amino acid sequence of SEQ ID NO:52, and an LC-CDR3 having the amino acid sequence of SEQ ID NO:58; or The V H-2 comprises an HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and an HC-CDR3 having the amino acid sequence of SEQ ID NO:

56.

12. The anti-sclerostin construct of claim 11, wherein the V L-2 comprises a HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, and the V L-2 comprises a LC-CDR1 having the amino acid sequence of SEQ ID NO: 57, a LC-CDR2 having the amino acid sequence of SEQ ID NO: 58, and a LC-CDR3 having the amino acid sequence of SEQ ID NO:

59.

15. The V Ｈ－２ has an HC-CDR1 having the amino acid sequence of SEQ ID NO: 66, an HC-CDR2 having the amino acid sequence of SEQ ID NO: 67, and HC-CDR3 having the amino acid sequence of NO:68, The V Ｌ－２ LC-CDR1 having the amino acid sequence of SEQ ID NO: 69, LC-CDR2 having the amino acid sequence of SEQ ID NO: 70, and The anti-sclerostin construct of claim 11, comprising an LC-CDR3 having the amino acid sequence of NO:

71.

16. The second antibody portion may be a full length antibody, a Fab, a Fab', a (Fab') ２ 12. The anti-sclerostin construct of claim 10 or 11, which is an Fv, a single chain Fv (scFv) fragment, scFv-scFv, a miniature antibody, a diabody or an sdAb.

17. The second antibody portion is a full-length antibody comprising two heavy chains, two light chains, and an Fc fragment, and the anti-sclerostin antibody portion is a V Ｌ V fused with Ｈ or a single chain Fv (scFv) fragment comprising the second antibody portion is a single chain Fv (scFv) fragment comprising a VH-2 and a VL-2, and the anti-sclerostin antibody portion is a full length antibody comprising two heavy chains, two light chains and an Fc fragment; The anti-sclerostin construct of claim 16.

18. the scFv fragment is fused to one or two heavy chains of the full-length antibody, or The anti-sclerostin construct of claim 17 , wherein the scFv fragment is fused to one or two light chains of the full-length antibody.

19. The scFv fragment is fused to the N-terminus of one or two heavy or light chains of a full-length antibody, or The anti-sclerostin construct of claim 17 , wherein the scFv fragment is fused to the C-terminus of one or two heavy or light chains of the full-length antibody.

20. the scFv fragment is fused to the full-length antibody via a first linker; or The anti-sclerostin construct of any one of claims 17 to 19, wherein the scFv fragment is fused to the full-length antibody without a linker.

21. The anti-sclerostin construct of claim 20, wherein the first linker is a GS linker selected from SEQ ID NOs: 74-84.

22. The V Ｈ is connected to the V via a second linker Ｌ or the V H-2 is fused to the V L-2 via a second linker; or said V H-2 is fused to said V L-2 without a linker; The anti-sclerostin construct of any one of claims 17 to 21.

23. The scFv fragment has from the N-terminus to the C-terminus Ｈ , a second linker and V Ｌ or The scFv fragment comprises, from N-terminus to C-terminus, a V H-2 , a second linker, and a V L-2 ; The anti-sclerostin construct of claim 22.

24. The scFv fragment has from the N-terminus to the C-terminus Ｌ , a second linker, V Ｈ、 and, optionally, a C-terminal alanine residue; or The scFv fragment comprises, from N-terminus to C-terminus, V L-2 , a second linker, V H-2 , and optionally a C-terminal alanine residue. The anti-sclerostin construct of claim 22.

25. The anti-sclerostin construct of claim 23 or 24, wherein the second linker comprises the amino acid sequence of SEQ ID NO: 76 or 77.

26. The anti-sclerostin construct of any one of claims 17 to 25, wherein the scFv is a disulfide bond stabilized scFv ("dsscFv").

27. The dsscFv comprises: a) a V Ｈ Or V Ｈ－２ and b) a G44C mutation in V according to the numbering of SEQ ID NO:

61. Ｌ Or V Ｌ－２ The anti-sclerostin construct of claim 26, comprising a G100C mutation in

28. The construct comprises: 1) two heavy chains fused to the anti-sclerostin antibody moiety or a second antibody moiety, each heavy chain comprising the amino acid sequence of SEQ ID NO:91, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:93; 2) two heavy chains fused to the anti-sclerostin antibody moiety or a second antibody moiety, each heavy chain comprising the amino acid sequence of SEQ ID NO: 92, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 93; 3) two heavy chains fused to the anti-sclerostin antibody moiety or a second antibody moiety, each heavy chain comprising the amino acid sequence of SEQ ID NO:94, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:96; 4) two heavy chains fused to the anti-sclerostin antibody moiety or a second antibody moiety, each heavy chain comprising the amino acid sequence of SEQ ID NO:95, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:96; 5) two heavy chains fused to the anti-sclerostin antibody moiety or a second antibody moiety, each heavy chain comprising the amino acid sequence of SEQ ID NO: 97, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 99; 6) fused to the anti-sclerostin antibody portion or a second antibody portion, each heavy chain having SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO:98 and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO:99; 7) two heavy chains fused to the anti-sclerostin antibody moiety or a second antibody moiety, each heavy chain comprising the amino acid sequence of SEQ ID NO: 100, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 102; 8) two heavy chains fused to the anti-sclerostin antibody moiety or a second antibody moiety, each heavy chain comprising the amino acid sequence of SEQ ID NO: 101, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 102; 9) two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 103, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 104, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 10) two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 103, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 105, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 11) two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 106, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 107, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 12) a heavy chain comprising the amino acid sequence of SEQ ID NO: 106, and a light chain comprising the amino acid sequence of SEQ ID NO: 108, fused to the anti-sclerostin antibody portion or a second antibody portion; 13) two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 109, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 110, fused to the anti-sclerostin antibody portion or a second antibody portion; 14) two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 109, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 111, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 15) two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 112, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 113, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 16) two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 112, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 114, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 17) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 115, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 119; 18) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 116, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 119; 19) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 117, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 119; 20) fused to the anti-sclerostin antibody portion or a second antibody portion, each heavy chain having SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 118, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 119; 21) The anti-sclerostin antibody moiety or a second antibody moiety is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 120, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 122; 22) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 121, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 122; 23) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 123, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 125; 24) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 124, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 125; 25) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 126 and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 129; 26) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 127, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 129; 27) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 128, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 129; 28) Two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 133, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 130, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 29) Two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 133, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 131, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 30) Two heavy chains, each of which comprises the amino acid sequence of SEQ ID NO: 133, and two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 132, fused to the anti-sclerostin antibody moiety or a second antibody moiety; 31) The anti-sclerostin antibody moiety or a second antibody moiety is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 177, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 125; 32) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 178, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 125; 33) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 178, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 122; 34) The anti-sclerostin antibody portion or a second antibody portion fused thereto, each heavy chain having SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 179 and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 122; or 35) The anti-sclerostin antibody portion or the second antibody portion is fused to each heavy chain of SEQ ID NO: two heavy chains comprising the amino acid sequence of SEQ ID NO: 180, and two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 122; The anti-sclerostin construct of any one of claims 17 to 27, comprising:

29. The construct comprises: a) a first polypeptide comprising a first light chain, the first light chain comprising from N-terminus to C-terminus: i) V Ｌ ii) a first polypeptide comprising a first light chain constant domain ("first CL domain"); b) a second polypeptide comprising a first heavy chain, said first heavy chain comprising from N-terminus to C-terminus i) V Ｈ ii) a first heavy chain constant domain ("first CH1 domain"); and iii) a second polypeptide comprising a first Fc domain; c) a third polypeptide comprising a second heavy chain, the second heavy chain comprising from N-terminus to C-terminus: i) V Ｈ－２ ii) a second heavy chain constant domain (a "second CH1 domain"); and iii) a third polypeptide comprising a second Fc domain; and d) a fourth polypeptide comprising a second light chain, the second light chain comprising from N-terminus to C-terminus: i) V Ｌ－２ ii) a fourth polypeptide comprising a second light chain constant domain ("second CL domain"); 16. The anti-sclerostin construct of any one of claims 11 to 15, comprising:

30. either the first Fc domain or the second Fc domain comprises a T366W mutation and optionally a S354C mutation; 30. The anti-sclerostin construct of claim 29, wherein the other Fc domain comprises a T366S mutation, a L368A mutation, a Y407V mutation and optionally a Y349C mutation, the numbering being based on the EU index.

31. i) a first CH1 domain and a first CL domain, or ii) a second CH1 domain and a second CL domain, a) a CH1 domain in which the amino acid at position 141 has been replaced by a cysteine ​​and the cysteine ​​at position 131 or 220 has been replaced by a non-cysteine ​​amino acid; and a CL domain in which the amino acid at position 116 has been replaced by a cysteine ​​and the cysteine ​​at position 214 has been replaced by a non-cysteine ​​amino acid; b) a CH1 domain in which the amino acid at position 168 has been replaced by a cysteine ​​and the cysteine ​​at position 131 or 220 has been replaced by a non-cysteine ​​amino acid; and a CL domain in which the amino acid at position 164 is replaced by a cysteine ​​and the cysteine ​​at position 214 is replaced by a non-cysteine ​​amino acid; c) a CH1 domain in which the amino acid at position 126 has been replaced by a cysteine ​​and the cysteine ​​at position 131 or 220 has been replaced by a non-cysteine ​​amino acid; and a CL domain in which the amino acid at position 121 is replaced by a cysteine ​​and the cysteine ​​at position 214 is replaced by a non-cysteine ​​amino acid; d) a CH1 domain in which the amino acid at position 128 has been replaced by a cysteine ​​and the cysteine ​​at position 131 or 220 has been replaced by a non-cysteine ​​amino acid; and a CL domain in which the amino acid at position 118 has been replaced by a cysteine ​​and the cysteine ​​at position 214 has been replaced by a non-cysteine ​​amino acid; e) a CH1 domain in which the amino acid at position 170 has been replaced by a cysteine ​​and the cysteine ​​at position 131 or 220 has been replaced by a non-cysteine ​​amino acid; and a CL domain in which the amino acid at position 176 has been replaced by a cysteine ​​and the cysteine ​​at position 214 has been replaced by a non-cysteine ​​amino acid; f) a CH1 domain in which the amino acid at position 171 has been replaced by a cysteine ​​and the cysteine ​​at position 131 or 220 has been replaced by a non-cysteine ​​amino acid; and a CL domain in which the amino acid at position 162 is replaced by a cysteine ​​and the cysteine ​​at position 214 is replaced by a non-cysteine ​​amino acid; g) a CH1 domain in which the amino acid at position 173 has been replaced by a cysteine ​​and the cysteine ​​at position 131 or 220 has been replaced by a non-cysteine ​​amino acid; and a CL domain in which the amino acid at position 160 has been replaced by a cysteine ​​and the cysteine ​​at position 214 has been replaced by a non-cysteine ​​amino acid; 31. The anti-sclerostin construct of claim 29 or claim 30, selected from the group consisting of:

32. 1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 135; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 134; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 136; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 137; 2) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 135; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 134; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 138; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 139; 3) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 135; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 134; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 140; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 141; 4) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 135; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 134; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 142; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 143; 5) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 135; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 134; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 144; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 145; 6) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 146; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 148; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 149; 7) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 146; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 150; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 151; 8) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 146; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 152; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 153; 9) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 146; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 154; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 155; 10) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 146; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 156; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 157; 11) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 146; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 158; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 159; 12) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 146; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 160; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 161; 13) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 146; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 162; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 163; 14) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 171; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 172; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 163; 15) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 173; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 174; the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 163; 16) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 147; the second polypeptide comprises the amino acid sequence of SEQ ID NO: 175; the third polypeptide comprises the amino acid sequence of SEQ ID NO: 176; The fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 163; The anti-sclerostin construct of any one of claims 29 to 31.

33. The anti-sclerostin construct of claim 9 , wherein the second portion comprises a half-life extending moiety.

34. The half-life extending moiety is an Fc fragment.

34. The anti-sclerostin construct of claim 33.

35. The anti-sclerostin construct of any one of claims 6, 17-32, and 34, wherein the Fc fragment is one selected from the group consisting of Fc fragments of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof.

36. 36. The anti-sclerostin construct of claim 35, wherein the Fc fragment is one selected from an IgG1, IgG2, IgG3, IgG4 Fc fragment, and combinations and hybrids thereof.

37. 37. The anti-sclerostin construct of claim 35 or claim 36, wherein the Fc fragment comprises a H435R mutation and a Y436F mutation.

38. The anti-sclerostin construct of any one of claims 35 to 37, wherein the Fc fragment has a reduced effector function compared to a corresponding wild-type Fc fragment.

39. The Fc fragment comprises: a) enhanced effector function compared to the corresponding wild-type Fc fragment, and / or b) enhanced FcRn binding affinity compared to the corresponding wild-type Fc fragment; 38. The anti-sclerostin construct of any one of claims 35 to 37, comprising:

40. The anti-sclerostin construct of claim 9, wherein the construct is an antibody-drug conjugate or an antibody fusion protein.

41. 41. The anti-sclerostin construct of claim 40, wherein the second portion comprises a drug, the drug being one or more selected from parathyroid hormone (PTH), a selective estrogen receptor modulator (SERM), a bisphosphonate, a prostaglandin E (PGE) receptor agonist, VEGF, TGFβ, a growth factor (myostatin), calcitonin, and combinations thereof.

42. 42. A pharmaceutical composition comprising an anti-sclerostin construct of any one of claims 1 to 41 and a pharma- ceutically acceptable carrier.

43. 43. The pharmaceutical composition of claim 42, further comprising a drug, the drug being one or more selected from parathyroid hormone (PTH), selective estrogen receptor modulators (SERMs), VEGF, TGFβ, growth factors (myostatin), calcitonin, and combinations thereof.

44. 42. An isolated nucleic acid molecule encoding the anti-sclerostin construct of any one of claims 1-41.

45. A vector comprising the isolated nucleic acid molecule of claim 44.

46. 46. ​​An isolated host cell comprising the isolated nucleic acid molecule of claim 44 or the vector of claim 45.

47. a) culturing the isolated host cell of claim 46 under conditions effective to express the anti-sclerostin construct or a portion thereof; and b) obtaining an expressed anti-sclerostin construct or a portion thereof from said host cell; A method for preparing an anti-sclerostin construct comprising:

48. A pharmaceutical composition comprising an anti-sclerostin construct of any one of claims 1 to 41 or a pharmaceutical composition of claim 42 or 43 for use in a method for treating and / or preventing a disease or condition in an individual, comprising administering to the individual an effective amount of an anti-sclerostin construct of any one of claims 1 to 41 and / or a pharmaceutical composition of claim 42 or 43.

49. The agent or pharmaceutical composition of claim 48, wherein the disease or condition is a bone-related or cartilage-related disease, a bone marrow or hematological disease, a rare disease of the musculoskeletal system, a muscle-related disease or cancer.

50. 50. The agent or pharmaceutical composition of claim 49, wherein the bone-related disease comprises osteogenesis imperfecta, osteoporosis or osteopenia (male and female), osteonecrosis, delayed bone union, non-union of fractures, multiple myeloma, multiple myeloma-related bone disease, primary bone tumors and bone metastases of malignant tumors, inflammatory or infectious bone diseases, osteomalacia, hypercalcemia, Paget's disease, bone loss due to immobilization, glucocorticoid-induced bone loss, inflammation-induced bone loss including arthritis-induced bone loss, osteoporosis / osteopenia and weightlessness-induced bone loss in astronauts, or other diseases or conditions associated with a) loss of bone mass or quality or both, and / or b) abnormalities in bone structure and quality.

51. The agent or pharmaceutical composition of claim 50, wherein the bone-related disease refers to osteoporosis or osteopenia.

52. The agent or pharmaceutical composition according to claim 50, wherein the bone-related disease refers to osteogenesis imperfecta.

53. The agent or pharmaceutical composition according to claim 50, wherein the bone-related disease refers to multiple myeloma and multiple myeloma-related bone disease.

54. The agent or pharmaceutical composition of claim 49, wherein the cartilage-related disease comprises chondromatosis, chondrodysplasia, achondroplasia, dysplasia epiphyseal dysplasia, chondrodystrophic myoankylosis, cortical parachondroma, knee joint cartilage tear, osteofibrous dysplasia, osteoarthritis, osteogenesis imperfecta, hypophosphatemic rickets or osteochondrodystrophy.

55. The agent or pharmaceutical composition of claim 49, wherein the muscle-related disease refers to sarcopenia and cancer-related sarcopenia.

56. 44. A method for promoting healing following bone or joint surgery in an individual comprising administering to the individual an effective amount of an anti-sclerostin construct of any one of claims 1 to 41 and / or a pharmaceutical composition of claim 42 or 43.

57. The agent or pharmaceutical composition of any one of claims 48 to 56, wherein the anti-sclerostin construct is administered to the individual by subcutaneous injection, intravenous injection, intramuscular injection, oral or parenteral administration.

58. The agent or pharmaceutical composition according to any one of claims 48 to 56, wherein the use further comprises administering a second drug or therapy.

59. 59. The agent or pharmaceutical composition of claim 58, wherein the second drug comprises an anti-DKK1 antibody.

60. 59. The agent or pharmaceutical composition of claim 58, wherein the second drug comprises an anti-RANKL antibody.

61. 59. The agent or drug composition of claim 58, wherein the second drug or therapy comprises a drug selected from parathyroid hormone (PTH), selective estrogen receptor modulators (SERMs), bisphosphonates, prostaglandin E (PGE) receptor agonists, VEGF, TGFβ, growth factors (myostatin), and calcitonin.

62. The agent or pharmaceutical composition according to any one of claims 48 to 61, wherein the individual refers to a human.

Images