Anti-sclerostin antibodies and their use

Abstract

This invention provides an anti-sclerostin antibody and a method for using the same. [Solution] The present invention provides an isolated multispecific antibody that binds to sclerostin, comprising at least two different variable regions for the antibody to bind to at least two different epitopes of sclerostin, and which binds to sclerostin with higher affinity at neutral pH than at acidic pH, and a method for treating an individual with bone-related disease using the multispecific antibody.

Description

Technical Field

[0001] The present invention relates to anti-sclerostin antibodies and methods of using the same.

Background Art

[0002] Reduction of bone mass can be caused by a very diverse set of conditions and can lead to significant medical problems. For example, osteoporosis is a debilitating disease in humans, characterized by a marked decrease in bone mass and bone mineral density (BMD) of the skeleton in affected individuals, a structural deterioration of the bone, such as breakdown of the bone microstructure and a corresponding increase in bone fragility (i.e., decrease in bone strength), and increased susceptibility to fractures. In general, osteoporosis in humans is preceded by clinical osteopenia, a condition found in approximately 25 million people in the United States. An additional 7 to 8 million patients in the United States are diagnosed with clinical osteoporosis. The frequency of osteoporosis in the human population increases with age. In Caucasians, osteoporosis is more common in women, who account for 80% of the pool of osteoporosis patients in the United States. The increased skeletal bone fragility and susceptibility to fractures in the elderly is exacerbated by the higher risk of accidental falls in this population. Hip, wrist, and vertebral fractures are one of the most common injuries associated with osteoporosis. Hip fractures are particularly unpleasant for patients, costly, and for women, correlate with high mortality and morbidity.

[0003] Treatments for osteoporosis exist in the form of lifestyle modifications and drug therapies, but currently available treatments are limited in number and effectiveness, often associated with undesirable side effects, and are not widely accepted by patients (see, for example, Non-Patent Document 1). Numerous absorption inhibitors, including calcitonin, bisphosphonates, estrogen replacement, and selective estrogen receptor modulators (SERMs), prevent further bone loss but do not rebuild bone once it has been lost. Anabolic agents that increase bone mass and mineral density and restore bone structure are available in the form of human PTH(1-34). However, these treatments require daily subcutaneous injections, often for a year or more, which leads to incomplete patient compliance.

[0004] Sclerostin, the product of the SOST gene, is a 213-amino acid secreted glycoprotein. Sclerostin is a member of the cystine knot-containing superfamily. Sclerostin is associated with the DAN / Kerberos protein family and directly interferes with BMP signaling by inhibiting the binding of BMP to its receptor and thus inhibiting the BMP signaling cascade (see, e.g., Non-Patent Literature 2). Sclerostin mRNA expression is detected primarily in bone and kidney in adults. Sclerostin protein is primarily detectable in bone. Expression within bone is limited to osteocytes, which are mature, well-differentiated osteogenic cells.

[0005] Sclerostin is a potent negative regulator of osteogenesis in humans and mice. Lack of SOST expression leads to indurated ossification (see, e.g., Non-Patent Document 3; Non-Patent Document 4). Patients suffer from lifelong abnormal bone proliferation, resulting in increased bone mineral density and strength. This phenotype can be reproduced in SOST-deficient mice, and SOST overexpression leads to osteopenia. SOST is downregulated by PTH during osteogenesis, suggesting that some of the anabolic effects of PTH may be mediated by SOST (see, e.g., Non-Patent Document 5).

[0006] Due to its role as a potent negative regulator of bone formation, sclerostin is a desirable target for therapeutic interventions for disorders or conditions that would benefit from an increase in at least one of bone mass, bone mineral density, bone mineral content, and bone strength, such as osteoporosis. For example, anti-sclerostin antibodies described in Patent Documents 1, 2, 3, 4, 5, and 6 have been shown to bind to sclerostin and inhibit sclerostin activity in vitro and in vivo, including sclerostin activity associated with the negative regulation of bone formation. However, further improvements in the efficacy and convenience of agents that bind to sclerostin and antagonist its activity are needed in the art. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2006 / 119062 [Patent Document 2] WO2006 / 119107 [Patent Document 3] WO2008 / 115732 [Patent Document 4] WO2009 / 039175 [Patent Document 5] WO2009 / 047356 [Patent Document 6] WO2012 / 145417 [Non-patent literature]

[0008] [Non-Patent Document 1] Khosla and Riggs (1995) Mayo Clin Proc 70(10): 978-982 [Non-Patent Document 2] Avsian-Kretchmer and Hsueh (2004) Mol Endocrinol 18(1): 1-12 [Non-Patent Document 3] Balemans et al (2001) Hum Mol Genet 10(5): 537-543 [Non-Patent Document 4] Brunkow et al (2001) Am J Hum Genet 68(3): 577-589 [Non-Patent Document 5] Keller and Kneissel (2005) Bone 37(2): 148-158 [Overview of the project]

[0009] technical challenges This invention provides an anti-sclerostin antibody and a method for using the same.

[0010] How to solve the problem In some embodiments, the isolated anti-sclerostin antibody of the present invention is a multispecific antibody. In some embodiments, the isolated anti-sclerostin antibody of the present invention comprises at least two different variable regions. In some embodiments, the isolated anti-sclerostin antibody of the present invention binds to at least two different epitopes of sclerostin. In some embodiments, the isolated anti-sclerostin antibody of the present invention binds to sclerostin with higher affinity at neutral pH than at acidic pH.

[0011] In some embodiments, the isolated anti-sclerostin antibody of the present invention forms an immune complex with sclerostin. In some embodiments, the immune complex comprises at least two antibody molecules of the present invention and at least two sclerostin molecules.

[0012] In some embodiments, the isolated anti-sclerostin antibody of the present invention binds to the same epitope as the anti-sclerostin antibody comprising the VH (heavy chain variable region) sequence of SEQ ID NO: 7 and the VL (light chain variable region) sequence of SEQ ID NO: 15. In further embodiments, the isolated anti-sclerostin antibody of the present invention binds to the same epitope as the anti-sclerostin antibody comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 27.

[0013] In some embodiments, the isolated anti-sclerostin antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 60): positions 1, 3, 4, 5, 6, 7, and 8. In a further embodiment, the isolated anti-sclerostin antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following positions in the hypervariable region (HVR): (a) in HVR-H1 (SEQ ID NO: 32): positions 1, 2, and 4; (b) in HVR-H2 (SEQ ID NO: 37): position 9; (c) in HVR-H3 (SEQ ID NO: 43): positions 2 and 9; (d) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 59): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 62): positions 1, 3, 4, 5, 6, 7, and 8.

[0014] In some embodiments, the isolated anti-sclerostin antibody of the present invention has inhibitory activity against sclerostin.

[0015] In some embodiments, the isolated anti-sclerostin antibody of the present invention contains a common light chain.

[0016] In some embodiments, the isolated anti-sclerostin antibody of the present invention is a monoclonal antibody. In some embodiments, the isolated anti-sclerostin antibody of the present invention is a human antibody, a humanized antibody, or a chimeric antibody. In a further embodiment, the isolated anti-sclerostin antibody of the present invention is a full-length IgG1 antibody. In a further embodiment, the isolated anti-sclerostin antibody of the present invention is an antibody fragment that binds to sclerostin. In a further embodiment, the isolated anti-sclerostin antibody of the present invention is a bispecific antibody.

[0017] In some embodiments, the isolated anti-sclerostin antibody of the present invention comprises a first variable region containing the VH sequence of SEQ ID NO: 101 or 102 and one VL sequence from SEQ ID NOs: 98-100, 105, and a second variable region containing the VH sequence of SEQ ID NO: 103 or 104 and one VL sequence from SEQ ID NOs: 98-100, 105. In further embodiments, the VL sequence of the first variable region and the VL sequence of the second variable region are identical.

[0018] In some embodiments, the isolated anti-sclerostin antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following HVR positions: (a) in HVR-H2 (SEQ ID NO: 34): positions 5 and 8; (b) in HVR-H3 (SEQ ID NO: 38): positions 2, 5, 7, and 13; (c) in HVR-L1 (SEQ ID NO: 52): positions 4 and 7; (d) in HVR-L2 (SEQ ID NO: 56): positions 1 and 2; and (e) in HVR-L3 (SEQ ID NO: 60): position 1.

[0019] The present invention also provides isolated nucleic acids encoding the anti-sclerostin antibody of the present invention. The present invention also provides host cells containing the nucleic acid of the present invention. The present invention also provides a method for producing antibodies, comprising the step of culturing the host cells of the present invention so that antibodies are produced.

[0020] The present invention also provides a pharmaceutical formulation comprising the anti-sclerostin antibody of the present invention and a pharmaceutically acceptable carrier.

[0021] The present invention also provides methods for treating individuals having bone-related diseases. In some embodiments, the method comprises administering an effective amount of the anti-sclerostin antibody of the present invention to an individual. The present invention also provides methods for treating diseases or conditions associated with elevated levels of sclerostin in an individual. In some embodiments, the method comprises administering an effective amount of the anti-sclerostin antibody of the present invention to an individual. The present invention also provides methods for increasing bone formation and / or inhibiting bone resorption in an individual. In some embodiments, the method comprises administering an effective amount of the anti-sclerostin antibody of the present invention to an individual in order to increase bone formation and / or inhibit bone resorption. The present invention also provides methods for increasing bone mineral density in an individual. In some embodiments, the method comprises administering an effective amount of the anti-sclerostin antibody of the present invention to an individual in order to increase bone mineral density.

[0022] More specifically, the present invention provides the following: [1] An isolated multispecific antibody that binds to sclerostin, comprising at least two different variable regions for binding to at least two different epitopes of sclerostin, and binding to sclerostin with higher affinity at neutral pH than at acidic pH. [2] Forms an immune complex with sclerostin, The immune complex comprises at least two antibody molecules and at least two sclerostin molecules. [1] A multispecific antibody. [3] A multispecific antibody according to [1] or [2], wherein at least one of two variable regions binds to the same epitope as the anti-sclerostin antibody containing the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 15. [4] A multispecific antibody, one of any of [1] to [3], wherein at least one of two variable regions binds to the same epitope as the anti-sclerostin antibody containing the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 27. [5] A multispecific antibody from any of [1] to [4] wherein at least one of two variable regions includes a VH containing the amino acid sequence of SEQ ID NO: 7 and a VL containing the amino acid sequence of SEQ ID NO: 15, and at least one amino acid is substituted at the following positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 60): positions 1, 3, 4, 5, 6, 7, and 8. [6] A multispecific antibody from any of [1] to [5] wherein at least one of two variable regions comprises a VH containing the amino acid sequence of SEQ ID NO: 23 and a VL containing the amino acid sequence of SEQ ID NO: 27, and at least one amino acid is substituted at the following positions: (a) in HVR-H1 (SEQ ID NO: 32): positions 1, 2, and 4; (b) in HVR-H2 (SEQ ID NO: 37): position 9; (c) in HVR-H3 (SEQ ID NO: 43): positions 2 and 9; (d) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 59): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 62): positions 1, 3, 4, 5, 6, 7, and 8. [7] A multispecific antibody from any one of [1] to [6] that has inhibitory activity against sclerostin. [8] A multispecific antibody from any one of [1] to [7], in which at least two different variable regions are contained in a common light chain. [9] A bispecific antibody comprising a first variable region containing the VH sequence of SEQ ID NO: 101 or 102 and one VL sequence from SEQ ID NOs: 98-100 and 105, and a second variable region containing the VH sequence of SEQ ID NO: 103 or 104 and one VL sequence from SEQ ID NOs: 98-100 and 105.

[10] A bispecific antibody of [9] in which the VL sequence of the first variable region and the VL sequence of the second variable region are identical. A pharmaceutical preparation comprising one antibody from [1] to

[10] and a pharmaceutically acceptable carrier.

[12] A method for treating an individual having a bone-related disease, comprising the step of administering an effective amount of any one of the antibodies [1] to

[10] to the individual.

[13] A method for treating a disease or condition associated with an increased level of sclerostin in an individual, comprising the step of administering an effective amount of one of the antibodies [1] to

[10] to the individual.

[14] A method for increasing bone formation and / or inhibiting bone resorption in an individual, comprising the step of administering an effective amount of any one antibody from [1] to

[10] to the individual in order to increase bone formation and / or inhibit bone resorption.

[15] A method for increasing bone mineral density in an individual, comprising the step of administering to the individual an effective amount of one of the antibodies [1] to

[10] in order to increase bone mineral density. This invention also provides the following:

[16] Isolated anti-sclerostin antibodies comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 60): positions 1, 3, 4, 5, 6, 7, and 8.

[17] Isolated anti-sclerostin antibodies comprising VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following positions in the hypervariable region (HVR): (a) in HVR-H1 (SEQ ID NO: 32): positions 1, 2, and 4; (b) in HVR-H2 (SEQ ID NO: 37): position 9; (c) in HVR-H3 (SEQ ID NO: 43): positions 2 and 9; (d) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 59): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 62): positions 1, 3, 4, 5, 6, 7, and 8.

[18] An antibody of

[16] or

[17] that binds to sclerostin with higher affinity at neutral pH than at acidic pH.

[19] One of the antibodies

[16] to

[18] that has inhibitory activity against sclerostin. A pharmaceutical formulation comprising one of the antibodies

[20]

[16] ~

[19] and a pharmaceutically acceptable carrier.

[21] A method for treating an individual having a bone-related disease, comprising the step of administering an effective amount of any one of the antibodies

[16] to

[19] to the individual.

[22] A method for treating a disease or condition associated with an increased level of sclerostin in an individual, comprising the step of administering an effective amount of any one of the antibodies

[16] to

[19] to the individual.

[23] A method for increasing bone formation and / or inhibiting bone resorption in an individual, comprising the step of administering to the individual an effective amount of any one antibody from

[16] to

[19] in order to increase bone formation and / or inhibit bone resorption.

[24] A method for increasing bone mineral density in an individual, comprising the step of administering to the individual an effective amount of one of the antibodies

[16] to

[19] in order to increase bone mineral density. This invention also provides the following:

[25] A method for producing a common VL to be shared between two different VHs, comprising the following steps: (1) A step of providing a first variable region (V1) comprising a first VH (VH1) and a first VL (VL1), and a second variable region (V2) comprising a second VH (VH2) and a second VL (VL2), wherein V1 has binding activity to a first antigen (Ag1) and V2 has binding activity to a second antigen (Ag2); and (2) A step of constructing a modified VL (mVL) by replacing an amino acid residue at a certain position in HVR-L1, HVR-L2, or HVR-L3 of VL1 with an amino acid residue at the corresponding position in HVR-L1, HVR-L2, or HVR-L3 of VL2, in accordance with Kabat numbering.

[26] The method of

[25] further includes the following steps: (3) Repeat step (2) for the amino acids at other positions until all amino acids at positions in HVR-L1, HVR-L2, and HVR-L3 of VL1 are replaced.

[27] The method of

[26] further includes the following steps: (2') The process of constructing a modified VL (mVL) by replacing an amino acid residue at a certain position in HVR-L1, HVR-L2, or HVR-L3 of VL2 with an amino acid residue at the corresponding position in HVR-L1, HVR-L2, or HVR-L3 of VL1, according to Kabat numbering; and (3') The process of repeating step (2') for the amino acids at other positions until all amino acids at positions in HVR-L1, HVR-L2, and HVR-L3 of VL2 are replaced.

[28] One of the following methods, further including the steps below: (4) A step of measuring the binding activity of the mVL constructed in the above step to Ag1 or Ag2 when combined with VH1 or VH2, respectively.

[29] The method of

[28] further includes the following steps: (5) A step of selecting a preferred amino acid residue at a certain position within HVR-L1, HVR-L2, and HVR-L3 from two amino acid residues based on the binding activity of mVL measured in step (4), wherein one of the two amino acid residues is an amino acid residue (AA1) located at the corresponding position in VL1, and the other is an amino acid residue (AA2) located at the corresponding position in VL2; (6) A step of repeating step (5) for at least two different locations within HVR-L1, HVR-L2, and HVR-L3; and (7) A step of constructing a novel VL (nVL) containing the amino acid residues selected in steps (5) and (6) at those positions within HVR-L1, HVR-L2, and HVR-L3.

[30] The method of

[29] , wherein step (5) is repeated until a preferred amino acid residue is selected at all positions in HVR-L1, HVR-L2, and HVR-L3.

[31] A method according to

[29] or

[30] in which an amino acid sequence of any one FR selected from FR-L1, FR-L2, FR-L3, and FR-L4 of an nVL is selected from two amino acid sequences, one of which is the amino acid sequence of the corresponding FR of VL1 and the other is the amino acid sequence of the corresponding FR of VL2.

[32] Either of the following methods, VL1 and VL2 are variable regions derived from the κ light chain:

[25] -

[31] .

[33] Either of the following methods, VL1 and VL2 are variable regions derived from the λ light chain:

[25] -

[31] .

[34] Any one of the methods

[25] to

[33] wherein the amino acid length of one HVR selected from HVR-L1, HVR-L2, and HVR-L3 of VL1 is the same as the amino acid length of the corresponding HVR of VL2.

[35] Any one of the methods

[25] to

[34] wherein the amino acid length of any one FR selected from FR-L1, FR-L2, FR-L3, and FR-L4 of VL1 is the same as the amino acid length of the corresponding FR of VL2.

[36] Any one of the methods

[25] to

[35] wherein the amino acid sequence of any one FR selected from FR-L1, FR-L2, FR-L3, and FR-L4 of VL1 has 50% or more identity when compared with the amino acid sequence of the corresponding FR of VL2. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 shows the in vitro neutralizing activity of the anti-sclerostin antibody mabA and its variants (mabA_pH1, mabA_NpH1, mabA_pH2, and mabA_NpH2), as described in Example 4. [Figure 2] Figure 2 shows the in vitro neutralizing activity of the anti-sclerostin antibody mabA and its variants (mabA_pH3 and mabA_NpH3), as described in Example 4. [Figure 3]Figure 3 shows the in vitro neutralizing activity of the anti-sclerostin antibody mabA and the biparatopic anti-sclerostin antibodies (mabB / / mabA_pH1, mabB / / mabA_pH2, mabB / / mabA_pH3, and mabB / / mabA) as described in Example 4. [Figure 4] Figure 4 shows a typical image of the immune complex between the biparatopic anti-sclerostin antibody (mabB / / mabA) and the sclerostin antigen, as observed by transmission electron microscopy, as described in Example 5. [Figure 5a] Figure 5a shows chromatograms obtained by applying size exclusion chromatography to the biparatopic anti-sclerostin antibody alone, the sclerostin antigen alone, and the immunocomplex of the biparatopic anti-sclerostin antibody and the sclerostin antigen, as described in Example 5. For the biparatopic antibody, mabB / / mabA was used. [Figure 5b] Figure 5b shows chromatograms obtained by applying size exclusion chromatography to the biparatopic anti-sclerostin antibody alone, the sclerostin antigen alone, and the immunocomplex of the biparatopic anti-sclerostin antibody and the sclerostin antigen, as described in Example 5. For the biparatopic antibody, mabB / / mabA_pH1 was used. [Figure 5c] Figure 5c shows chromatograms obtained by applying size exclusion chromatography to the biparatopic anti-sclerostin antibody alone, the sclerostin antigen alone, and the immunocomplex of the biparatopic anti-sclerostin antibody and the sclerostin antigen, as described in Example 5. For the biparatopic antibody, mabB / / mabA-pH2 was used. [Figure 6] Figure 6 shows the Biacore analysis of biparatopic antibodies (mabB / / mabA_pH1, mabB / / mabA_pH2, mabB / / mabA) as described in Example 5. Binding activity to FcγRIIB is measured in the absence of the antigen (referred to as Ab alone) and in the presence of the antigen (referred to as IC). [Figure 7] Figure 7 shows the time course of plasma concentrations of anti-sclerostin antibodies (mabA, mabA_pH1, mabA_pH2, mabA_NpH1, and mabA_NpH2) administered intravenously to cynomolgus monkeys, as described in Example 6. [Figure 8] Figure 8 shows the time course of plasma concentrations of sclerostin after intravenous administration of anti-sclerostin antibodies (mabA, mabA_pH1, mabA_pH2, mabA_NpH1, and mabA_NpH2) to cynomolgus monkeys, as described in Example 6. [Figure 9] Figure 9 shows the time course of plasma concentrations of osteocalcin, a bone formation marker, after intravenous administration of anti-sclerostin antibodies (mabA_pH1, mabA_pH2, mabA_NpH1, and mabA_NpH2) to cynomolgus monkeys, as described in Example 7. [Figure 10] Figure 10 shows the time course of plasma concentrations of anti-sclerostin antibodies (mabA, mabA_pH3, and mabA_NpH3) administered intravenously to normal rats, as described in Example 8. [Figure 11a] Figure 11a shows the in vivo efficacy of anti-sclerostin antibodies (mabA, mabA_pH3, and mabA_NpH3) against bone mineral density (BMD) of the lumbar spine in normal rats, as described in Example 8. Data represent mean + SE. **: p<0.01, ***: p<0.001 (comparison with vehicle by Williams analysis). $$: p<0.01, $$$: p<0.001 (comparison with vehicle by t-test analysis). ++: p<0.01, +++: p<0.001 (comparison with mabA_NpH3-SG2 at the same dosage by t-test analysis). [Figure 11b]Figure 11b shows the in vivo efficacy of anti-sclerostin antibodies (mabA, mabA_pH3, and mabA_NpH3) against bone mineral density (BMD) of the right femur in normal rats, as described in Example 8. Data represent mean + SE. **: p<0.01, ***: p<0.001 (comparison with vehicle by Williams analysis). $$: p<0.01, $$$: p<0.001 (comparison with vehicle by t-test analysis). ++: p<0.01, +++: p<0.001 (comparison with mabA_NpH3-SG2 at the same dosage by t-test analysis). [Figure 12] Figure 12 shows the time course of plasma concentrations of anti-sclerostin antibodies (mabB / / mabA_pH3-BS01, mabB / / mabA_pH3-BS02, mabA_pH3-SG2, and mabA-hG2) administered intravenously to SCID mice, as described in Example 9. [Figure 13] Figure 13 shows the time course of plasma sclerostin concentrations after intravenous administration of anti-sclerostin antibodies (mabB / / mabA_pH3-BS01 and mabB / / mabA_pH3-BS02), as described in Example 9, to SCID mice. [Figure 14] Figure 14 shows the in vivo efficacy of anti-sclerostin antibodies (mabB / / mabA_pH3-BS01, mabA_pH3-SG2, and mabA-hG2) against lumbar spine bone mineral density (BMD) in SCID mice, as described in Example 9. Data represent mean + SE. *: p<0.05, **: p<0.01, ***: p<0.001 (comparison with vehicle by Williams analysis). +: p<0.5, ++: p<0.01, +++: p<0.001 (comparison with the same dose of mabA_pH3-SG2 or mabA_hG2 by t-test analysis). [Figure 15] Figure 15 shows a schematic diagram of the CDR shuffling method as described in Example 10. The CDRs at the same positions in the light chains of mabA and mabB were exchanged (shuffled) to subsequently construct variant light chains of AAB, ABA, ABB, BAA, BAB, and BBA. [Figure 16] Figure 16 shows the binding of CDR shuffling anti-sclerostin antibody variants to human sclerostin, as described in Example 10. Each of the light chains of mabA, mabB, AAB, ABA, ABB, BAA, BAB, BBA, abL063, abL081, and abL083 was combined with either the heavy chain of mabA (black circles) or the heavy chain of mabB (white circles), and the binding activity of the resulting antibodies to human sclerostin was measured by SPR analysis. Each plot is normalized by the antibody capture level. [Figure 17] Figure 17 shows a schematic diagram of the first step of the residue shuffling method as described in Example 10. The amino acid residues at the same position in the CDR of the mabA and mabB light chains were exchanged (shuffled) to construct the variant mabA light chain and the variant mabB light chain. [Figure 18] Figure 18 shows a schematic diagram of the subsequent steps of the per-residue shuffling method, as described in Example 10. Pairs of light chain variants, mabA and mabB, each having a single mutation at the same position, were evaluated and compared with each other. A "per-residue shuffling" light chain variant was designed by selecting the better amino acid residue at each position and combining the selected residues. [Figure 19] Figure 19 shows the binding of each residue-shuffling anti-sclerostin antibody variant to human sclerostin, as described in Example 10. The light chains of abL063, abL081, and abL083 were each combined with either the mabA heavy chain (black circles) or the mabB heavy chain (white circles), and the binding activity of the resulting antibodies to human sclerostin was measured by SPR analysis. Each plot is normalized by the antibody capture level. [Figure 20a]Figure 20a shows chromatograms obtained by applying pI-modified biparatopic anti-sclerostin antibody and homodimer antibody to cation exchange chromatography, as described in Example 11. The chromatograms include (i) a biparatopic antibody containing the mabA heavy chain variant, the mabB heavy chain variant, and the common light chain variant, (ii) a homodimer antibody containing the mabA heavy chain variant and the common light chain variant, and (iii) a homodimer antibody containing the mabB heavy chain variant and the common light chain variant. The biparatopic antibody used in Figure 20a was amH848 / bsH638 / abL152. [Figure 20b] Figure 20b shows chromatograms obtained by applying pI-modified biparatopic anti-sclerostin antibodies and homodimer antibodies to cation exchange chromatography, as described in Example 11. The chromatograms include (i) biparatopic antibodies containing the mabA heavy chain variant, the mabB heavy chain variant, and the common light chain variant, (ii) homodimer antibodies containing the mabA heavy chain variant and the common light chain variant, and (iii) homodimer antibodies containing the mabB heavy chain variant and the common light chain variant. The biparatopic antibodies used in Figure 20b were amH848 / bsH656 / abL152. [Figure 20c] Figure 20c shows chromatograms obtained by applying pI-modified biparatopic anti-sclerostin antibodies and homodimer antibodies to cation exchange chromatography, as described in Example 11. The chromatograms include (i) biparatopic antibodies containing the mabA heavy chain variant, the mabB heavy chain variant, and the common light chain variant, (ii) homodimer antibodies containing the mabA heavy chain variant and the common light chain variant, and (iii) homodimer antibodies containing the mabB heavy chain variant and the common light chain variant. The biparatopic antibodies used in Figure 20c were amH852 / bsH638 / abL152. [Figure 20d]Figure 20d shows chromatograms obtained by applying pI-modified biparatopic anti-sclerostin antibodies and homodimer antibodies to cation exchange chromatography, as described in Example 11. The chromatograms include (i) biparatopic antibodies containing the mabA heavy chain variant, the mabB heavy chain variant, and the common light chain variant, (ii) homodimer antibodies containing the mabA heavy chain variant and the common light chain variant, and (iii) homodimer antibodies containing the mabB heavy chain variant and the common light chain variant. The biparatopic antibodies used in Figure 20d were amH852 / bsH656 / abL152. [Figure 21] Figure 21 shows the in vitro neutralizing activity of pI-modified biparatopic anti-sclerostin antibodies (amH848 / bsH638 / abL152, amH848 / bsH656 / abL152, amH852 / bsH638 / abL152, and amH852 / bsH656 / abL152) and anti-sclerostin antibody mabB, as described in Example 11. [Figure 22a] Figure 22a shows Biacore sensorgrams of anti-sclerostin biparatopic antibodies against human sclerostin (h-SOST) and cynomolgus sclerostin (cy-SOST) at pH 7.4, as described in Example 12. amH848 / bsH638 / abL152 were used as the biparatopic antibodies. [Figure 22b] Figure 22b shows Biacore sensorgrams of anti-sclerostin biparatopic antibodies against human sclerostin (h-SOST) and cynomolgus sclerostin (cy-SOST) at pH 7.4, as described in Example 12. amH848 / bsH656 / abL152 were used as the biparatopic antibodies. [Figure 22c]Figure 22c shows Biacore sensorgrams of anti-sclerostin biparatopic antibodies against human sclerostin (h-SOST) and cynomolgus sclerostin (cy-SOST) at pH 7.4, as described in Example 12. amH852 / bsH638 / abL152 were used as the biparatopic antibodies. [Figure 22d] Figure 22d shows Biacore sensorgrams of anti-sclerostin biparatopic antibodies against human sclerostin (h-SOST) and cynomolgus sclerostin (cy-SOST) at pH 7.4, as described in Example 12. amH852 / bsH656 / abL152 were used as the biparatopic antibodies. [Modes for carrying out the invention]

[0024] Description of the manner The methods and procedures described or cited herein are generally well understood, and refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Conventional techniques, such as those widely used by those skilled in the art, are commonly employed by those skilled in the art, as described in Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0025] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide general guidance for many of the terms used herein. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0026] For the purpose of interpreting this Spec., the following definitions apply, and wherever applicable, a term used in the singular also includes the plural, and vice versa. It should be understood that the terms used herein are intended solely to describe a particular aspect and not to limit it. In the event of any conflict between the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

[0027] In the spirit of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from the human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived” from the human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0028] "Affinity" refers to the strength of the combined non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding affinity are described below.

[0029] An "affinity-matured" antibody is an antibody that, compared to a parent antibody without modifications, has one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody to the antigen.

[0030] The term "anti-sclerostin antibody" or "antibody that binds to sclerostin" refers to an antibody that can bind to sclerostin with sufficient affinity, and as a result, is useful as a diagnostic and / or therapeutic agent when it targets sclerostin. In one embodiment, the degree of binding of an anti-sclerostin antibody to unrelated non-sclerostin proteins is less than approximately 10% of the antibody's binding to sclerostin, as measured (e.g., by radioimmunoassay (RIA)). In a particular embodiment, the antibody that binds to sclerostin has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, anti-sclerostin antibodies bind to sclerostin epitopes that are conserved among sclerostins from different species.

[0031] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0032] An "antibody fragment" refers to a molecule other than the complete antibody, containing a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments, but not limited to these, include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0033] An antibody that binds to the same epitope as a reference antibody is an antibody that, in a competitive assay, blocks the binding of that reference antibody to its antigen by 50% or more. Conversely, the reference antibody blocks the binding of the aforementioned antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0034] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0035] The "class" of an antibody refers to the type of constant domain or constant region present in the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

[0036] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or interferes with the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, [[ID=Radioisotopes of Pb and Lu; chemotrephines or chemotherapy agents (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nucleases and their fragments; antibiotics; toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including their fragments and / or variants); and various antitumor agents or anticancer agents as disclosed below.

[0037] "Effector function" refers to the biological activity that varies depending on the antibody isotype, stemming from the Fc region of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0038] The “effective dose” of a drug (for example, a pharmaceutical formulation) refers to the amount in the required dosage and over the required period of time that is effective in achieving the desired therapeutic or prophylactic outcome.

[0039] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody targeting that antigen and contains specific amino acids that are in direct contact with the antibody. Epitope determinants can include a group of chemically active surface molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can possess specific three-dimensional structural properties and / or specific charge properties. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on that target antigen in a complex mixture of proteins and / or macromolecules.

[0040] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0041] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0042] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0043] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0044] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0045] The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al. As described above. In another embodiment, for VH, the subgroup is subgroup III by Kabat et al. As described above.

[0046] A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0047] As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0048] An "immunoconjugate" is an antibody that has been conjugated to one or more heterologous molecules (the heterologous molecules may include, but are not limited to, cytotoxic agents).

[0049] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0050] "Isolated" antibodies are those separated from the components of their original environment. In some embodiments, antibodies are purified to a purity of over 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0051] "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0052] "Isolated nucleic acids encoding anti-sclerostin antibodies" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, and includes nucleic acid molecules borne on one or more vectors, and nucleic acid molecules present at one or more locations within a host cell.

[0053] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any possible mutant antibodies (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0054] A "naked antibody" is an antibody that is not conjugated with a different part (e.g., a cytotoxic part) or a radioactive label. Naked antibodies may be present in pharmaceutical preparations.

[0055] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ).

[0056] The term “package insert” is used to refer to instructions for use that are typically included in the commercial packaging of therapeutic products and contain information about indications, usage, dosage, method of administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic products.

[0057] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced where necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0058] The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office (Washington DC, 20559) and registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may also be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, or with, or relative to, a given amino acid sequence B (or, a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to, a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0059] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained therein can exert its effect, and which does not contain additional elements that are toxic to an unacceptable degree to the person to whom the preparation is administered.

[0060] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0061] As used herein, the term “sclerostin” refers to any native sclerostin from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses both “full-length” sclerostin that has not undergone processing and any form of sclerostin resulting from processing within cells. The term also encompasses naturally occurring variants of sclerostin, such as splice variants and allele variants. The amino acid sequence of exemplary human sclerostin is shown in SEQ ID NO: 1. The amino acid sequences of exemplary cynomolgus monkey, rat, and mouse sclerostin are shown in SEQ ID NOs: 2, 3, and 4, respectively.

[0062] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0063] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0064] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0065] II. Compositions and Methods In one aspect, the present invention is based in part on an anti-sclerostin antibody and a method of using the same. In a particular embodiment, an antibody that binds to sclerostin is provided. The antibody of the present invention is useful, for example, for the diagnosis or treatment of bone-related diseases. In another aspect, the present invention is partly based on a method for producing common VLs. Such a method is useful, for example, for producing multispecific antibodies such as bispecific antibodies.

[0066] A. Exemplary anti-sclerostin antibody In one aspect, the present invention provides an isolated antibody that binds to sclerostin. In a particular aspect, an anti-sclerostin antibody variant is provided which is prepared by introducing an amino acid modification to an antibody comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 15 (mabA variant). In a particular aspect, an anti-sclerostin antibody variant is also provided which is prepared by introducing an amino acid modification to an antibody comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 27 (mabB variant). In a particular aspect, the anti-sclerostin antibody of the present invention is a multispecific antibody comprising at least two different variable regions. In a particular aspect, the anti-sclerostin antibody of the present invention is a multiparatope antibody comprising at least two different variable regions for which the antibody binds to at least two different epitopes on the sclerostin molecule.

[0067] In some embodiments, one of two distinct variable regions of the multispecific antibody of the present invention may be selected from the variable region of the mabA variant. In some embodiments, one of two distinct variable regions of the multispecific antibody of the present invention may be selected from the variable region of the mabB variant. In a particular embodiment, one of two distinct variable regions of the multispecific antibody binds to the same epitope of an antibody (mabA) containing the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 15. In a particular embodiment, one of two distinct variable regions of the multispecific antibody binds to the same epitope of an antibody (mabB) containing the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 27. In a further embodiment, the multispecific antibody of the present invention includes a variable region derived from either one of the mabA variants and a variable region derived from either one of the mabB variants. In a particular embodiment, the anti-sclerostin antibody is a bispecific antibody or a biparatopic antibody.

[0068] In some embodiments, the anti-sclerostin antibody of the present invention forms a complex with the antigen sclerostin (also referred to herein as an antigen-antibody complex or immune complex). In some embodiments, the complex comprises at least two antibody molecules of the present invention. In further embodiments, the complex comprises at least two antigen molecules. In certain embodiments, the complex comprises two antibody molecules of the present invention and two antigen molecules, where each antibody molecule is bound to two antigen molecules, and each antigen molecule is bound to two antibody molecules.

[0069] In some embodiments, the anti-sclerostin antibody of the present invention is taken up into cells. In certain embodiments, the uptake of the antibody into cells is enhanced when the antibody is complexed with an antigen compared to when the antibody is not complexed with an antigen. Enhanced uptake of the antigen-antibody complex into cells may result in enhanced clearance of the antigen from plasma when the antibody is administered to a subject. In another embodiment, the clearance of sclerostin from plasma is enhanced when the anti-sclerostin antibody of the present invention is administered to a subject.

[0070] In some embodiments, the anti-sclerostin antibodies of the present invention are taken up into cells through interactions between the Fc region of the antibody and Fc receptors on the surface of cells. In certain embodiments, the Fc receptor may be an Fcγ receptor (FcγR), which includes, for example, FcγRI including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII including isoform FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2).

[0071] An immune complex containing two or more antibody molecules can bind more strongly to Fc receptors on the cell surface and be taken up into the cell more efficiently than an immune complex containing only one antibody molecule, due to the avidity effect mediated by multiple Fc regions within the complex. In some embodiments, the anti-sclerostin antibody of the present invention comprises at least two different variable regions that bind to different epitopes on a sclerostin molecule, and two or more antibody molecules and two or more sclerostin molecules bind to each other to form an immune complex. In certain embodiments, the anti-sclerostin antibody of the present invention comprises two different variable regions that bind to different epitopes on a sclerostin molecule, and two antibody molecules and two sclerostin molecules bind to each other to form an immune complex.

[0072] In some embodiments, the anti-sclerostin antibodies of the present invention bind to sclerostin with higher affinity at neutral pH than at acidic pH. In another aspect, the present invention provides anti-sclerostin antibodies exhibiting pH-dependent binding to sclerostin. As used herein, the expression “pH-dependent binding” means “reduced binding at acidic pH compared to binding at neutral pH,” and both expressions may be interchangeable. For example, an anti-sclerostin antibody “having pH-dependent binding properties” includes an antibody that binds to sclerostin with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibody of the present invention binds to sclerostin with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or higher affinity at neutral pH than at acidic pH.

[0073] When the antigen is a soluble protein, the antibody can have a longer half-life in plasma than the antigen itself and can act as a carrier for the antigen. Therefore, antibody binding to the antigen can lead to an extension of the antigen's half-life in plasma (i.e., reduced clearance of the antigen from plasma). This is due to the reuse of the antigen-antibody complex by FcRn via the endosomal pathway in cells (Roopenian and Akilesh (2007) Nat Rev Immunol 7(9): 715-725). However, antibodies with pH-dependent binding properties, which bind to the antigen in a neutral extracellular environment while releasing the antigen into the acidic endosomal compartment after entering the cell, are expected to have superior properties in terms of antigen neutralization and clearance compared to their pH-independent binding counterparts (Igawa et al (2010) Nature Biotechnol 28(11); 1203-1207; Devanaboyina et al (2013) mAbs 5(6): 851-859; International Patent Application Publication No. WO 2009 / 125825).

[0074] Antibodies possessing both of the aforementioned properties, i.e., antibodies that bind to an antigen in a pH-dependent manner and form an immune complex containing two or more antibody molecules, are expected to have even better properties for highly accelerated elimination of antigens from plasma.

[0075] For the purposes of this disclosure, the “affinity” of an antibody against sclerostin is expressed by the antibody’s KD. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value of an antibody that binds to an antigen, the weaker its binding affinity to that particular antigen. Thus, as used herein, the expression “higher affinity at neutral pH than at acidic pH” (or the equivalent expression “pH-dependent binding”) means that the KD of an antibody at acidic pH is greater than the KD of an antibody at neutral pH. For example, in the context of the present invention, if the KD of an antibody that binds to sclerostin at acidic pH is at least twice as high as the KD of the antibody that binds to sclerostin at neutral pH, the antibody is considered to bind to sclerostin with higher affinity at neutral pH than at acidic pH. Accordingly, the present invention includes an antibody that binds to sclerostin at an acidic pH with a KD of at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater than the KD of the antibody that binds to sclerostin at a neutral pH. In another embodiment, the KD value of the antibody at a neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 It may be M or less. In another embodiment, the KD value of the antibody at an acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 It can be M or a larger value.

[0076] The binding characteristics of an antibody to a specific antigen may be expressed by the antibody's kd. The antibody's kd refers to the dissociation rate constant of the antibody to a specific antigen, and is the reciprocal of seconds (i.e., sec). -1The kd value is expressed as ). An increase in the kd value means that the antibody binds to its antigen more weakly. Therefore, the present invention includes antibodies that bind to sclerostin at an acidic pH with a higher kd value than at a neutral pH. The present invention includes antibodies that bind to sclerostin at an acidic pH with a kd value at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater than the kd of the antibody that binds to sclerostin at a neutral pH. In another embodiment, the kd value of the antibody at a neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 It may be 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s or a value greater than that.

[0077] In certain examples, "binding at acidic pH that is lower than binding at neutral pH" is expressed by the ratio of the antibody's KD value at acidic pH to its KD value at neutral pH (or vice versa). For example, if an antibody exhibits an acid / neutral KD ratio of 2 or higher, the antibody may be considered to exhibit "binding to sclerostin at acidic pH that is lower than binding at neutral pH" for the purposes of the present invention. In certain exemplary embodiments, the acid / neutral KD ratio for the antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment, the KD value of the antibody at neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12It may be M or less. In another embodiment, the KD value of the antibody at an acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 It can be M or a larger value.

[0078] In certain examples, "reduced binding at acidic pH compared to binding at neutral pH" is expressed by the ratio of the antibody's kd value at acidic pH to its kd value at neutral pH (or vice versa). For example, if an antibody exhibits an acid / neutral kd ratio of 2 or greater, the antibody may be considered to exhibit "reduced binding to sclerostin at acidic pH compared to binding at neutral pH" for the purposes of the present invention. In certain exemplary embodiments, the acid / neutral kd ratio for the antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment, the kd value of the antibody at neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 It may be 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s or a value greater than that.

[0079] As used herein, the term "acidic pH" refers to a pH range of 4.0 to 6.5. The term "acidic pH" includes pH values ​​of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In certain circumstances, the "acidic pH" is 5.8.

[0080] As used herein, the term "neutral pH" refers to a pH of 6.7 to 10.0. The term "neutral pH" includes pH values ​​of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In certain circumstances, "neutral pH" is 7.4.

[0081] The KD and kd values ​​expressed herein may be measured using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions (see, for example, Example 3 herein). The KD and kd values ​​can be measured at 25°C or 37°C.

[0082] In a particular embodiment, the anti-sclerostin antibody of the present invention has inhibitory activity against sclerostin. In another embodiment, the anti-sclerostin antibody of the present invention blocks sclerostin signaling via cell surface receptors such as low-density lipoprotein receptor-associated proteins 5 and 6 (LRP5 and LRP6).

[0083] In certain embodiments, the anti-sclerostin antibody of the present invention binds to sclerostin derived from multiple species. In certain embodiments, the anti-sclerostin antibody binds to sclerostin derived from humans and non-human animals. In certain embodiments, the anti-sclerostin antibody binds to sclerostin derived from humans, mice, rats, and monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, and baboons).

[0084] MabA variant In one aspect, the present invention provides an anti-sclerostin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 127; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132.

[0085] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 127. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 127. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 127 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 127, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 126; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 127. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 69; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 70. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 118; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 120. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 36; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 41. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 116; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 118; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 120. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 118; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 120.

[0086] In a further embodiment, the antibody comprises (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 8-14, 101, or 102; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 8-14, 101, or 102; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 8-14, 101, or 102. In a further embodiment, the antibody comprises (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 10; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 10; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 10. In a further embodiment, the antibody comprises (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 101; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 101; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 101. In a further embodiment, the antibody comprises (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 102; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 102; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 102.

[0087] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132. In another embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 73. In one embodiment, the antibody comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 53; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 56; and (c) HVR-L3 containing an amino acid sequence selected from SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 109; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 112; and (c) HVR-L3 containing an amino acid sequence selected from SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 110; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 113; and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 110; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 111; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115; and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 60.

[0088] In one embodiment, the antibody comprises (a) HVR-L1 derived from VL sequences of sequence numbers 16-22, 98-100, or 105; (b) HVR-L2 derived from VL sequences of sequence numbers 16-22, 98-100, or 105; and (c) HVR-L3 derived from VL sequences of sequence numbers 16-22, 98-100, or 105. In one embodiment, the antibody comprises (a) HVR-L1 derived from VL sequences of sequence number 18; (b) HVR-L2 derived from VL sequences of sequence number 18; and (c) HVR-L3 derived from VL sequences of sequence number 18. In one embodiment, the antibody comprises (a) HVR-L1 derived from VL sequences of sequence number 98; (b) HVR-L2 derived from VL sequences of sequence number 98; and (c) HVR-L3 derived from VL sequences of sequence number 98. In one embodiment, the antibody comprises (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 99; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 99; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 99. In one embodiment, the antibody comprises (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 100; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 100; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 100. In one embodiment, the antibody comprises (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 105; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 105; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 105.

[0089] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 124, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 127; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132.

[0090] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 127; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 132. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 69; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 70; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 73. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 120; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 53; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 56; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 120; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 111; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60.In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 120; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 111; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60.

[0091] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 8-14, 101, or 102; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 8-14, 101, or 102; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 8-14, 101, or 102; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 16-22, 98-100, or 105; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 16-22, 98-100, or 105; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 16-22, 98-100, or 105. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 10; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 10; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 10; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 18; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 18; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 18. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 101; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 101; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 101; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 105; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 105; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 105. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 102; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 102; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 102; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 105; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 105; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 105.

[0092] In a particular embodiment, one or more amino acids of the above-mentioned anti-sclerostin antibody are substituted at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 60): positions 1, 3, 4, 5, 6, 7, and 8. In one embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 60): positions 1, 3, 4, 5, 6, 7, and 8. In a further embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following HVR positions: (a) in HVR-H2 (SEQ ID NO: 34): positions 5 and 8; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 4 and 7; (e) in HVR-L2 (SEQ ID NO: 56): positions 1 and 2; and (f) in HVR-L3 (SEQ ID NO: 60): position 1.In a further embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, and 5; (d) in HVR-L1 (SEQ ID NO: 52): positions 4, 5, 7, 8, 9, and 11; and (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, and 6.

[0093] In certain embodiments, one or more amino acid substitutions in the anti-sclerostin antibodies provided herein are conservative substitutions. In certain embodiments, one or more of the following substitutions may be made in any combination (Note: The abbreviation "AxB" below means that amino acid A at position x (number) is substituted with amino acid B, where A and B are single-letter amino acid abbreviations used in the art): (a) In HVR-H1 (SEQ ID NO: 31): D1S; (b) In HVR-H2 (SEQ ID NO: 34): N3M; N5H; G8H or R; A9Y; Y11L; N12K; (c) In HVR-H3 (SEQ ID NO: 34) (d) In HVR-L1 (sequence number: 52): G2H or E; D4S; D5H or E; Y7H; D13H; (e) In HVR-L2 (sequence number: 56): Y1H or W; T2H or A; R4T; L5R; L6W or E; S7T; and (f) In HVR-L3 (sequence number: 60): Q1H; G3Y; D4S or H; T5D; L6Y; P7H; Y8W. In one embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 31): D1S; (b) in HVR-H2 (SEQ ID NO: 34): N3M; N5H; G8H or R; A9Y; Y11L; N12K; (c) in HVR-H3 (SEQ ID NO: 38): G2H or E;D4S;D5H or E;Y7H;D13H;(d) In HVR-L1 (Sequence ID: 52): R1K;Q4H or E;D5G;I6V;S7H;N8T or D;Y9A;L10V;N11A;(e) In HVR-L2 (Sequence ID: 56): Y1H or W;T2H or A;R4T;L5R;L6W or E;S7T; and (f) In HVR-L3 (Sequence ID: 60): Q1H;G3Y;D4S or H;T5D;L6Y;P7H;Y8W.In a further embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H2 (SEQ ID NO: 34): N5H;G8H; (c) in HVR-H3 (SEQ ID NO: 38): G2H;D5H;Y7H;D13H; (d) in HVR-L1 (SEQ ID NO: 52): Q4H;S7H; (e) in HVR-L2 (SEQ ID NO: 56): Y1H;T2H; and (f) in HVR-L3 (SEQ ID NO: 60): Q1H. In a further embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 31): D1S; (b) in HVR-H2 (SEQ ID NO: 34): N3M; N5H; G8R; A9Y; Y11L; N12K; (c) in HVR-H3 (SEQ ID NO: 38): G2E; D4S; D5E; (d) in HVR-L1 (SEQ ID NO: 52): Q4E; D5G; S7H; N8T or D; Y9A; N11A; and (e) in HVR-L2 (SEQ ID NO: 56): Y1W; T2A; R4T; L5R; L6W or E.

[0094] All possible substitution combinations described above are included in the consensus sequences of sequence numbers 124, 126, 127, 130, 131, and 132 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively.

[0095] In any of the embodiments described above, the anti-sclerostin antibody is humanized. In one embodiment, the anti-sclerostin antibody comprises HVR in any of the embodiments described above and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another embodiment, the anti-sclerostin antibody comprises HVR in any of the embodiments described above and further comprises VH or VL containing an FR sequence. In a further embodiment, the anti-sclerostin antibody comprises the following heavy-chain or light-chain variable domain FR sequences: for the heavy-chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO: 45, FR2 comprises the amino acid sequence of SEQ ID NO: 47 or 48, FR3 comprises the amino acid sequence of SEQ ID NO: 49 or 134, and FR4 comprises the amino acid sequence of SEQ ID NO: 51. Regarding the light chain variable domains, FR1 contains the amino acid sequence of SEQ ID NO: 65, FR2 contains the amino acid sequence of SEQ ID NO: 66, FR3 contains the amino acid sequence of SEQ ID NO: 67, and FR4 contains the amino acid sequence of SEQ ID NO: 68.

[0096] In another aspect, the anti-sclerostin antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-sclerostin antibody containing such sequence retains the ability to bind to sclerostin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 10. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VH sequence in SEQ ID NO: 10, including the post-translational modifications of said sequence. In certain embodiments, VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 31, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 36, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 41.

[0097] In another aspect, the anti-sclerostin antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 101. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-sclerostin antibody containing such sequence retains the ability to bind to sclerostin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 101. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VH sequence in SEQ ID NO: 101, including the post-translational modifications of said sequence. In certain embodiments, VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 116, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 118, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 120.

[0098] In another aspect, the anti-sclerostin antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the anti-sclerostin antibody containing such sequence retains the ability to bind to sclerostin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 102. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VH sequence in SEQ ID NO: 102, including the post-translational modifications of said sequence. In certain embodiments, VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 31, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 118, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 120.

[0099] In another aspect, an anti-sclerostin antibody is provided comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 18. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-sclerostin antibody comprising such sequence retains the ability to bind to sclerostin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 18. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VL sequence in SEQ ID NO: 18, including the post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 53, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 56, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60.

[0100] In another aspect, an anti-sclerostin antibody is provided comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 105. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-sclerostin antibody comprising such sequence retains the ability to bind to sclerostin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 105. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VL sequence in SEQ ID NO: 105, including the post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 111, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 115, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60.

[0101] In another aspect, an anti-sclerostin antibody is provided comprising VH in any of the above embodiments and VL in any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 7-14, 101, and 102, and any one of SEQ ID NOs: 15-22, 98-100, and 105, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NOs: 10 and 18, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NOs: 101 and 105, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NOs: 102 and 105, including post-translational modifications of said sequences.

[0102] In certain embodiments, the anti-sclerostin antibody of the present invention is not an antibody containing the VH and VL sequences in SEQ ID NO: 7 and SEQ ID NO: 15, respectively.

[0103] MabB Variant In one aspect, the present invention provides an anti-sclerostin antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 129; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132.

[0104] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 129. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 129. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 129 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 129, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 128; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 125; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 129. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 74; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 37; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 75. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 125; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 121. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 37; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 43. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 119; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 121. In a further embodiment, the antibody comprises (a) HVR-H1 having the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 having the amino acid sequence of SEQ ID NO: 37; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO: 121.

[0105] In a further embodiment, the antibody comprises (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 24-26, 103, or 104; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 24-26, 103, or 104; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 24-26, 103, or 104. In a further embodiment, the antibody comprises (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 24; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 24; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 24. In a further embodiment, the antibody comprises (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 103; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 103; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 103. In a further embodiment, the antibody comprises (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 104; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 104; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 104.

[0106] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132. In another embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 55; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 59; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76. In one embodiment, the antibody comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 55; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 59; and (c) HVR-L3 containing an amino acid sequence selected from SEQ ID NO: 63. In one embodiment, the antibody comprises (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 109; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 112; and (c) HVR-L3 containing an amino acid sequence selected from SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 110; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 113; and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 110; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 60. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 111; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115; and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 60.

[0107] In one embodiment, the antibody comprises (a) HVR-L1 derived from VL sequences of SEQ ID NO: 28-30, 98-100, or 105; (b) HVR-L2 derived from VL sequences of SEQ ID NO: 28-30, 98-100, or 105; and (c) HVR-L3 derived from VL sequences of SEQ ID NO: 28-30, 98-100, or 105. In one embodiment, the antibody comprises (a) HVR-L1 derived from VL sequences of SEQ ID NO: 28; (b) HVR-L2 derived from VL sequences of SEQ ID NO: 28; and (c) HVR-L3 derived from VL sequences of SEQ ID NO: 28. In one embodiment, the antibody comprises (a) HVR-L1 derived from VL sequences of SEQ ID NO: 98; (b) HVR-L2 derived from VL sequences of SEQ ID NO: 98; and (c) HVR-L3 derived from VL sequences of SEQ ID NO: 98. In one embodiment, the antibody comprises (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 99; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 99; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 99. In one embodiment, the antibody comprises (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 100; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 100; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 100. In one embodiment, the antibody comprises (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 105; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 105; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 105.

[0108] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 129; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 132.

[0109] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 129; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 132. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 74; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 55; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 59; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 55; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 59; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63. In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 119; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 111; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60.In another aspect, the present invention provides antibodies comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 111; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60.

[0110] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 24-26, 103, or 104; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 24-26, 103, or 104; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 24-26, 103, or 104; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 28-30, 98-100, or 105; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 28-30, 98-100, or 105; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 28-30, 98-100, or 105. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 24; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 24; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 24; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 28; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 28; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 28. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 103; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 103; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 103; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 105; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 105; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 105. In another aspect, the present invention provides an antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 104; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 104; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 104; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 105; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 105; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 105.

[0111] In a particular embodiment, one or more amino acids of the above-mentioned anti-sclerostin antibody are substituted at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 32): positions 1, 2, and 4; (b) in HVR-H2 (SEQ ID NO: 37): position 9; (c) in HVR-H3 (SEQ ID NO: 43): positions 2 and 9; (d) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 59): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 62): positions 1, 3, 4, 5, 6, 7, and 8. In one embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 32): positions 1, 2, and 4; (b) in HVR-H2 (SEQ ID NO: 37): position 9; (c) in HVR-H3 (SEQ ID NO: 43): positions 2 and 9; (d) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 59): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 62): positions 1, 3, 4, 5, 6, 7, and 8. In a further embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following positions in HVR: (a) in HVR-H1 (SEQ ID NO: 32): positions 1 and 2; (b) in HVR-H3 (SEQ ID NO: 43): position 2; and (c) in HVR-L3 (SEQ ID NO: 62): positions 4 and 7.In a further embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 32): positions 1 and 4; (b) in HVR-H2 (SEQ ID NO: 37): position 9; (c) in HVR-H3 (SEQ ID NO: 43): position 9; (d) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, and 10; and (e) in HVR-L2 (SEQ ID NO: 59): positions 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 62): positions 3, 4, 5, 6, and 8.

[0112] In certain embodiments, one or more amino acid substitutions in the anti-sclerostin antibodies provided herein are conservative substitutions. In certain embodiments, one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (sequence number: 32): D1H;T2H;Q4M; (b) in HVR-H2 (sequence number: 37): T9H; (c) in HVR-H3 (sequence number: 43): D2H;F9Y; (d) in HVR-L1 (sequence number: 55): K1R;Q4H or E;D5G;V6I;H7S;T8N or D;A9Y;V10L;A11N; (e) in HVR-L2 (sequence number: 59): W1H or Y;A2H or T;T4R;R5L;W6L or E;T7S; and (f) in HVR-L3 (sequence number: 62): Q1H;Y3G;S4D or H;D5T;Y6L;P7H;W8Y. In one embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 32): D1H; T2H; Q4M; (b) in HVR-H2 (SEQ ID NO: 37): T9H; (c) in HVR-H3 (SEQ ID NO: 43): D2H; F9Y;(d) In HVR-L1 (Sequence ID: 55): K1R;Q4H or E;D5G;V6I;H7S;T8N or D;A9Y;V10L;A11N;(e) In HVR-L2 (Sequence ID: 59): W1H or Y;A2H or T;T4R;R5L;W6L or E;T7S; and (f) In HVR-L3 (Sequence ID: 62): Q1H;Y3G;S4D or H;D5T;Y6L;P7H;W8Y. In a further embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 32): D1H; T2H; (b) in HVR-H3 (SEQ ID NO: 43): D2H; (c) in HVR-L3 (SEQ ID NO: 62): S4H; P7H.In a further embodiment, the antibody of the present invention comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 32): D1H;Q4M; (b) in HVR-H2 (SEQ ID NO: 37): T9H; (c) in HVR-H3 (SEQ ID NO: 43): F9Y; (d) in HVR-L1 (SEQ ID NO: 55): K1R;Q4E;D5G;V6I;H7S;T8D;A9Y;V10L; (e) in HVR-L2 (SEQ ID NO: 59): R5L;W6L or E;T7S; and (f) in HVR-L3 (SEQ ID NO: 62): Y3G;S4D;D5T;Y6L;W8Y.

[0113] All possible substitution combinations described above are included in the consensus sequences of sequence numbers 128, 125, 129, 130, 131, and 132 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively.

[0114] In any of the embodiments described above, the anti-sclerostin antibody is humanized. In one embodiment, the anti-sclerostin antibody comprises HVR in any of the embodiments described above and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another embodiment, the anti-sclerostin antibody comprises HVR in any of the embodiments described above and further comprises VH or VL containing an FR sequence. In a further embodiment, the anti-sclerostin antibody comprises the following heavy-chain or light-chain variable domain FR sequences: for the heavy-chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO: 46 or 133, FR2 comprises the amino acid sequence of SEQ ID NO: 48, FR3 comprises the amino acid sequence of SEQ ID NO: 50, and FR4 comprises the amino acid sequence of SEQ ID NO: 51 or 135. Regarding the light chain variable domains, FR1 contains the amino acid sequence of SEQ ID NO: 65, FR2 contains the amino acid sequence of SEQ ID NO: 66, FR3 contains the amino acid sequence of SEQ ID NO: 67, and FR4 contains the amino acid sequence of SEQ ID NO: 68.

[0115] In another aspect, the anti-sclerostin antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the anti-sclerostin antibody containing such sequence retains the ability to bind to sclerostin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 24. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VH sequence in SEQ ID NO: 24, including the post-translational modifications of said sequence. In certain embodiments, VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 37, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 43.

[0116] In another aspect, the anti-sclerostin antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the anti-sclerostin antibody containing such sequence retains the ability to bind to sclerostin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 103. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VH sequence in SEQ ID NO: 103, including the post-translational modifications of said sequence. In certain embodiments, VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 119, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 121.

[0117] In another aspect, the anti-sclerostin antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 104. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the anti-sclerostin antibody containing such sequence retains the ability to bind to sclerostin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 104. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VH sequence in SEQ ID NO: 104, including the post-translational modifications of said sequence. In certain embodiments, VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 117, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 37, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 121.

[0118] In another aspect, an anti-sclerostin antibody is provided comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 28. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-sclerostin antibody comprising such sequence retains the ability to bind to sclerostin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 28. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VL sequence in SEQ ID NO: 28, including the post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 55, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 59, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 63.

[0119] In another aspect, an anti-sclerostin antibody is provided comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 105. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-sclerostin antibody comprising such sequence retains the ability to bind to sclerostin. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 105. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-sclerostin antibody includes the VL sequence in SEQ ID NO: 105, including the post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 111, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 115, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60.

[0120] In another aspect, an anti-sclerostin antibody is provided, comprising VH in any of the above embodiments and VL in any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences in any one of SEQ ID NOs: 23-26, 103, and 104, and any one of SEQ ID NOs: 27-30, 98-100, and 105, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NOs: 24 and 28, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NOs: 103 and 105, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NOs: 104 and 105, including post-translational modifications of said sequences.

[0121] In certain embodiments, the anti-sclerostin antibody of the present invention is not an antibody containing the VH and VL sequences in SEQ ID NO: 23 and SEQ ID NO: 27, respectively.

[0122] In a further aspect, the present invention provides antibodies that bind to the same epitopes as the anti-sclerostin antibodies provided herein. In another aspect, the present invention provides antibodies that compete with the anti-sclerostin antibodies provided herein in terms of binding to sclerostin. For example, in a particular embodiment, an antibody is provided that binds to the same epitopes as the anti-sclerostin antibody comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 15 and / or competes with it in terms of binding to sclerostin. For example, in a particular embodiment, an antibody is provided that binds to the same epitopes as the anti-sclerostin antibody comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 27 and / or competes with it in terms of binding to sclerostin. In a particular embodiment, an antibody is provided that binds to the loop structure of sclerostin comprising amino acids 109-134 (CGPARLLPNAIGRGKWWRPSGPDFRC) of SEQ ID NO: 1. In a particular embodiment, an antibody is provided that binds to the cystine knot structure of sclerostin, which contains four polypeptides of Sequence ID No. 1, with amino acids 74-87 (DVSEYSCRELHFTR), 96-113 (SAKPVTELVCSGQCGPAR), 124-140 (WWRPSGPDFRCIPDRYR), and 161-172 (LVASCKCKRLTR), all of which are disulfide-bonded to each other.

[0123] In one aspect, the anti-sclerostin antibody of the present invention is a multispecific antibody or multiparatopic antibody comprising at least two different variable regions. In certain embodiments, the anti-sclerostin antibody is a bispecific antibody or biparatopic antibody. Any variable region of the anti-sclerostin antibody provided herein, or any combination thereof, can be used in the multispecific antibody or multiparatopic antibody. In some embodiments, the multispecific antibody or multiparatopic antibody of the present invention comprises a variable region derived from any one of the mabA variants and a variable region derived from any one of the mabB variants.

[0124] In one embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 126; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 127; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 131; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 132, and the second variable region comprises (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 128; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 125; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 129; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 131; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 132. In a further embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 69; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 70; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 73, and the second variable region comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 74; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 55; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 59; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76.In a further embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 120; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60, and the second variable region comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 125; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60.

[0125] In one embodiment, the multispecific antibody of the present invention has a first variable region that is (a) HVR-H1 derived from any one VH sequence among SEQ ID NOs: 8-14, 101, 102; (b) HVR-H2 derived from any one VH sequence among SEQ ID NOs: 8-14, 101, 102; (c) HVR-H3 derived from any one VH sequence among SEQ ID NOs: 8-14, 101, 102; (d) HVR-L1 derived from any one VL sequence among SEQ ID NOs: 16-22, 98-100, 105; (e) HVR-L2 derived from any one VL sequence among SEQ ID NOs: 16-22, 98-100, 105; and (f) HVR-L An antibody comprising 3, wherein the second variable region comprises (a) HVR-H1 derived from any one VH sequence among SEQ ID NOs: 24-26, 103, and 104; (b) HVR-H2 derived from any one VH sequence among SEQ ID NOs: 24-26, 103, and 104; (c) HVR-H3 derived from any one VH sequence among SEQ ID NOs: 24-26, 103, and 104; (d) HVR-L1 derived from any one VL sequence among SEQ ID NOs: 28-30, 98-100, and 105; (e) HVR-L2 derived from any one VL sequence among SEQ ID NOs: 28-30, 98-100, and 105; and (f) HVR-L3 derived from any one VL sequence among SEQ ID NOs: 28-30, 98-100, and 105.In a further embodiment, the multispecific antibody of the present invention has a first variable region that is (a) HVR-H1 derived from any one VH sequence of SEQ ID NOs: 8 to 14; (b) HVR-H2 derived from any one VH sequence of SEQ ID NOs: 8 to 14; (c) HVR-H3 derived from any one VH sequence of SEQ ID NOs: 8 to 14; (d) HVR-L1 derived from any one VL sequence of SEQ ID NOs: 16 to 22; (e) HVR-L2 derived from any one VL sequence of SEQ ID NOs: 16 to 22; and (f) HVR derived from any one VL sequence of SEQ ID NOs: 16 to 22 An antibody that contains -L3 and whose second variable region contains (a) HVR-H1 derived from any one VH sequence among SEQ ID NOs: 24 to 26; (b) HVR-H2 derived from any one VH sequence among SEQ ID NOs: 24 to 26; (c) HVR-H3 derived from any one VH sequence among SEQ ID NOs: 24 to 26; (d) HVR-L1 derived from any one VL sequence among SEQ ID NOs: 28 to 30; (e) HVR-L2 derived from any one VL sequence among SEQ ID NOs: 28 to 30; and (f) HVR-L3 derived from any one VL sequence among SEQ ID NOs: 28 to 30.In a further embodiment, the multispecific antibody of the present invention has a first variable region that is (a) HVR-H1 derived from any one VH sequence of SEQ ID NO: 101 or 102; (b) HVR-H2 derived from any one VH sequence of SEQ ID NO: 101 or 102; (c) HVR-H3 derived from any one VH sequence of SEQ ID NO: 101 or 102; (d) HVR-L1 derived from any one VL sequence of SEQ ID NO: 98-100 or 105; (e) HVR-L2 derived from any one VL sequence of SEQ ID NO: 98-100 or 105; and (f) HV derived from any one VL sequence of SEQ ID NO: 98-100 or 105 An antibody containing R-L3 and having a second variable region comprising (a) HVR-H1 derived from any one VH sequence among SEQ ID NOs: 103 and 104; (b) HVR-H2 derived from any one VH sequence among SEQ ID NOs: 103 and 104; (c) HVR-H3 derived from any one VH sequence among SEQ ID NOs: 103 and 104; (d) HVR-L1 derived from any one VL sequence among SEQ ID NOs: 98-100 and 105; (e) HVR-L2 derived from any one VL sequence among SEQ ID NOs: 98-100 and 105; and (f) HVR-L3 derived from any one VL sequence among SEQ ID NOs: 98-100 and 105.

[0126] In one embodiment, the multispecific antibody of the present invention comprises a first variable region comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is at the following HVR positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 60): positions 1, 3, 4, 5, 6, 7, and The antibody is substituted at position 8, and the second variable region comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following HVR positions: (a) HVR-H1 (SEQ ID NO: 32): positions 1, 2, and 4; (b) HVR-H2 (SEQ ID NO: 37): position 9; (c) HVR-H3 (SEQ ID NO: 43): positions 2 and 9; (d) HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) HVR-L2 (SEQ ID NO: 59): positions 1, 2, 4, 5, 6, and 7; and (f) HVR-L3 (SEQ ID NO: 62): positions 1, 3, 4, 5, 6, 7, and 8.In a further embodiment, the multispecific antibody of the present invention comprises a first variable region comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is at the following HVR positions: (a) in HVR-H2 (SEQ ID NO: 34): positions 5 and 8; (b) in HVR-H3 (SEQ ID NO: 38): positions 2, 5, 7, and 13; (c) in HVR-L1 (SEQ ID NO: 52): positions 4 and 7; (d) in HVR-L2 (SEQ ID NO: 56): positions 1 and 2; Rabini (e) HVR-L3 (SEQ ID NO: 60) is substituted at position 1, and the second variable region includes VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following HVR positions: (a) HVR-H1 (SEQ ID NO: 32) at positions 1 and 2; (b) HVR-H3 (SEQ ID NO: 43) at position 2; and (c) HVR-L3 (SEQ ID NO: 62) at positions 4 and 7.In a further embodiment, the multispecific antibody of the present invention comprises a first variable region comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following HVR positions: (a) HVR-H1 (SEQ ID NO: 31): position 1; (b) HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) HVR-H3 (SEQ ID NO: 38): positions 2, 4, and 5; (d) HVR-L1 (SEQ ID NO: 52): positions 4, 5, 7, 8, 9, and 11; and (e) HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, and 6. The antibody comprises a second variable region VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following HVR positions: (a) HVR-H1 (SEQ ID NO: 32): positions 1 and 4; (b) HVR-H2 (SEQ ID NO: 37): position 9; (c) HVR-H3 (SEQ ID NO: 43): position 9; (d) HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, and 10; (e) HVR-L2 (SEQ ID NO: 59): positions 5, 6, and 7; and (f) HVR-L3 (SEQ ID NO: 62): positions 3, 4, 5, 6, and 8.

[0127] In one embodiment, the multispecific antibody of the present invention comprises a first variable region comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein one or more of the following substitutions are present: (a) in HVR-H1 (SEQ ID NO: 31): D1S; (b) in HVR-H2 (SEQ ID NO: 34): N3M; N5H; G8H or R; A9Y; Y11L; N12K; (c) in HVR-H3 (SEQ ID NO: 38): G2H or E;D4S;D5H or E;Y7H;D13H;(d) In HVR-L1 (sequence number: 52): R1K;Q4H or E;D5G;I6V;S7H;N8T or D;Y9A;L10V;N11A;(e) In HVR-L2 (sequence number: 56): Y1H or W;T2H or A;R4T;L5R;L6W or E;S7T; and (f) In HVR-L3 (sequence number: 60): Q1H;G3Y;D4S or H;T5D;L6Y; P7H;Y8W may be performed in any combination, and the second variable region includes VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, where one or more of the following substitutions: (a) in HVR-H1 (SEQ ID NO: 32): D1H;T2H;Q4M; (b) in HVR-H2 (SEQ ID NO: 37): T9H; (c) in HVR-H3 (SEQ ID NO: 43): D2H;F9Y; (d) HVR- In L1 (SEQ ID NO: 55): K1R;Q4H or E;D5G;V6I;H7S;T8N or D;A9Y;V10L;A11N; (e) HVR-L2 (SEQ ID NO: 59): W1H or Y;A2H or T;T4R;R5L;W6L or E;T7S; and (f) HVR-L3 (SEQ ID NO: 62): Q1H;Y3G;S4D or H;D5T;Y6L;P7H;W8Y, these antibodies may be performed in any combination.In a further embodiment, the multispecific antibody of the present invention comprises a first variable region comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein one or more of the following substitutions are present: (a) in HVR-H2 (SEQ ID NO: 34): N5H;G8H; (c) in HVR-H3 (SEQ ID NO: 38): G2H;D5H;Y7H;D13H; (d) in HVR-L1 (SEQ ID NO: 52): Q4H;S7H; (e) in HVR-L2 (SEQ ID NO: 56): Y1H;T2H; and (f In HVR-L3 (SEQ ID NO: 60), Q1H may be performed in any combination, and the second variable region comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein one or more of the following substitutions may be performed in any combination: (a) in HVR-H1 (SEQ ID NO: 32): D1H;T2H; (b) in HVR-H3 (SEQ ID NO: 43): D2H; and (c) in HVR-L3 (SEQ ID NO: 62): S4H;P7H.In a further embodiment, the multispecific antibody of the present invention comprises a first variable region comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 31): D1S; (b) in HVR-H2 (SEQ ID NO: 34): N3M;N5H;G8R;A9Y;Y11L;N12K; (c) in HVR-H3 (SEQ ID NO: 38): G2E;D4S;D5E; (d) in HVR-L1 (SEQ ID NO: 52): Q4E;D5G;S7H;N8T or D;Y9A;N11A; and (e) in HVR-L2 (SEQ ID NO: 56): Y1W;T2A;R4T;L5R;L6W or E The antibody is characterized in that the second variable region comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 32): D1H;Q4M; (b) in HVR-H2 (SEQ ID NO: 37): T9H; (c) in HVR-H3 (SEQ ID NO: 43): F9Y; (d) in HVR-L1 (SEQ ID NO: 55): K1R;Q4E;D5G;V6I;H7S;T8D;A9Y;V10L; (e) in HVR-L2 (SEQ ID NO: 59): R5L;W6L or E;T7S; and (f) in HVR-L3 (SEQ ID NO: 62): Y3G;S4D;D5T;Y6L;W8Y.

[0128] In one embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises a VH sequence and a VL sequence from any one of SEQ ID NOs: 7-14, 101, and 102 and any one of SEQ ID NOs: 15-22, 98-100, and 105, respectively, and the second variable region comprises a VH sequence and a VL sequence from any one of SEQ ID NOs: 23-26, 103, and 104 and any one of SEQ ID NOs: 27-30, 98-100, and 105, respectively. In a further embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises a VH sequence and a VL sequence from any one of SEQ ID NOs: 7-14 and any one of SEQ ID NOs: 15-22, respectively, and the second variable region comprises a VH sequence and a VL sequence from any one of SEQ ID NOs: 23-26 and any one of SEQ ID NOs: 27-30, respectively. In a further embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises a VH sequence and a VL sequence from either SEQ ID NO: 101 or 102 and either SEQ ID NO: 98-100 or 105, respectively, and the second variable region comprises a VH sequence and a VL sequence from either SEQ ID NO: 103 or 104 and either SEQ ID NO: 98-100 or 105, respectively.

[0129] In one embodiment, the multispecific antibody of the present invention is an antibody in which a first variable region binds to the same epitope as an anti-sclerostin antibody containing the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 15, or competes with it for binding to sclerostin, and a second variable region binds to the same epitope as an anti-sclerostin antibody containing the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 27, or competes with it for binding to sclerostin.

[0130] In one aspect, the multispecific antibody of the present invention is an antibody in which at least two different variable regions include a common light chain. The common light chain can bind to each of at least two different heavy chains, and thus at least two different variable regions are formed. In some embodiments, the common light chain is shared by mabA and mabB. Such a light chain can bind to both the heavy chain of mabA and the heavy chain of mabB, and thus two different variable regions, such as mabA and mabB, are formed, respectively.

[0131] The common light chain of mabA and mabB In one embodiment, the present invention provides a common light chain comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60. In another embodiment, the common light chain comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60. In a particular embodiment, the common light chain comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 109; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 60. In certain embodiments, the common light chain includes (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 110; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 113; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60. In certain embodiments, the common light chain includes (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 110; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60. In certain embodiments, the common light chain includes (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 111; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 115; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60. In one embodiment, the common light chain includes (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 98; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 98; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 98. In one embodiment, the common light chain includes (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 99; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 99; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 99. In one embodiment, the common light chain includes (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 100; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 100; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 100.In one embodiment, the common light chain includes (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 105; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 105; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 105.

[0132] In certain embodiments, one or more amino acids of the common light chain described above are substituted at the following HVR positions: (a) in HVR-L1 (SEQ ID NO: 52): positions 4, 5, 7, 8, 9, and 11; and (b) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, and 6. In one embodiment, the common light chain of the present invention comprises a VL having the amino acid sequence of SEQ ID NO: 15, where at least one amino acid is substituted at the following HVR positions: (a) in HVR-L1 (SEQ ID NO: 52): positions 4, 5, 7, 8, 9, and 11; and (b) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, and 6.

[0133] In certain embodiments, one or more amino acids of the common light chain described above are substituted at the following HVR positions: (a) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, and 10; (b) in HVR-L2 (SEQ ID NO: 59): positions 5, 6, and 7; and (c) in HVR-L3 (SEQ ID NO: 62): positions 3, 4, 5, 6, and 8. In one embodiment, the common light chain of the present invention comprises a VL having the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following HVR positions: (a) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, and 10; (b) in HVR-L2 (SEQ ID NO: 59): positions 5, 6, and 7; and (c) in HVR-L3 (SEQ ID NO: 62): positions 3, 4, 5, 6, and 8.

[0134] In certain embodiments, one or more amino acid substitutions of the common light chain are conserved substitutions provided herein. In certain embodiments, one or more of the following substitutions may be made in any combination: (a) in HVR-L1 (SEQ ID NO: 52): Q4E;D5G;S7H;N8T or D;Y9A;N11A; and (b) in HVR-L2 (SEQ ID NO: 56): Y1W;T2A;R4T;L5R;L6W or E. In one embodiment, the common light chain of the present invention comprises a VL having the amino acid sequence of SEQ ID NO: 15, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-L1 (SEQ ID NO: 52): Q4E;D5G;S7H;N8T or D;Y9A;N11A; and (b) in HVR-L2 (SEQ ID NO: 56): Y1W;T2A;R4T;L5R;L6W or E.

[0135] In certain embodiments, one or more amino acid substitutions of the common light chain are conserved substitutions provided herein. In certain embodiments, one or more of the following substitutions may be made in any combination: (a) in HVR-L1 (SEQ ID NO: 55): K1R;Q4E;D5G;V6I;H7S;T8D;A9Y;V10L; (b) in HVR-L2 (SEQ ID NO: 59): R5L;W6L or E;T7S; and (c) in HVR-L3 (SEQ ID NO: 62): Y3G;S4D;D5T;Y6L;W8Y. In one embodiment, the common light chain of the present invention comprises a VL having the amino acid sequence of SEQ ID NO: 27, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-L1 (SEQ ID NO: 55): K1R;Q4E;D5G;V6I;H7S;T8D;A9Y;V10L; (b) in HVR-L2 (SEQ ID NO: 59): R5L;W6L or E;T7S; and (c) in HVR-L3 (SEQ ID NO: 62): Y3G;S4D;D5T;Y6L;W8Y.

[0136] All possible substitution combinations described above are included in the consensus sequences of sequence numbers 122, 123, and 60 for HVR-L1, HVR-L2, and HVR-L3, respectively.

[0137] In any of the embodiments described above, the common light chain is humanized. In one embodiment, the common light chain comprises an HVR in any of the embodiments described above and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another embodiment, the common light chain comprises an HVR in any of the embodiments described above and further comprises a VL containing an FR sequence. In a further embodiment, the common light chain comprises the following light chain variable domain FR sequences: FR1 comprises the amino acid sequence of SEQ ID NO: 65, FR2 comprises the amino acid sequence of SEQ ID NO: 66, FR3 comprises the amino acid sequence of SEQ ID NO: 67, and FR4 comprises the amino acid sequence of SEQ ID NO: 68.

[0138] In another aspect, a common light chain is provided that includes a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 105. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 105. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the common light chain includes the VL sequence in SEQ ID NO: 105, including post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 111; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 115; and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60.

[0139] In certain embodiments, the common light chain of the present invention is not a light chain containing the VL sequence in SEQ ID NO: 15 or SEQ ID NO: 27.

[0140] In one embodiment, the multispecific antibody of the present invention comprises a first variable region including (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 124; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 118; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 120; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60, and a second variable region The antibody comprises (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 117; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 125; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 121; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 60, wherein the VL sequence of the first variable region and the VL sequence of the second variable region are identical.

[0141] In one embodiment, the multispecific antibody of the present invention comprises a first variable region which includes (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 101 or 102; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 101 or 102; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 101 or 102; (d) HVR-L1 derived from any one VL sequence of SEQ ID NO: 98-100 or 105; (e) HVR-L2 derived from any one VL sequence of SEQ ID NO: 98-100 or 105; and (f) HVR-L3 derived from any one VL sequence of SEQ ID NO: 98-100 or 105, and a second variable region which An antibody comprising (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 103 or 104; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 103 or 104; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 103 or 104; (d) HVR-L1 derived from any one VL sequence among SEQ ID NO: 98-100 or 105; (e) HVR-L2 derived from any one VL sequence among SEQ ID NO: 98-100 or 105; and (f) HVR-L3 derived from any one VL sequence among SEQ ID NO: 98-100 or 105, wherein the VL sequence of the first variable region and the VL sequence of the second variable region are identical.

[0142] In one embodiment, the multispecific antibody of the present invention comprises a first variable region comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following HVR positions: (a) HVR-H1 (SEQ ID NO: 31): position 1; (b) HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) HVR-H3 (SEQ ID NO: 38): positions 2, 4, and 5; (d) HVR-L1 (SEQ ID NO: 52): positions 4, 5, 7, 8, 9, and 11; and (e) HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, and 6, and the second variable region comprises the amino acid sequence of SEQ ID NO: 23 The antibody comprises a VH containing an acid sequence and a VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following HVR positions: (a) HVR-H1 (SEQ ID NO: 32): positions 1 and 4; (b) HVR-H2 (SEQ ID NO: 37): position 9; (c) HVR-H3 (SEQ ID NO: 43): position 9; (d) HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, and 10; (e) HVR-L2 (SEQ ID NO: 59): positions 5, 6, and 7; and (f) HVR-L3 (SEQ ID NO: 62): positions 3, 4, 5, 6, and 8, and the VL sequence of the first variable region and the VL sequence of the second variable region are identical.

[0143] In one embodiment, the multispecific antibody of the present invention comprises a first variable region comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 31): D1S; (b) in HVR-H2 (SEQ ID NO: 34): N3M, N5H, G8R, A9Y, Y11L, N12K; (c) in HVR-H3 (SEQ ID NO: 38): G2E, D4S, D5E; (d) in HVR-L1 (SEQ ID NO: 52): Q4E, D5G, S7H, N8T or D, Y9A, N11A; and (e) in HVR-L2 (SEQ ID NO: 56): Y1W, T2A, R4T, L5R, L6W or E, and the second variable region comprises SEQ ID NO: 23 The antibody comprises a VH containing the amino acid sequence of SEQ ID NO: 27 and a VL containing the amino acid sequence of SEQ ID NO: 27, wherein one or more of the following substitutions may be made in any combination: (a) in HVR-H1 (SEQ ID NO: 32): D1H;Q4M; (b) in HVR-H2 (SEQ ID NO: 37): T9H; (c) in HVR-H3 (SEQ ID NO: 43): F9Y; (d) in HVR-L1 (SEQ ID NO: 55): K1R, Q4E, D5G, V6I, H7S, T8D, A9Y, V10L; (e) in HVR-L2 (SEQ ID NO: 59): R5L, W6L or E, T7S; and (f) in HVR-L3 (SEQ ID NO: 62): Y3G, S4D, D5T, Y6L, W8Y, and the VL sequence of the first variable region and the VL sequence of the second variable region are identical.

[0144] In one embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises a VH sequence and a VL sequence from any one of SEQ ID NOs: 101 or 102 and SEQ ID NOs: 98-100, 105, respectively, and the second variable region comprises a VH sequence and a VL sequence from any one of SEQ ID NOs: 103 or 104 and SEQ ID NOs: 98-100, 105, respectively, and the VL of the first variable region and the VL of the second variable region are the same. In a further embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises a VH sequence and a VL sequence from SEQ ID NOs: 101 and SEQ ID NOs: 105, respectively, and the second variable region comprises a VH sequence and a VL sequence from SEQ ID NOs: 103 and SEQ ID NOs: 105, respectively. In a further embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises the VH sequence and the VL sequence in SEQ ID NO: 101 and SEQ ID NO: 105, respectively, and the second variable region comprises the VH sequence and the VL sequence in SEQ ID NO: 104 and SEQ ID NO: 105, respectively. In a further embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises the VH sequence and the VL sequence in SEQ ID NO: 102 and SEQ ID NO: 105, respectively, and the second variable region comprises the VH sequence and the VL sequence in SEQ ID NO: 103 and SEQ ID NO: 105, respectively. In a further embodiment, the multispecific antibody of the present invention is an antibody in which the first variable region comprises the VH sequence and the VL sequence in SEQ ID NO: 102 and SEQ ID NO: 105, respectively, and the second variable region comprises the VH sequence and the VL sequence in SEQ ID NO: 104 and SEQ ID NO: 105, respectively.

[0145] In a further aspect of the present invention, the anti-sclerostin antibody according to any of the above embodiments is a monoclonal antibody comprising a chimeric, humanized, or human antibody. In one embodiment, the anti-sclerostin antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody or another antibody class or isotype as defined herein.

[0146] In further contexts, anti-sclerostin antibodies in any of the above embodiments may, alone or in combination, incorporate any of the features described in items 1 to 7 below.

[0147] 1. Antibody affinity In certain embodiments, the antibodies provided herein are ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0148] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a gradual increase in the concentration of the unlabeled antigen. 125 I) Fab is equilibrated with a labeled antigen, and then the bound antigen is captured by a plate coated with anti-Fab antibody. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., as in the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although this incubation may be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate for 10 minutes on a TOPCOUNT® gamma counter (Packard). Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0149] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions (0.78nM to 500nM) of Fab in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. Binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the coupling and dissociation sensorgrams using a simple one-to-one Langmuir coupling model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on It is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on velocity is 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If it exceeds this, the ON rate can be determined by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C in the presence of gradually increasing concentrations of antigen using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0150] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, see WO93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion on the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting extended in vivo half-lives, see U.S. Patent No. 5,869,046.

[0151] A diabody is an antibody fragment containing two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0152] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).

[0153] Antibody fragments can be produced by various methods, including, but are not limited to, the proteolytic digestion of complete antibodies and production by recombinant host cells (e.g., Escherichia coli or phages) as described herein.

[0154] 3. Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass of the parent antibody has been changed. A chimeric antibody also includes its antigen-binding fragment.

[0155] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually contains one or more variable domains, in which the HVR (e.g., CDR (or a portion thereof)) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody optionally contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues originated) to restore or improve the specificity or affinity of the antibody, for example.

[0156] Humanized antibodies and their preparation methods have been reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes resurfacing); Further details can be found in Dall'Acqua et al., Methods 36:43-60 (2005) (which describes FR shuffling), as well as in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (which describes a "guided selection" approach for FR shuffling).

[0157] The human framework regions that can be used for humanization are not limited to these, but include: framework regions selected using the "best fit" method (see Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) 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 of FR libraries (Baca et al., J. Biol. Chem. 272:10678-10684 (1997)). (and see Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0158] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various methods known in the art. Human antibodies are outlined in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0159] Human antibodies may be prepared by administering immunogens to transgenic animals modified to produce fully human antibodies or fully human antibodies with human variable regions in response to antigen challenge (loading). Such animals typically contain all or part of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is randomly incorporated extrachromosomally or within the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions from complete antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0160] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (See, for example, 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).) Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in 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).

[0161] Human antibodies can also be generated by isolating selected Fv clone variable domain sequences from a human-derived phage display library. Such variable domain sequences can then be combined with a desired human constant domain. A method for selecting human antibodies from an antibody library is described below.

[0162] 5. Antibodies derived from libraries The antibodies of the present invention may be isolated by screening a combinatorial library for antibodies exhibiting one or more desired activities. For example, various methods are known in the art for generating phage display libraries and for screening such libraries for antibodies possessing desired binding properties. Such methods have been reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further, for example, 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, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. This is described in 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).

[0163] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The phages typically present antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunosource without requiring the construction of hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self-antigens without immunization. Finally, naive libraries can also be synthesized synthetically, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), by cloning the pre-reorganization V-gene segment from stem cells and using PCR primers containing random sequences that encode the hypervariable CDR3 region and achieve in vitro rearrangement. Patent documents describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, as well as U.S. Patent Application Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0164] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0165] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one binding specificity is to sclerostin and the other is to any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of sclerostin. A bispecific antibody may be used to localize a cytotoxic agent to cells expressing sclerostin. A bispecific antibody may be prepared as a full-length antibody or as an antibody fragment.

[0166] Methods for producing multispecific antibodies are not limited to these, but include, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole techniques (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create Fc heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); creating antibodies with two specificities using a leucine zipper (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); producing bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368). (See 1994); and may also be prepared by preparing a trispecific antibody as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0167] Modified antibodies containing three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0168] In this specification, an antibody or fragment also includes a “dual-acting Fab” or “DAF” which includes one antigen-binding site that binds to sclerostin and another different antigen (see, for example, U.S. Patent Application Publication No. 2008 / 0069820).

[0169] 7. Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are also considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the antibody, and / or insertions into the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen-binding ability).

[0170] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial modifications are provided in Table 1 under the heading "Exemplary Substitutions" and are described in detail below, with reference to the classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retained / improved antigen-binding, reduced immunogenicity, or improved ADCC or CDC.

[0171] (Table 1) TIFF2026095400000001.tif170170

[0172] Amino acids can be grouped according to their common side-chain characteristics: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: Aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) Residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: Tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitution refers to replacing one member of one class with one of another.

[0173] One type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting mutants, and those selected for further study, will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution mutant is an affinity-matured antibody, which can be appropriately produced using, for example, a phage display-based affinity-mature technique (e.g., one described herein). Briefly, one or more HVR residues are mutated, and the mutant antibody is displayed on a phage and screened for specific biological activity (e.g., binding affinity).

[0174] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL may be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by any variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Next, a secondary library is prepared. This library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach that randomizes several HVR residues (e.g., 4-6 residues at a time). 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 often targeted.

[0175] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within an HVR. Such modifications may be, for example, outside the antigen-contact residue of the HVR. In certain embodiments of the mutant VH and VL sequences described above, each HVR is either unmodified or contains only one, two, or three amino acid substitutions.

[0176] A useful method for identifying antibody residues or regions that can be targeted for mutational introduction is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one or a group of target residues (e.g., charged residues, e.g., arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively, the crystal structure of the antigen-antibody complex may be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from the list of substitution candidates. Mutants may be screened to determine whether they possess the desired properties.

[0177] Amino acid sequence insertions include not only the insertion of single or multiple amino acid residues within a sequence, but also the fusion of polypeptides ranging in length from one to over 100 residues at the amino and / or carboxyl terminals. An example of terminal insertion is an antibody with a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include those in which an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody is fused to the N- or C-terminus of the antibody.

[0178] b) Glycosylated mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Adding or removing glycosylation sites to an antibody can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0179] If the antibody contains an Fc region, the carbohydrate to which it is attached may be modified. Native antibodies produced by mammalian cells typically contain branched, bifurcated oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the bifurcated oligosaccharide structure. In some embodiments, the modification of oligosaccharides in the antibody of the present invention may be carried out to produce antibody variants with specific improved properties.

[0180] In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability among multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications concerning "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 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. 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 Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., particularly Example 11) and knockout cell lines, such as alpha-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 WO2003 / 085107).

[0181] Further antibody variants are provided having a bifid oligosaccharide, for example, in which a bifid branched oligosaccharide attached to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0182] c) Fc region mutant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0183] In certain embodiments, antibody variants possessing some, but not all, effector functions are also within consideration of the present invention, such effector functions making the antibody a desirable candidate for application when its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity measurements can be performed to confirm reduced / deficient CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements may be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) while maintaining FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent 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); and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive measurement methods may be used (see, for example, ACT1® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. Furthermore, CDC measurements may be performed to evaluate complement activation (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)). In addition, FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0184] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant with alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581), and Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327.

[0185] Certain antibody variants exhibiting increased or decreased binding affinity to FcRs have been described. (See U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0186] In certain embodiments, the antibody variant includes an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residue in EU numbering) of the Fc region).

[0187] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement-dependent cell injury (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0188] Antibodies with increased half-life and increased binding affinity to the neonatal Fc receptor (FcRn: which plays a role in transferring maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994))) are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions therein that increase the binding affinity of the Fc region to FcRn. Such Fc variants include those involving substitutions at one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (for example, substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0189] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0190] d) Cysteine-modified antibody variant In certain embodiments, it would be desirable to produce cysteine-modified antibodies (e.g., "thioMAbs") in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the residues to be substituted occur in accessible sites of the antibody. By substituting these residues with cysteine, a reactive thiol group is located in an accessible site of the antibody, and this reactive thiol group may be used to conjugate the antibody to other parts (such as a drug part or a linker-drug part) to create an immunoconjugate as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-modified antibodies may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0191] e) antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties 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 (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if one or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used in derivatization can be determined based on considerations such as the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for therapy under specified conditions, although these are not limited to these.

[0192] In another embodiment, a conjugate is provided of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, but is not limited thereto, and includes wavelengths that heat the non-protein moiety to a temperature that does not harm normal cells but kills cells adjacent to the antibody-non-protein moiety.

[0193] B. Method and configuration of rearrangement For example, as described in U.S. Patent No. 4,816,567, antibodies can be produced using recombinant methods or compositions. In one embodiment, an isolated nucleic acid encoding the anti-sclerostin antibody described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and an amino acid sequence containing VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence containing VH of the antibody (e.g., transformed). In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method for producing an anti-sclerostin antibody is provided, comprising culturing host cells containing the nucleic acid encoding the antibody as described above under conditions suitable for the expression of the anti-sclerostin antibody, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0194] For the recombinant production of anti-sclerostin antibodies, nucleic acids encoding the antibody (e.g., those described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0195] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, for the expression of antibody fragments in Escherichia coli.) After expression, antibodies may be isolated from bacterial cell paste into soluble fractions and further purified.

[0196] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns, are suitable cloning or expression hosts for antibody-coding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0197] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0198] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0199] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). These include MRC5 cells and FS4 cells, as described in [reference]. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0200] For example, antibodies with pH-dependent properties can be obtained by using screening and / or mutagenesis methods, such as those described in WO2009 / 125825. Screening methods may include any steps for identifying antibodies with pH-dependent binding properties within a population of antibodies specific to a particular antigen. In certain embodiments, a screening method may include measuring one or more binding parameters (e.g., KD or kd) of individual antibodies within an initial antibody population at both acidic and neutral pH. Antibody binding parameters may be measured, for example, by surface plasmon resonance or other analytical methods that allow for quantitative or qualitative evaluation of the antibody's binding properties to a particular antigen. In certain embodiments, a screening method may include identifying antibodies that bind to the antigen at two or more acidic / neutral KD ratios. Alternatively, a screening method may include identifying antibodies that bind to the antigen at two or more acidic / neutral kd ratios.

[0201] In another embodiment, the mutagenesis method may include the step of incorporating amino acid deletions, substitutions, or additions to the heavy and / or light chains of an antibody in order to enhance the pH-dependent binding of the antibody to an antigen. In a particular embodiment, mutagenesis may be carried out within one or more variable domains of the antibody, e.g., one or more HVRs (e.g., CDRs). For example, mutagenesis may include the step of substituting one amino acid within one or more HVRs (e.g., CDRs) of the antibody for another amino acid. In a particular embodiment, mutagenesis may include the step of substituting one or more amino acids within at least one HVR (e.g., CDR) of the antibody for histidine. In a particular embodiment, “enhanced pH-dependent binding” means that the mutant version of the antibody exhibits a larger or greater acid / neutral KD ratio than the original “parent” (i.e., less pH-dependent) version of the antibody before mutagenesis. In a particular embodiment, the mutant version of the antibody has an acid / neutral KD ratio of 2 or more. Or, the mutant version of the antibody has an acid / neutral KD ratio of 2 or more.

[0202] Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. The relevant antigen is converted to an immunogenic protein in the immunized species, for example, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soy trypsin inhibitor, and a bifunctional substance or derivatizer, for example, maleimide benzoyl sulfosuccinimide (conjugation via cysteine ​​residue), N-hydroxysuccinimide (conjugation via lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR(where R and R 1 It may be useful to conjugate them using different alkyl groups.

[0203] Animals (usually non-human mammals) are immunized to an antigen, immunogenic conjugate, or derivative by intradermal injection at multiple sites of a solution containing, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) combined with three times the volume of Freund's complete adjuvant. After one month, the animals are boost-immunized with 1 / 5 to 1 / 10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, blood is collected from the animals and the serum is assayed for antibody titer. The animals are boost-immunized until the titer reaches a plateau. Preferably, the animals are boost-immunized with conjugates that are the same antigen but conjugated to a different protein and / or conjugated via a different crosslinking reagent. The conjugates can also be prepared as protein fusions in recombinant cell cultures. Coagulants such as alum are also preferably used to enhance the immune response.

[0204] Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for naturally occurring potential mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates the characteristic of an antibody that is not a mixture of distinct antibodies.

[0205] For example, monoclonal antibodies can be produced using the hybridoma method first described in Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described hereinabove to induce lymphocytes that are capable of producing or have the ability to produce antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0206] The immunizing agent typically includes the antigen protein or a fusion variant thereof. Generally, peripheral blood lymphocytes (PBL) are used when cells of human origin are desired, and spleen cells or lymph node cells are used when a non-human mammalian source is desired. Thereafter, the lymphocytes are fused with an immortalized cell line using a suitable fusogen such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0207] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells thus produced are seeded and grown in a suitable culture medium preferably containing one or more substances that inhibit the proliferation or survival of unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas would typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that inhibit the proliferation of HGPRT-deficient cells.

[0208] Preferred immortalized myeloma cells are those that efficiently fuse, facilitate stable and high-level antibody production by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Among these, mouse myeloma strains, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, California, USA, and SP-2 cells (and their derivatives, e.g., X63-Ag8-653) available from the American Type Culture Collection in Manassas, Virginia, USA, are preferred. For the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described (Kozbor et al. J. Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0209] The culture medium in which hybridoma cells are proliferating is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis as described in Munson, Anal Biochem. 107(1):220-239 (1980).

[0210] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, these clones can be subcloned by limiting dilution and grown using standard methods (Goding, cited above). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors within mammals.

[0211] Monoclonal antibodies secreted by subclones can be appropriately isolated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0212] Multispecific antibodies can be appropriately prepared, for example, by substituting an amino acid side chain present in one Fc region with a larger side chain (knob; meaning "projection") and substituting an amino acid side chain present in the Fc region with a smaller side chain (hole; meaning "void"), thereby placing the knob within the hole. This can facilitate efficient association between Fc regions having different amino acid sequences (WO1996 / 027011; Ridgway et al., Prot. Eng. 9:617-621 (1996); Merchant et al., Nat. Biotech. 16, 677-681 (1998)).

[0213] Other known techniques may be used to promote association between heterogeneous Fc regions. Specifically, this can be achieved by introducing electrostatic repulsion at the interface of the CH2 or CH3 domains of the Fc region to suppress unintended homogeneous association between Fc regions (WO2006 / 106905). Furthermore, association between heterogeneous Fc regions can be efficiently induced using a strand exchange engineered domain (SEED) CH3 heterodimer (Davis et al., Prot. Eng. Des. & Sel., 23:195-202 (2010)). In addition, heterodimerized antibody production techniques using the association of CH1 and CL and VH and VL of antibodies can also be used (WO2011 / 028952). Similar to the methods described in WO2008 / 119353 and WO2011 / 131746, it is also possible to produce heterodimerized antibodies by pre-preparing two homodimerized antibodies, incubating them under reducing conditions that cause dissociation, and then reassociating them. Similar to the method described by Strop (J. Mol. Biol. 420:204-219 (2012)), it is also possible to produce heterodimerized antibodies by introducing charged residues such as Lys, Arg, Glu, and Asp so that electrostatic repulsion is introduced into the CH3 domain. Furthermore, similar to the method described in WO2012 / 058768, it is also possible to produce heterodimerized antibodies by modifying the CH2 and CH3 domains.

[0214] A method has been reported for efficiently separating and purifying heterodimerized antibodies from homodimerized antibodies using ion-exchange chromatography by introducing amino acid modifications into the variable regions of the two types of antibody heavy chains that create a difference in isoelectric point between homodimerized and heterodimerized antibodies (WO2007 / 114325). Another method has been reported for purifying heterodimerized antibodies using protein A chromatography by constructing heterodimerized antibodies containing two types of heavy chains derived from mouse IgG2a, which binds to protein A, and rat IgG2b, which does not bind to protein A (WO1998 / 050431 and WO1995 / 033844). Furthermore, heterodimerized antibodies can be efficiently purified using protein A chromatography by substituting amino acid residues at positions 435 and 436 (EU numbering) located at the protein A binding site of the antibody heavy chain with amino acids such as Tyr or His to result in different protein A binding affinities.

[0215] In one aspect, the present invention provides a method for producing a common VL. Generally, one type of VL specifically binds to only one type of VH to form an antibody variable region, but a common VL can bind to at least two different types of VH to form at least two different variable regions. For example, a common VL can be used when constructing a multispecific antibody, such as a bispecific antibody. The present invention also provides a method for producing a multispecific antibody containing a common VL.

[0216] In one embodiment, the present invention provides a method for producing a common VL shared between two different VHs. The method may include the following steps: (1) A step of preparing a first variable region (V1) comprising a first VH (VH1) and a first VL (VL1), and a second variable region (V2) comprising a second VH (VH2) and a second VL (VL2), wherein V1 has binding activity to a first antigen (Ag1) and V2 has binding activity to a second antigen (Ag2); and (2) A step of constructing a modified VL (mVL) by replacing an amino acid residue at a certain position in HVR-L1, HVR-L2, or HVR-L3 of VL1 with an amino acid residue at the corresponding position in HVR-L1, HVR-L2, or HVR-L3 of VL2, in accordance with Kabat numbering. Ag1 and Ag2 may be the same antigen or different antigens. Furthermore, V1 and V2 may bind to the same epitopes or different epitopes. For example, Ag1 and Ag2 may be the same antigen, but V1 and V2 may bind to different epitopes on the same antigen. mVL construction can be carried out, for example, using a recombination method in which nucleic acids encoding VL1 and VL2 are prepared, and the nucleic acid encoding VL1 is mutated by replacing the codon of an amino acid residue at a certain position in HVR-L1, HVR-L2, or HVR-L3 of VL1 with the codon of an amino acid residue at the corresponding position in HVR-L1, HVR-L2, or HVR-L3 of VL2, according to Kabat numbering. The mutated nucleic acid can be introduced into host cells, and the host cells can be cultured to produce mVLs to obtain them.

[0217] In one embodiment, the method further includes the following steps: (3) Repeat step (2) for the amino acids at other positions until all amino acids at positions in HVR-L1, HVR-L2, and HVR-L3 of VL1 are replaced. In steps (2) and (3), amino acid substitution may be performed at positions where both VL1 and VL2 have amino acid residues, but the types of amino acid residues in VL1 and VL2 are different. If an amino acid residue at a certain position in VL1 is identical to a residue at a corresponding position in VL2, the substitution at that position may be omitted. If there is no corresponding position in VL2, the substitution at that position may be omitted.

[0218] In one embodiment, the method further includes the following steps: (2') The process of constructing a modified VL (mVL) by replacing an amino acid residue at a certain position in HVR-L1, HVR-L2, or HVR-L3 of VL2 with an amino acid residue at the corresponding position in HVR-L1, HVR-L2, or HVR-L3 of VL1, according to Kabat numbering; and (3') The process of repeating step (2') for the amino acids at other positions until all amino acids at positions in HVR-L1, HVR-L2, and HVR-L3 of VL2 are replaced. mVL construction can be carried out, for example, using a recombination method in which nucleic acids encoding VL1 and VL2 are prepared, and the nucleic acid encoding VL2 is mutated by replacing the codon of an amino acid residue at a certain position in HVR-L1, HVR-L2, or HVR-L3 of VL2 with the codon of an amino acid residue at the corresponding position in HVR-L1, HVR-L2, or HVR-L3 of VL1, according to Kabat numbering. The mutated nucleic acid can be introduced into host cells, and the host cells can be cultured to produce mVLs to obtain them. In steps (2') and (3'), amino acid substitution may be performed at positions where both VL1 and VL2 have amino acid residues, but the types of amino acid residues in VL1 and VL2 are different. If an amino acid residue at a certain position in VL2 is identical to a residue at a corresponding position in VL1, the substitution at that position may be omitted. If there is no corresponding position in VL1 in VL1, the substitution at that position may be omitted.

[0219] In one embodiment, the method further includes the following steps: (4) A step of measuring the binding activity of the mVL constructed in the above step to Ag1 or Ag2 when combined with VH1 or VH2, respectively. The binding activity of mVL in combination with VH1 or VH2 can be measured, for example, by performing a binding assay on a modified variable region containing mVL and either VH1 or VH2. A modified variable region (mV1) containing mVL and VH1 can be obtained by preparing nucleic acids encoding mVL and VH1, introducing these nucleic acids into host cells, and culturing the host cells to produce mV1. A modified variable region (mV2) containing mVL and VH2 can be obtained by preparing nucleic acids encoding mVL and VH2, introducing these nucleic acids into host cells, and culturing the host cells to produce mV2. Binding assays for mV1 or mV2 can be performed by known methods such as ELISA and Biacore.

[0220] In one embodiment, the method further includes the following steps: (5) A step of selecting a preferred amino acid residue at a certain position within HVR-L1, HVR-L2, and HVR-L3 from two amino acid residues based on the binding activity of mVL measured in step (4), wherein one of the two amino acid residues is an amino acid residue (AA1) located at the corresponding position in VL1, and the other is an amino acid residue (AA2) located at the corresponding position in VL2; (6) A step of repeating step (5) for at least two different locations within HVR-L1, HVR-L2, and HVR-L3; and (7) A step of constructing a novel VL (nVL) containing the amino acid residues selected in steps (5) and (6) at those positions within HVR-L1, HVR-L2, and HVR-L3. A preferred amino acid residue at a particular position within HVR-L1, HVR-L2, and HVR-L3 can be selected by evaluating whether the presence of that amino acid residue at that position is essential for antigen binding. In an exemplary scenario where an mVL is constructed by replacing an amino acid residue (AA1) at a particular position in VL1 with another amino acid residue (AA2) at a corresponding position in VL2, if such an mVL loses its binding activity to Ag1 when combined with VH1, then AA1 can be presumed to be essential for antigen binding, and it is preferable to select AA1 as the amino acid residue at that position. Conversely, if the aforementioned mVL maintains its binding activity to Ag1 when combined with VH1, then AA1 can be presumed to be not essential for antigen binding and can be replaced with AA2. In that case, selecting AA2 as the amino acid residue at that position would be preferable because it would almost certainly increase the binding activity of the mVL to Ag2 when combined with VH2. Alternatively, if the aforementioned mVL exhibits moderate binding activity to Ag1 when combined with VH1, it can be assumed that both AA1 and AA2 are possible candidates, and depending on the situation, either AA1 or AA2 can be selected as the amino acid residue at that position. In steps (5) and (6), amino acid selection may be performed at positions where both VL1 and VL2 have amino acid residues, but the types of amino acid residues in VL1 and VL2 are different from each other. When an amino acid residue at a certain position in VL1 is identical to a residue at a corresponding position in VL2, that amino acid residue is automatically selected as the preferred amino acid residue at that position. If there is no corresponding position in VL2 in VL1, or no corresponding position in VL1 in VL2, selection at that position can be omitted, or a new position can be created in the nVL and an amino acid residue present only in VL1 or VL2 can be incorporated therein. Furthermore, step (5) can be repeated until preferred amino acid residues are selected at all positions in HVR-L1, HVR-L2, and HVR-L3. The amino acid sequence of nVL can be designed by concatenating the amino acid sequences of HVR (HVR-L1, HVR-L2, and HVR-L3) and FR (FR-L1, FR-L2, FR-L3, and FR-L4). The amino acid sequences of HVR-L1, HVR-L2, and HVR-L3 can be determined to include the amino acid residues selected in steps (5) and (6) in their respective positions. The amino acid sequence of any one FR selected from FR-L1, FR-L2, FR-L3, and FR-L4 can be selected from two amino acid sequences, one of which is the amino acid sequence of the corresponding FR of VL1 and the other is the amino acid sequence of the corresponding FR of VL2. Such methodologies for constructing modified antibodies having FRs derived from different antibodies (referred to as "FR shuffling") are known in the art (see, for example, US2004 / 0044187). The construction of nVL can be carried out, for example, using recombinant methods such as preparing nucleic acids encoding HVRs (HVR-L1, HVR-L2, and HVR-L3) and FRs (FR-L1, FR-L2, FR-L3, and FR-L4) and ligating them to construct a nucleic acid encoding nVL. nVL can be obtained by introducing the nucleic acid into a host cell and culturing the host cell so that nVL is produced.

[0221] In one aspect, both VL1 and VL2 are variable regions derived from the κ light chain. In a further aspect, both VL1 and VL2 belong to the same subgroup of the κ light chain, such as human VK1, VK2, VK3, VK4, VK5, VK6, and VK7. In another aspect, both VL1 and VL2 are variable regions derived from the λ light chain. In a further aspect, both VL1 and VL2 belong to the same subgroup of the λ light chain, such as human VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, and VL9.

[0222] In one aspect, the amino acid length of any one HVR selected from HVR-L1, HVR-L2, and HVR-L3 of VL1 is the same as the amino acid length of the corresponding HVR of VL2. In a further aspect, the amino acid lengths of HVR-L1, HVR-L2, and HVR-L3 of VL1 are respectively the same as the amino acid lengths of HVR-L1, HVR-L2, and HVR-L3 of VL2.

[0223] In one aspect, the amino acid sequence of any one HVR selected from HVR-L1, HVR-L2, and HVR-L3 of VL1 has 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% identity when compared with the amino acid sequence of the corresponding HVR of VL2.

[0224] In one embodiment, the amino acid length of any one FR selected from FR-L1, FR-L2, FR-L3, and FR-L4 of VL1 is the same as the amino acid length of the corresponding FR of VL2. In a further embodiment, the amino acid lengths of FR-L1, FR-L2, FR-L3, and FR-L4 of VL1 are the same as the amino acid lengths of FR-L1, FR-L2, FR-L3, and FR-L4 of VL2, respectively.

[0225] In one embodiment, the amino acid sequence of any one FR selected from FR-L1, FR-L2, FR-L3, and FR-L4 of VL1 has 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity when compared with the amino acid sequence of the corresponding FR of VL2.

[0226] In one embodiment, the method further includes the following steps: (8) A step of producing a multispecific antibody that includes at least two variable regions, namely, one of which is a variable region containing VH1 and nVL that binds to Ag1, and the other of which is a variable region containing VH2 and nVL that binds to Ag2. Such multispecific antibodies can be produced, for example, by using a recombinant method that involves preparing nucleic acids encoding VH1, VH2, and nVL, introducing these nucleic acids into host cells, and culturing the host cells to produce multispecific antibodies. Each of VH1 and VH2 may be attached to the heavy chain constant region. nVL may be attached to the light chain constant region.

[0227] Measurement method (assay) The anti-sclerostin antibodies provided herein may be identified, screened, or have their physical / chemical properties and / or biological activity elucidated by various assay methods known in the art.

[0228] 1. Combined measurement methods and other measurement methods In one aspect, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA and Western blotting.

[0229] In another context, a competitive assay may be used to identify antibodies that compete with the anti-sclerostin antibodies described herein in terms of binding to sclerostin. In certain embodiments, if such a competitive antibody is present in excess, it will inhibit (e.g., reduce) the binding of the reference antibody to sclerostin by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some examples, binding will be inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such a competitive antibody will bind to the same epitope (e.g., a linear or conformational epitope) that is bound by the anti-sclerostin antibodies described herein. Detailed illustrative methods for mapping antibody-bound epitopes are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0230] In an exemplary competition assay, immobilized sclerostin is incubated in a solution containing a first labeled antibody that binds to sclerostin and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to sclerostin. The second antibody may be present in the hybridoma supernatant. As a control, immobilized sclerostin is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to sclerostin, any excess unbound antibody is removed and the amount of label bound to the immobilized sclerostin is measured. If the amount of label bound to the immobilized sclerostin is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to sclerostin. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0231] In another context, antibodies that bind to the same epitope as the anti-sclerostin antibodies provided herein, or that compete with the anti-sclerostin antibodies provided herein for binding to sclerostin, can be identified using a sandwich assay. A sandwich assay involves the use of two antibodies, each capable of binding to a different immunogenic moiety or epitope of the protein to be detected. In a sandwich assay, a first antibody immobilized on a solid support binds to the test sample analyte, and then a second antibody binds to the analyte, thus forming an insoluble ternary complex. See U.S. Patent No. 4,376,110 of David & Greene. The second antibody may be labeled by a detectable moiety itself (direct sandwich assay), or it may be measured using an anti-immunoglobulin antibody labeled by a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. An antibody that binds to sclerostin simultaneously with the anti-sclerostin antibodies provided herein may be determined to be an antibody that binds to a different epitope than the anti-sclerostin antibody. Therefore, an antibody that does not bind to sclerostin at the same time as the anti-sclerostin antibody provided herein may be determined to be an antibody that binds to the same epitope as the anti-sclerostin antibody, or an antibody that competes with the anti-sclerostin antibody for binding to sclerostin.

[0232] 2.Activity measurement method In one aspect, a method for identifying biologically active anti-sclerostin antibodies is provided. This biological activity may include, for example, inhibitory activity against sclerostin. Furthermore, antibodies possessing such biological activity in vivo and / or in vitro are provided.

[0233] In certain embodiments, the antibodies of the present invention are tested for such biological activity. In various aspects, anti-sclerostin antibodies can neutralize sclerostin in MC3T3 cell-based mineralization assays. Mineralization by osteoblast lineage cells, primary cells, or cell lines in culture is used as an in vitro model of bone formation. An exemplary cell-based mineralization assay is described in U.S. Patent Publication No. 20070110747. MC3T3-E1 cells (Sudo et al., J. Cell Biol., 96:191-198 (1983)) and subclones of its original cell line can form minerals in culture when grown in the presence of a differentiation agent. For MC3T3-E1 cells, sclerostin can inhibit one or more of the events leading to mineral deposition, including mineral deposition (i.e., sclerostin inhibits mineralization). Anti-sclerostin antibodies that can neutralize the inhibitory activity of sclerostin enable mineralization of cultures in the presence of sclerostin, resulting in a statistically significant increase, for example, in the deposition of calcium phosphate (measured as calcium), compared to the amount of calcium measured in the sclerostin-only (i.e., no antibody) treatment group.

[0234] In another context, anti-sclerostin antibodies can neutralize sclerostin in cell-based assays, such as the bone-specific alkaline phosphatase assay described in International Patent Publication No. WO2008 / 115732. The bone-specific alkaline phosphatase assay is based on sclerostin's ability to reduce alkaline phosphatase levels induced by BMP-4 and Wnt3a stimulation in the multipotent mouse cell line C2C12. The neutralizing anti-sclerostin antibody mediates a dose-dependent increase in alkaline phosphatase activity in this assay.

[0235] In another context, anti-sclerostin antibodies can neutralize sclerostin in cell-based Wnt signaling assays in the HEK293 cell line, such as the Wnt assay described in International Patent Publication No. WO2009 / 047356, which involves induction of a Wnt1-mediated STF reporter gene. Alternatively, anti-sclerostin antibodies can neutralize sclerostin in BMP2-induced mineralization assays in MC3T3 cells, such as the mineralization assay described in International Patent Publication No. WO2009 / 047356.

[0236] In certain embodiments, inhibition of sclerostin activity includes a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more of sclerostin activity in the assay compared to a negative control under similar conditions. In some embodiments, it refers to inhibition of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of sclerostin activity.

[0237] In certain situations, the anti-sclerostin antibody of the present invention is taken up into cells, and in particular, the uptake of the antibody is enhanced when the antibody binds to an antigen to form an immune complex. Antibody uptake into cells can be observed by cell imaging analysis. Fluorescently labeled antibodies are brought into contact with cells expressing the Fc receptor (e.g., FcγR) in the absence and presence of the antigen, and the fluorescence intensity of the resulting cells is measured using an imaging analyzer. Cells useful for such assays may express endogenous Fc receptors or may be genetically modified transiently or stably to express transgenes encoding the Fc receptor. If an increased fluorescence intensity is detected in the presence of the antigen compared to the absence of the antigen, it is determined that the uptake of the test antibody into cells is enhanced when the test antibody forms a complex with the antigen.

[0238] In another context, whether an antibody can form an immune complex with an antigen, particularly an immune complex containing two or more antibody molecules, can be assessed by methods such as size exclusion (gel filtration) chromatography, ultracentrifugation, light scattering, electron microscopy, or mass spectrometry (Mol Immunol (2002) 39:77-84, Mol Immunol (2009) 47:357-364). These methods can estimate the molecular size of the immune complex formed in the presence of the antigen. When the molecular size of the immune complex is larger than that estimated from a single antibody molecule, the antibody is determined to be capable of forming an immune complex containing two or more antibody molecules. In another context, the formation of immune complexes can be detected by binding assays to Fc receptors (e.g., FcγR) using, for example, ELISA, FACS, or SPR (surface plasmon resonance assay; e.g., using Biacore) (J Biol Chem (2001) 276(9):6591-6604; J Immunol Methods (1982) 50:109-114; J Immunol (2010) 184(4):1968-1976; mAbs (2009) 1(5):491-504). These methods utilize the property that immune complexes containing two or more antibody molecules can bind to Fc receptors more strongly than antibody molecules alone or immune complexes containing only one antibody molecule. When an antibody binds to the Fc receptor in the presence of an antigen with higher affinity compared to the absence of the antigen, that antibody is determined to be capable of forming an immune complex containing two or more antibody molecules.

[0239] C. Immunoconjugate The present invention also provides an immunoconjugate comprising one or more cytotoxic agents (e.g., chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins, or fragments thereof) or radioisotopes) conjugated with the anti-sclerostin antibodies of this specification.

[0240] In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including, but not limited to, the following: These include: meitansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235B1); auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patents Nos. 5,635,483, 5,780,588, and 7,498,298); drastatin; calicheamycin or its derivatives (see U.S. Patents Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342) (1993); and see Lode et al., Cancer Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and see U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0241] In another embodiment, the immunoconjugate includes antibodies described herein, conjugated to enzymatically active toxins or fragments thereof, including, but not limited to, diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crocin, saponaria officinalis inhibitor, geronin, mitogellin, restrictosin, phenomycin, enomycin, and trichothecene.

[0242] In another embodiment, the immunoconjugate comprises an antibody described herein that has been conjugated to a radioactive atom to form a radioactive conjugate. Various radioisotopes are available for the production of radioactive conjugates. For example, 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Contains radioactive isotopes of Pb and Lu. When using a radioactive conjugate for detection, the radioactive conjugate contains radioactive atoms (e.g., Tc-99m or) for scintigraphy. 123 I) or may include spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging or MRI) (e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron).

[0243] Antibody and cytotoxic agent conjugates can be prepared using a variety of bifunctional protein conjugates. Examples include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyl HCl adipiimidoate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionuclides to antibodies. See WO94 / 11026. Linkers may be “cleavable linkers” that facilitate the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0244] The immunoconjugates or ADCs described herein expressly consider conjugates prepared using crosslinking reagents, including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0245] D. Methods and compositions for diagnosis and detection In certain embodiments, any of the anti-sclerostin antibodies provided herein are useful for detecting the presence of sclerostin in a biological sample. As used herein, the term “detection” encompasses quantitative or qualitative detection. In certain embodiments, a biological sample includes cells or tissues, such as serum, whole blood, plasma, biopsy samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, breast milk, colostrum, mammary secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, ascites, ophthalmic lens solution, and mucus.

[0246] In one embodiment, an anti-sclerostin antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of sclerostin in a biological sample is provided. In a particular embodiment, the method comprises contacting a biological sample with the anti-sclerostin antibody described herein under conditions in which binding of the anti-sclerostin antibody to sclerostin is permitted, and detecting whether a complex has been formed between the anti-sclerostin antibody and sclerostin. Such a method may be an in vitro or in vivo method. In one embodiment, the anti-sclerostin antibody is used to select subjects suitable for treatment using the anti-sclerostin antibody, for example, when sclerostin is a biomarker for patient selection.

[0247] Exemplary disorders that can be diagnosed using the antibodies of the present invention include achondroplasia, basocranial dysplasia, enchondromatosis, fibrous dysplasia, Gaucher disease, hypophosphatemic rickets, Marfan syndrome, hereditary multiple exostosis, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteomatosis, sclerosing lesions, pseudoarthrosis, suppurative osteomyelitis, periodontal disease, antiepileptic drug-induced bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism, weight-loss-induced bone loss, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative bone disorders, oral bone loss, osteonecrosis of the jaw, and juvenile Paget's disease. Thosh disease, meroleostosis, metabolic bone disease, mastocytosis, sickle cell anemia / disease, organ transplant-related bone loss, kidney transplant-related bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidosis, Fabry disease, Turner syndrome, Down syndrome, Klinefelter syndrome, leprosy, Perthes disease, adolescent idiopathic scoliosis, infant-onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson's disease, ischemic bone disease (such as Legg-Calvé-Perthes disease and focal migratory osteoporosis), anemia, steroid use Conditions caused by the following: glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, nutritional deficiencies, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory state, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory bowel disease, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy-related bone loss, diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism, motor inhibition or inactivity, reflex sympathetic dystrophy syndrome, focal osteoporosis, osteomalacia, arthritis This includes bone-related diseases such as bone loss associated with surgery, HIV-related bone loss, bone loss associated with growth hormone reduction, bone loss associated with cystic fibrosis, bone loss associated with chemotherapy, tumor-induced bone loss, cancer-related bone loss, bone loss due to hormone deprivation, multiple myeloma, drug-induced bone loss, anorexia nervosa, disease-related facial bone loss, disease-related cranial bone loss, disease-related jawbone loss, disease-related skull loss, age-related bone loss, age-related facial bone loss, age-related cranial bone loss, age-related jawbone loss, age-related skull loss, and bone loss associated with space travel.

[0248] In certain embodiments, labeled anti-sclerostin antibodies are provided. Labeling includes, but is not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, high-electron-density labels, chemiluminescent labels, and radioactive labels) as well as moieties indirectly detectable through, for example, enzymatic reactions or intermolecular interactions (e.g., enzymes or ligands). Exemplary labels, but are not limited to, include: radioactive isotopes. 32 P, 14 C, 125 I, 3 H and 131 I, fluorescent phosphopoides such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, luciferases such as dansyl, umbelliferone, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase, enzymes linked to oxidizing pigment precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin-labeled, bacteriophage-labeled, stable free radicals, and similar substances.

[0249] E. Pharmaceutical preparations The pharmaceutical formulations of anti-sclerostin antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing an antibody of the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and Examples of pharmaceutically acceptable carriers herein include: m-cresol, etc.; low molecular weight (less than approximately 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP) (e.g., human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Specific exemplary sHASEGPs and their uses (including rHuPH20) are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0250] An exemplary lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Aqueous aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0251] The formulations herein may contain one or more active ingredients if necessary for the specific indication being treated. Preferably, these ingredients have complementary activities that do not adversely affect each other. Examples include: bone resorption inhibitors, osteogenic agents (i.e., anabolic agents), estrogen receptor modulators (including, but not limited to, raloxifene, bazedoxifene, and rasofoxifene), drugs having inhibitory effects on osteoclasts, bisphosphonates (including, but not limited to, alendronate sodium (FOSAMAX®), risedronate, ibandronate sodium (BONIVA®), and zoledronic acid (RECLAST®)); estrogens or estrogen analogs; anti-RANKL inhibitors such as anti-RANK ligand (RANKL) antibodies (e.g., PROLIA®); vitamin D or vitamin D derivatives or mimics thereof; calcium sources, cathepsin K (cat-K) inhibitors (e.g., odanacatib), tiborone, calcitonin, or calcitriol; and hormone supplements. It is desirable to further provide a second bone-strengthening agent selected from the group consisting of suppositories, parathyroid hormone (PTH) or its peptide fragments, PTH-related proteins (PTHrp), bone morphogenetic proteins (e.g., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, and / or BMP-15), osteogenin, NaF, PGE2 agonists, statins, strontium ranelate, sclerostin inhibitors (e.g., anti-sclerostin antibodies described in U.S. Patent No. 7,592,429 or No. 7,872,106, etc.), anti-DKKl antibodies or inhibitors, Forteo® (teriparatide), Preotact®, or Protelos®. Such active ingredients are present in a suitably combined quantity that is effective for the intended purpose.

[0252] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by, for example, a droplet formation (coacervation) technique or interfacial polymerization, or into a colloidal drug delivery system (e.g., liposomes, albumin spheres, microemulsions, nanoparticles, and nanocapsules), or into a macroemulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0253] A sustained-release formulation may be prepared. A preferred example of a sustained-release formulation is one comprising a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a fabricated product such as a film or microcapsule.

[0254] Preparations used for in vivo administration are typically sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0255] F. Therapeutic methods and therapeutic compositions Any of the anti-sclerostin antibodies provided herein may be used in therapeutic methods. In one aspect, an anti-sclerostin antibody is provided for use as a pharmaceutical. In a further aspect, an anti-sclerostin antibody is provided for use in the treatment of bone-related diseases and / or diseases or conditions associated with elevated levels of sclerostin. In a particular embodiment, an anti-sclerostin antibody is provided for use in a therapeutic method. In a particular embodiment, the present invention provides an anti-sclerostin antibody for use in a method for treating an individual having a bone-related disease and / or a disease or condition associated with elevated levels of sclerostin, the method comprising the step of administering an effective amount of an anti-sclerostin antibody to the individual. In one such embodiment, the method further comprises the step of administering an effective amount of at least one additional therapeutic agent (for example, as described below) to the individual. In a further embodiment, the present invention provides an anti-sclerostin antibody for use in (i) increasing bone formation, (ii) inhibiting bone resorption, and / or (iii) increasing bone mineral density. In a particular embodiment, the present invention provides an anti-sclerostin antibody for use in a method for (i) increasing bone formation, (ii) inhibiting bone resorption, and / or (iii) increasing bone mineral density in an individual, comprising the step of administering an effective amount of an anti-sclerostin antibody to the individual in order to (i) increase bone formation, (ii) inhibit bone resorption, and / or (iii) increase bone mineral density, respectively. In a further embodiment, the present invention provides an anti-sclerostin antibody for use in enhancing the clearance of sclerostin from plasma. In a particular embodiment, the present invention provides an anti-sclerostin antibody for use in a method for enhancing the clearance of sclerostin from plasma in an individual, comprising the step of administering an effective amount of an anti-sclerostin antibody to the individual in order to enhance the clearance of sclerostin from plasma. An anti-sclerostin antibody according to any of the above embodiments can form an immune complex containing at least two antibody molecules. Anti-sclerostin antibodies according to any of the above embodiments can also bind to sclerostin with higher affinity at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.8).The "individual" in any of the above embodiments is preferably a human being.

[0256] In a further aspect, the present invention provides the use of anti-sclerostin antibodies in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of bone-related diseases and / or diseases or conditions associated with elevated levels of sclerostin. In a further embodiment, the pharmaceutical is for use in a method of treating an individual having a bone-related disease and / or a disease or condition associated with elevated levels of sclerostin, the method comprising the step of administering an effective amount of the pharmaceutical to the individual. In one such embodiment, the method further comprises the step of administering an effective amount of at least one additional therapeutic agent (e.g., as described below) to the individual. In a further embodiment, the pharmaceutical is for (i) increasing bone formation, (ii) inhibiting bone resorption, and / or (iii) increasing bone mineral density. In a further embodiment, the pharmaceutical is for use in a method for (i) increasing bone formation, (ii) inhibiting bone resorption, and / or (iii) increasing bone mineral density in an individual, comprising the step of administering an effective amount of the pharmaceutical to the individual in order to (i) increase bone formation, (ii) inhibit bone resorption, and / or (iii) increase bone mineral density, respectively. In a further embodiment, the pharmaceutical is for enhancing the clearance of sclerostin from plasma. In a further embodiment, the pharmaceutical is for use in a method for enhancing the clearance of sclerostin from plasma in an individual, comprising the step of administering an effective amount of the pharmaceutical to the individual in order to enhance the clearance of sclerostin from plasma. Anti-sclerostin antibodies according to any of the above embodiments can form an immune complex containing at least two antibody molecules. Anti-sclerostin antibodies according to any of the above embodiments can also bind to sclerostin with higher affinity at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.8). An "individual" in any of the above-described forms may be a human being.

[0257] In a further aspect, the present invention provides a method for treating bone-related diseases and / or diseases or conditions associated with elevated levels of sclerostin. In one embodiment, the method comprises administering an effective amount of an anti-sclerostin antibody to an individual having such a bone-related disease and / or disease or condition associated with elevated levels of sclerostin. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (as described below) to the individual. The “individual” in any of the above embodiments may be a human.

[0258] In a further aspect, the present invention provides a method for (i) increasing bone formation, (ii) inhibiting bone resorption, and / or (iii) increasing bone mineral density in an individual. In one embodiment, the method comprises administering an effective amount of an anti-sclerostin antibody to an individual to (i) increase bone formation, (ii) inhibit bone resorption, and / or (iii) increase bone mineral density, respectively. In one embodiment, “individual” is a human.

[0259] In a further aspect, the present invention provides a method for enhancing the clearance of sclerostin from plasma in an individual. In one embodiment, the method comprises administering an effective amount of an anti-sclerostin antibody to an individual in order to enhance the clearance of sclerostin from plasma. The anti-sclerostin antibody according to any of the above embodiments can form an immune complex containing at least two antibody molecules. The anti-sclerostin antibody according to any of the above embodiments can also bind to sclerostin with higher affinity at a neutral pH (e.g., pH 7.4) than at an acidic pH (e.g., pH 5.8). In one embodiment, “individual” is a human.

[0260] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-sclerostin antibodies provided herein (for use, for example, in any of the therapeutic methods described above). In one embodiment, the pharmaceutical formulation comprises any of the anti-sclerostin antibodies provided herein and a pharmaceutically acceptable carrier. In a further embodiment, the pharmaceutical formulation is for the treatment of bone-related diseases and / or diseases or conditions associated with elevated levels of sclerostin. In one embodiment, the pharmaceutical formulation is administered to an individual having a bone-related disease and / or a disease or condition associated with elevated levels of sclerostin. In another embodiment, the pharmaceutical formulation comprises any of the anti-sclerostin antibodies provided herein and at least one additional therapeutic agent (for example, as described below). In a further embodiment, the pharmaceutical formulation is for (i) increasing bone formation, (ii) inhibiting bone resorption, and / or (iii) increasing bone mineral density. In a further embodiment, the pharmaceutical formulation is for enhancing the clearance of sclerostin from plasma. An anti-sclerostin antibody according to any of the above embodiments can form an immune complex containing at least two antibody molecules. An anti-sclerostin antibody according to any of the above embodiments can also bind to sclerostin with higher affinity at a neutral pH (e.g., pH 7.4) than at an acidic pH (e.g., pH 5.8). The "individual" according to any of the above embodiments is preferably a human.

[0261] In certain aspects, bone-related diseases include achondroplasia, basocranial dysplasia, enchondromatosis, fibrous dysplasia, Gaucher disease, hypophosphatemic rickets, Marfan syndrome, hereditary multiple exostosis, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteomycosis, sclerosing lesions, pseudoarthrosis, suppurative osteomyelitis, periodontal disease, antiepileptic drug-induced bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism, weight-loss-induced bone loss, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative bone disorders, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, and Mero Rheostomy, metabolic bone disease, mastocytosis, sickle cell anemia / disease, organ transplant-related bone loss, kidney transplant-related bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidosis, Fabry disease, Turner syndrome, Down syndrome, Klinefelter syndrome, leprosy, Perthes disease, adolescent idiopathic scoliosis, infant-onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson's disease, ischemic bone disease (such as Legg-Calvé-Perthes disease and focal migratory osteoporosis), anemia, steroid-induced Conditions caused by: glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal conditions, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory bowel syndrome, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy-related bone loss, diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism, motor depression or inactivity, reflex sympathetic dystrophy syndrome, focal osteoporosis, osteomalacia, joint replacement The group is selected from the following: surgery-related bone loss, HIV-related bone loss, bone loss associated with growth hormone reduction, bone loss associated with cystic fibrosis, chemotherapy-related bone loss, tumor-induced bone loss, cancer-related bone loss, bone loss due to hormone deprivation, multiple myeloma, drug-induced bone loss, anorexia nervosa, disease-related facial bone loss, disease-related cranial bone loss, disease-related jawbone loss, disease-related skull loss, age-related bone loss, age-related facial bone loss, age-related cranial bone loss, age-related jawbone loss, age-related skull loss, and bone loss associated with space travel.

[0262] In some embodiments, pharmaceuticals for increasing bone formation, inhibiting bone resorption, and / or increasing bone mineral density are useful in improving outcomes of orthopedic procedures, dental procedures, transplant surgeries, joint replacements, bone grafts, bone cosmetic surgeries, and bone repairs, such as fracture healing, nonunion, delayed union, and facial reconstruction. Such pharmaceuticals may be administered before, during, and / or after procedures, replacements, transplants, surgeries, or repairs.

[0263] In some embodiments, pharmaceuticals for increasing bone formation, inhibiting bone resorption, and / or increasing bone mineral density are useful for treating any fracture involving a gap between two bone segments. Exemplary interosseous gap defects include, but are not limited to, comminuted fractures, ununion fractures, partial skeletal defects, surgically resulting bone defects, surgically treated bone defects, and bone defects resulting from traumatic bone injury or disease (including, but not limited to, arthritis, tumor removal (excision), or infection removal). In some embodiments, interosseous gap defects are created by the removal of an infected bone segment or by the removal of cancer from bone due to bone cancer including, but not limited to, osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma, and chordoma. In some embodiments, interosseous gap defects are developmental deformities, for example, due to gene defects. In some embodiments, interosseous gap defects are created by the removal of a bone segment containing a benign tumor. Exemplary benign bone tumors include, but are not limited to, osteomas, osteoids, osteoblastomas, osteochondromas, enchondromas, chondromyxofibromas, aneurysmal bone cysts, solitary bone cysts, fibrous dysplasia, and giant cell tumors of bone.

[0264] Bone formation and / or bone mineral density can be measured using radiography (e.g., absorptiometry), single or dual-energy absorptiometry, quantitative computed tomography (QCT), ultrasound, and magnetic resonance imaging. Bone mass can also be calculated from body weight or by other means (see Gunness-Hey, Metab. Bone Dis. Relat. Res., 5:177-181 (1984)). Animal models that mimic human disease conditions such as osteoporosis and osteopenia are used in the art to test the effects of pharmaceutical compositions and methods on parameters such as bone loss, bone resorption, bone formation, bone strength, or bone mineralization. Examples of such models include the ovariectomized rat model (Kalu, Bone and Mineral, 15:175-191 (1991); Frost and Jee, Bone and Mineral, 18:227-236 (1992); and Jee and Yao, J. Musculoskelet. Neuronal Interact., 1:193-207 (2001)).

[0265] Alternatively, physiological responses to one or more sclerostin binders can be determined by monitoring bone marker levels. Bone markers are products of bone remodeling and are released by bone, osteoblasts, and / or osteoclasts. Fluctuations in bone marker levels indicate changes in bone remodeling. Markers indicating bone resorption (or osteoclast activity) include, for example, C-telopeptides (e.g., C-terminal telopeptide of type I collagen (CTX) or serum cross-linked C-telopeptide), N-telopeptides (N-terminal telopeptide of type I collagen (NTX)), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline, galactosylhydroxylysine, and tartrate-resistant acid phosphatases (e.g., serum tartrate-resistant acid phosphatase isoform 5b). Bone formation / mineralization markers include, but are not limited to, bone-specific alkaline phosphatase (BSAP), peptides released from the N-terminal and C-terminal extensions of type I procollagen (P1NP, P1CP), and osteocalcin.

[0266] In a further aspect, the present invention provides a method for preparing a pharmaceutical or pharmaceutical formulation, comprising the step of mixing one of the anti-sclerostin antibodies provided herein with a pharmaceutically acceptable carrier for use in, for example, any of the therapeutic methods described above. In one embodiment, the method for preparing a pharmaceutical or pharmaceutical formulation further comprises the step of adding at least one additional therapeutic agent to the pharmaceutical or pharmaceutical formulation.

[0267] The antibodies of the present invention may be used in therapy either alone or in combination with other agents. For example, the antibodies of the present invention may be administered concurrently with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agents may include bone resorption inhibitors, osteogenic agents (i.e., anabolic agents), estrogen receptor modulators (including, but not limited to, raloxifene, bazedoxifene, and rasofoxifene), drugs having an inhibitory effect on osteoclasts (including, but not limited to, alendronate sodium (FOSAMAX®), risedronate, ibandronate sodium (BONIVA®), and zoledronic acid (RECLAST®)); estrogens or estrogen analogs; anti-RANK ligand (RANKL) inhibitors such as anti-RANKL antibodies (e.g., PROLIA®); vitamin D or vitamin D derivatives or mimics thereof; calcium sources, cathepsin K (cat-K) inhibitors (e.g., odanacatib), tiborone, calcitonin , or calcitriol; hormone replacement therapy, parathyroid hormone (PTH) or its peptide fragments, PTH-related proteins (PTHrp), bone morphogenetic proteins (e.g., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, and / or BMP-15), osteogenin, NaF, PGE2 agonists, statins, strontium ranelate, sclerostin inhibitors (e.g., anti-sclerostin antibodies described in U.S. Patent No. 7,592,429 or No. 7,872,106, etc.), anti-DKKl antibodies or inhibitors, Forteo® (teriparatide), Preotact®, or Protelos®.

[0268] The combination therapies described above include combination administration (where two or more therapeutic agents are contained in the same or separate formulations) and individual administration, in which case the antibody of the present invention may be administered prior to, simultaneously with, and / or subsequently to the administration of the additional therapeutic agent. In one embodiment, the administration of the anti-sclerostin antibody and the administration of the additional therapeutic agent are performed within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days.

[0269] The antibodies (and any additional therapeutic agents) of the present invention may be administered by any preferred means, including parenteral administration, intrapulmonary administration, and nasal administration, and, if desired for local treatment, intrafocal administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be made by any preferred route, such as injection, including intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules, including single doses, repeated doses over various time points, bolus administration, and pulse infusion, are within consideration herein, but are not limited to these.

[0270] The antibodies of the present invention are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered from this perspective include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to healthcare professionals. The antibodies are formulated, optionally but not necessarily, with one or more agents already in use to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are typically used in the same doses and routes of administration as described herein, or at about 1 to 99% of the doses described herein, or in any dose and route deemed empirically / clinically appropriate.

[0271] For the prevention or treatment of a disease, the appropriate dose of the antibody of the present invention (when used alone or with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, the patient's medical history, clinical history and response to the antibody, and the discretion of the attending physician. The antibody is preferably administered to the patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, whether by single or multiple separate doses or by continuous infusion, an antibody dose of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to the patient. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg or more, depending on the factors described above. In the case of repeated administrations over several days or longer, treatment is usually maintained, depending on the situation, until the desired suppression of disease symptoms occurs. One exemplary dose of the antibody is in the range of approximately 0.05 mg / kg to approximately 10 mg / kg. Therefore, one or more doses (or any combination thereof) of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (for example, so that the patient receives approximately 2 to approximately 20, or for example, approximately 6, doses of antibody). One or more low doses may be administered after a high initial loading dose. The course of this therapy is readily monitored by conventional methods and measurements.

[0272] It will be understood that either of the above-described formulations or therapeutic methods may be carried out using the immunoconjugate of the present invention, either in place of or in addition to the anti-sclerostin antibody.

[0273] G.Product In another aspect of the present invention, a product is provided comprising equipment useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The product comprises a container and a label on the container or a document accompanying the container. Preferred containers include, for example, bottles, vials, syringes, and IV solution bags. Containers may be formed from a variety of materials, such as glass or plastic. A container may hold a composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a symptom, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous injection needle). At least one active ingredient in the composition is the antibody of the present invention. The label or document indicates that the composition is used to treat a selected symptom. The product further comprises (a) a first container comprising a composition containing the antibody of the present invention; and (b) a second container comprising a composition containing a further cytotoxic agent or other therapeutic agent. The product in this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other commercially or user-desirable equipment, such as other buffers, diluents, filters, needles, and syringes.

[0274] It will be understood that any of the above-mentioned products may contain the immunoconjugate of the present invention in place of or in addition to the anti-sclerostin antibody.

[0275] All patent and non-patent documents cited herein are incorporated herein by reference. [Examples]

[0276] The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be implemented.

[0277] Example 1: Expression and purification of recombinant human, cynomolgus monkey, rat, and mouse sclerostin Recombinant human sclerostin (NCBI GenBank accession number: NP_079513, SEQ ID NO: 1) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). The culture supernatant expressing human sclerostin was applied to a heparin Sepharose HP column (GE Healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. The fraction containing human sclerostin was pooled and the salt concentration was adjusted to 100 mM NaCl. The resulting sample was applied to an SP Sepharose HP cation exchange column (GE Healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. The fraction containing human sclerostin was pooled and subjected to Superdex 200 or Superdex 75 gel filtration column (GE Healthcare, Uppsala, Sweden). The fraction containing human sclerostin was pooled and stored at -150°C.

[0278] Recombinant cynomolgusal sclerostin (SEQ ID NO: 2), recombinant rat sclerostin (NCBI GenBank accession number: NP_085073.1, SEQ ID NO: 3), and recombinant mouse sclerostin (NCBI GenBank accession number: NP_077769.4, SEQ ID NO: 4) were expressed and purified using the exact same method as for the human counterparts.

[0279] Example 2: Generation of pH-dependent anti-sclerostin antibodies and biparatopic antibodies The amino acid sequences of the anti-sclerostin antibodies mabA (VH; SEQ ID NO: 7 and VL; SEQ ID NO: 15) and mabB (VH; SEQ ID NO: 23 and VL; SEQ ID NO: 27), both of which are publicly known in the art, were modified to impart pH-dependent antigen-binding activity to the antibodies. Antibody variants of mabA and mabB were generated as follows: A vast number of mutations and their combinations were tested, and several mutations were introduced into the variable region of mabA or mabB to improve antigen-binding properties, resulting in the optimized variable regions mabA_pH1 (VH; SEQ ID NO: 8 and VL; SEQ ID NO: 16), mabA_pH2 (VH; SEQ ID NO: 9 and VL; SEQ ID NO: 17), mabA_pH3 (VH; SEQ ID NO: 10 and VL; SEQ ID NO: 18), mabA_pH4 (VH; SEQ ID NO: 11). The following antibody variants were generated: mabA_NpH1 (VH; SEQ ID NO: 12 and VL; SEQ ID NO: 20), mabA_NpH2 (VH; SEQ ID NO: 13 and VL; SEQ ID NO: 21), mabA_NpH3 (VH; SEQ ID NO: 14 and VL; SEQ ID NO: 22), mabB_pH1 (VH; SEQ ID NO: 24 and VL; SEQ ID NO: 28), mabB_pH4 (VH; SEQ ID NO: 25 and VL; SEQ ID NO: 29), and mabB_pH5 (VH; SEQ ID NO: 26 and VL; SEQ ID NO: 30). The amino acid sequences of the antibody variants are summarized in Table 2. The gene encoding VH was combined with wild-type human IgG2 CH, hG2 (SEQ ID NO: 77), human IgG4 CH, hG4 (SEQ ID NO: 108), or modified human IgG CH variants, SG1 (SEQ ID NO: 78), SG2 (SEQ ID NO: 79), BS01b (SEQ ID NO: 80), and BS01a (SEQ ID NO: 81). The gene encoding VL was combined with wild-type human CL, SK1 (SEQ ID NO: 82), and each of these combinations was cloned into an expression vector.

[0280] (Table 2) Amino acid sequences of mabA and mabB variants TIFF2026095400000002.tif73170

[0281] For pharmacokinetic analysis, the anti-sclerostin antibody SCL0099-rbIgG (VH; SEQ ID NO: 83 and VL; SEQ ID NO: 84) was prepared as follows: 12-16 week old NZW rabbits were intradermally immunized with human sclerostin and / or monkey sclerostin (50-100 μg / administration / rabbit). This administration was repeated 4-5 times over 2 months. One week after the final immunization, spleens and blood were collected from the immunized rabbits. Antigen-specific B cells were stained with labeled antigens, sorted using an FCM cell sorter (FACS aria III, BD), and seeded at a density of 1 cell / well in 96-well plates with 25,000 cells / well of EL4 cells (European Collection of Cell Cultures) and 20-fold diluted activated rabbit T cell culture supernatant, and cultured for 7-12 days. EL4 cells were pre-treated with mitomycin C (Sigma, Cat No. M4287) for 2 hours and washed three times before use. Activated rabbit T cell culture supernatant was prepared by culturing rabbit thymocytes in RPMI-1640 medium containing phytohemagglutinin M (Roche, Cat No. 1 1082132-001), phorbol 12-myristate 13-acetate (Sigma, Cat No. P1585), and 2% FBS. After culturing, the B cell culture supernatant was collected for further analysis, and the pellet was cryopreserved. Several anti-sclerostin antibodies were selected, and the heavy chain variable region and light chain variable region were cloned together with the rabbit IgG constant region (rbIgG) (SEQ ID NO: 85) and the rabbit IgG constant region (rbIgk) (SEQ ID NO: 86), respectively. SCL0099-rbIgG was then selected for pharmacokinetic analysis. Anti-sclerostin antibodies SCL0122-SG110 (VH; SEQ ID NO: 87 and VL; SEQ ID NO: 88) were also prepared for pharmacokinetic analysis. VH and VL were combined with the modified human IgG CH variant, SG110 (SEQ ID NO: 89), and wild-type human CL, SK1 (SEQ ID NO: 82), respectively. Anti-sclerostin antibodies SCL0800-rbIgG (VH; SEQ ID NO: 90 and VL; SEQ ID NO: 91) were also prepared for pharmacokinetic analysis.VH and VL were combined with the rabbit IgG constant region (rbIgG) (SEQ ID NO: 85) and the rabbit Igk constant region (rbIgk) (SEQ ID NO: 86), respectively.

[0282] Recombinant antibodies were transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Purification of the antibody-expressing culture supernatant was performed using a conventional method with protein A. Further gel filtration was performed as needed.

[0283] Pairs of parental antibodies (Ab-A and Ab-B, listed in Table 3) were mixed in a 1:1 molar ratio in 1×PBS in the presence of 25 mM 2-mercaptoethylamine-HCl (2-MEA) and incubated at 37°C for 90 minutes to generate bispecific antibodies. The bispecific antibody products were purified by using protein A under standard conditions to remove 2-MEA.

[0284] (Table 3) Construction of biparatopic antibodies TIFF2026095400000003.tif72147

[0285] Example 3: Evaluation of pH dependence in Biacore The binding affinity of anti-sclerostin variants to human sclerostin, cynomolgus monkey (cyno) sclerostin, rat sclerostin, or mouse sclerostin was measured at pH 7.4 and pH 5.8 at 37°C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized on all flow cells of the CM4 sensor tip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in ACES buffer at pH 7.4 or pH 5.8 containing 20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, and 0.005% NaN3. Each antibody was captured on the sensor surface by anti-human Fc. Human sclerostin, cyno-sclerostin, rat sclerostin, or mouse sclerostin were injected at 1.25 nM and 5 nM or 0.5 nM and 2 nM for the pH 7.4 assay conditions, and at 5 nM or 2 nM for the pH 5.8 assay conditions. The sensor surface was regenerated with 3 MgCl2 in each cycle. Binding affinity was measured by processing the data and fitting it to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare).

[0286] pH-dependent interaction was assessed using a modified Biacore assay. Briefly, an additional dissociation phase at pH 5.8 was incorporated into the Biacore assay immediately following the dissociation phase at pH 7.4. This was done to evaluate the pH-dependent dissociation between the antibody and antigen from the complex formed at pH 7.4. The dissociation rate in pH 5.8 buffer was measured by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0287] Table 4 shows the binding affinity (KD) of anti-sclerostin variants to human sclerostin, cynomolgus monkey (cyno) sclerostin, rat sclerostin, or mouse sclerostin at pH 7.4 and pH 5.8. off 7.4>>5.8 refers to the dissociation rate at pH 5.8 for antibody / antigen complexes formed at pH 7.4.

[0288] (Table 4) Dynamic parameters of anti-sclerostin (SOST) antibody at pH 7.4 and pH 5.8 TIFF2026095400000004.tif23481Note: nd refers to a weak bond, where KD cannot be measured. * indicates a fast koff at pH 7.4, and koff at pH 5.8 is not measurable. # indicates slow dissociation, and the koff value at pH 5.8 is unmeasurable.

[0289] Example 4: Evaluation of in vitro neutralization effectiveness Wnt1-induced reporter gene assay Complete culture medium was prepared as follows: 50 mL of fetal bovine serum (Bovogen, Cat. SFBS-D), 5 mL of penicillin / streptomycin (Gibco, Cat. 15140-122), and 5 mL of MEM non-essential amino acids (Gibco, Cat. 11140-050) were added to 500 mL of DMEM (Gibco, Cat. 11965-092).

[0290] HEK293T cells (ATCC CRL11268) in complete medium were transfected 2 × 10⁶ cells per well the day before transfection. 4 Cells were seeded at a specific density (in 0.05 mL volumes) into 96-well plates (PerkinElmer Cat.600568).

[0291] The following day, 3-fold serial dilutions of the antibody were prepared in PBS, starting from 400 μg / mL. 4 μg / mL of sclerostin (monkey, rat, or mouse) was prepared in complete medium. 60 μL of serially diluted antibody and 60 μL of 4 μg / mL of sclerostin were mixed in a polypropylene plate and incubated at 37°C for 1 hour. 50 μL of the antibody-antigen mixture was added to a culture plate and incubated at 37°C for 30 minutes. The final concentrations of antibody and sclerostin in the culture medium were 100 μg / mL and 1 μg / mL, respectively.

[0292] The following plasmids were used in this assay: phWnt1, a human Wnt1 plasmid (nucleotide sequence of SEQ ID NO: 5, amino acid sequence of SEQ ID NO: 6) driven by a CAG promoter; pCXND3, an empty CAG vector; pTCF-Fluc (Promega pGL4.49), a firefly luciferase plasmid driven by a TCF / LEF response element; and pTK-Rluc (Promega pGL4.74), a sea urchin luciferase plasmid driven by a TK promoter.

[0293] Plasmid transfection was performed as follows: Fixed doses of plasmid were added to 5 mL / well of OptiMEM (Invitrogen, Cat. 31985-070). Specifically, for the control well, 0.1 ng pCXND3, 50 ng pTCF-Fluc, and 50 ng pTK-Rluc were added to 5 mL / well of OptiMEM (Invitrogen, Cat. 31985-070). 0.3 mL of FuGENE (Promega, Cat. E231A) per well was added to the diluted plasmid and thoroughly mixed. The DNA-lipid complex was then added to the cells and incubated in a 5% CO2 incubator at 37°C for longer than 16 hours. Each condition was tested twice.

[0294] The next day, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega, Cat. E2940). The measurement of luciferase activity was performed using a microplate reader, SpectraMax Paradigm (Molecular Devices).

[0295] The ratio of firefly to Renilla luciferase (F / R) was calculated for each well. The percent inhibition of sclerostin was defined as 100 × [(F / R MAb - F / R Wnt(+)SOST(+) ) / (F / R Wnt(+)SOST(-) - F / R Wnt(+)SOST(+) )]. In this specification, F / R Wnt(+)SOST(-) represents the F / R value in the presence of Wnt1 alone, which indicates the assumed maximum inhibition of sclerostin. F / R Mab represents the F / R value in the presence of the anti-sclerostin monoclonal antibody being evaluated, Wnt1, and sclerostin. F / R Wnt(+)SOST(+) represents the F / R value in the presence of Wnt1 and sclerostin, which indicates no inhibition of sclerostin.

[0296] The pH-dependent antibodies (mabA_pH1-SG2, mabA_pH2-SG2, and mabA_pH3-SG2) showed equivalent neutralizing activity compared to the parental mabA-hG2 (Figures 1 and 2).

[0297] The pH-dependent biparatopic antibodies (mabB / / mabA_pH1-BS01, mabB / / mabA_pH2-BS01, and mabB / / mabA_pH3-BS01) showed superior specific activity compared to mabA-hG2 in the Wnt1 reporter gene assay (Figure 3).

[0298] Example 5: Immune Complex Formation of Biparatopic Anti-Sclerostin Antibodies Antigen uptake assay The ability of antibodies to uptake antigens was evaluated by in vitro cell-based assays. MDCK cells expressing human Fcγ receptor IIB and human FcRn were established and cultured in EMEM (Sigma, Cat. M5650) containing 10% fetal bovine serum (Bovogen, Cat. SFBS-D), 2 mM GlutaMAX-1 (Invitrogen, Cat. 35050-061), and 1% penicillin / streptomycin (Invitrogen Cat. 15140).

[0299] MDCK cells are divided into 5 x 10⁶ wells. 4 Cells were seeded in 48-well plates at their normal cell density. Mouse sclerostin was labeled with the pH-sensitive fluorescent dye pHrodo-Red (Molecular Probes, Cat. P36600) according to the manufacturer's instructions. The antibody and labeled sclerostin were mixed in a 1:1 molar ratio in culture medium and incubated at 37°C for 1 hour. Immediately after incubation, the cell culture medium was aspirated and replaced with the antibody-antigen mixture, and the cell culture was then incubated at 37°C for 1.5 hours. MDCK cells were washed and detached with trypsin. MDCK cells were washed with culture medium and suspended in 0.2% FBS in PBS. The amount of antigen that had migrated internally was measured based on the fluorescence intensity detected by flow cytometry. The mean fluorescence intensity (MFI) in live cells was calculated using FlowJo software. mabB / / mabA_pH1-BS01, mabB / / mabA_pH2-BS01, and mabB / / mabA_pH3-BS01 showed enhanced antigen uptake compared to non-pH-dependent antibodies (mabB / / mabA_NpH3-BS01 and mabA_NpH3-SG2) (Table 5). These results are thought to be due to the antibody's ability to form large immune complexes with the antigen, resulting in accelerated uptake of the complex into cells, and the antibody's pH-dependent antigen-binding activity, resulting in accelerated release of the antigen within cells.

[0300] (Table 5) Amount of fluorescently labeled sclerostin taken up by cells TIFF2026095400000005.tif53165

[0301] Transmission electron microscopy Immunocomplex formation was confirmed by transmission electron microscopy. mabB / / mabA-BS01 and human sclerostin were mixed in a 1:1 molar ratio and incubated at 37°C for 1 hour. Immediately after incubation, the mixture was diluted to 1:1000 for imaging. Electron microscopy analysis was performed using an FEI Tecnai T12 electron microscope equipped with a CCD camera. Images of each grid were acquired at multiple scales to assess the overall distribution of the specimen. Microscopic observation and analysis were performed at Nanoimaging Services, Inc. Representative images are shown in Figure 4. Complexes composed of two antibody molecules and two antigen molecules were observed.

[0302] Size exclusion chromatography Size exclusion chromatography (SEC) was performed using an ACQUITY UPLC H-Class system (Waters) with an ACQUITY UPLC Protein BEH SEC column (450 Å, 1.7 μm, 4.6 mm × 150 mm) (Waters) in 50 mM Na2HPO4, 300 mM NaCl (pH 7.0) at a flow rate of 0.3 ml / min. Detection was performed using a UV detector (280 nm). For immunocomplex sample preparation, each antibody (1 μM) was mixed with human sclerostin (1 μM) in a 1:1 molar ratio and incubated in PBS at 25°C for 1 hour. Chromatograms for mabB / / mabA-BS01, mabB / / mabA_pH1-BS01, and mabB / / mabA_pH2-BS01 are shown in Figures 5a, 5b, and 5c, respectively. Chromatograms suggest that these biparatopic antibodies formed large immune complexes containing multiple antibody molecules when mixed with the antigen.

[0303] 5.4. Biacore analysis of FcγRIIB binding The binding of anti-sclerostin antibody / sclerostin immune complex to mouse FcγRIIB at pH 7.4 was determined using a Biacore T200 instrument (GE Healthcare) at 25°C. All antibodies and mouse FcγRIIB were prepared in ACES (pH 7.4) containing 20 mM ACES, 250 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 1 mg / ml BSA, 1 mg / ml carboxymethyl dextran (CMD), and 0.005% NaN3. Anti-histidine antibody (GE Healthcare) was immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). Mouse FcγRIIB was captured on flow cell 2 with the anti-histidine antibody, using flow cell 1 as the reference flow cell. The mouse FcγRIIB capture level was set to 400 resonance units (RU). All antibodies were injected into all flow cells at 100 nM. Immunoconjugates were prepared by mixing antibodies and human sclerostin in a 1:1 molar ratio and incubated at room temperature for 1 hour. In each cycle, the sensor surface was regenerated with 10 mM glycine HCl (pH 1.5).

[0304] The binding levels of anti-sclerostin antibodies or immune complexes to mouse FcγRIIB were monitored from the binding response. Binding levels were standardized against the corresponding mouse FcγRIIB capture levels. The results indicate that antibody binding to FcγRIIB was significantly enhanced when the antibody formed an immune complex with the antigen (Figure 6).

[0305] Example 6: In vivo pharmacokinetics and sweeping effect of pH-dependent anti-sclerostin antibody (monkey) In vivo trial using cynomolgus monkeys The in vivo pharmacokinetics of anti-sclerostin antibodies were evaluated in cynomolgus monkeys (Primetrics or Biological Resource Centre, Singapore). A dose level of 10 mg / kg was administered via cephalic or saphenous vein using a disposable syringe. Blood samples were collected before administration and at 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. Blood was immediately cooled on ice, and plasma was obtained by centrifugation at 4°C and 2500×g for 10 minutes. Plasma samples were stored in a -80°C freezer until measurement. The anti-sclerostin antibodies used were mabA-hG2, mabA_pH1-SG2, mabA_pH2-SG2, mabA_NpH1-SG2, and mabA_NpH2-SG2.

[0306] Measurement of anti-sclerostin antibody concentration in plasma by ELISA The concentration of anti-sclerostin antibody in cynomolgus monkey plasma was measured by ELISA. To prepare anti-human IgG immobilized plates, anti-human IgG k-chain antibody (Antibody Solutions) was dispensed into Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left overnight at 4°C. Calibration curve samples and cynomolgus monkey plasma samples were prepared at dilutions of 100-fold or more. The samples were then dispensed into anti-human IgG immobilized plates and left at room temperature for 1 hour. Next, HRP-labeled anti-human IgG antibody (SouthernBiotech) was added and allowed to react at room temperature for 30 minutes, followed by washing. Subsequently, ABTS ELISA HRP substrate (KPL) was added. The signal was measured using a plate reader at a wavelength of 405 nm. The anti-sclerostin antibody concentration was calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). The time-course plasma anti-sclerostin antibody concentrations measured by this method are shown in Figure 7. The results indicate that the pH-dependent anti-sclerostin antibodies mabA_pH1-SG2 and mabA_pH2-SG2 have extended PK profiles compared to the non-pH-dependent anti-sclerostin antibodies mabA_NpH1-SG2 and mabA_NpH2-SG2, respectively.

[0307] Measurement of total sclerostin concentration in plasma by electrochemiluminescence (ECL) The concentration of total sclerostin in cynomolgus monkey plasma was measured by ECL. Anti-sclerostin immobilized plates were prepared by distributing the anti-sclerostin antibody SCL0099-rbIgG onto an untreated MULTI-ARRAY 96-well plate (Meso Scale Discovery) and leaving it overnight at 4°C. Calibration curve samples and cynomolgus monkey plasma samples were prepared at least 16.7-fold dilutions. The samples were mixed with mabA-hG2 solution (20 μg / mL) and incubated at 37°C for 1 hour to ensure that all sclerostins formed complexes with the antibody. Subsequently, the samples were added to anti-sclerostin immobilized plates and incubated at 30°C for 1 hour. Next, SULFO TAG-labeled anti-human IgG antibody (SouthernBiotech) was added, and the plates were incubated at room temperature for 1 hour. Read Buffer T (×2) (Meso Scale Discovery) was immediately added to the plate, and the signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). Total sclerostin concentration was calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). The time-course plasma total sclerostin concentrations measured by this method are shown in Figure 8. The results indicate that the pH-dependent anti-sclerostin antibodies mabA_pH1-SG2 and mabA_pH2-SG2 have a 2-3-fold ability to reduce plasma sclerostin accumulation compared to the non-pH-dependent anti-sclerostin antibodies mabA_NpH1-SG2 and mabA_NpH2-SG2, respectively.

[0308] Example 7: In vivo PD efficacy of pH-dependent anti-sclerostin antibody (monkey) Evaluation of serum bone markers in monkeys Osteocalcin, a clinically used bone formation marker, was measured in the monkeys described in Example 6. Serum was collected weekly and measured using a commercially available ELISA kit (Biomedical Technologies Inc., Cat. J64816). Each serum was diluted 10-fold with the specified sample buffer before measurement. Osteocalcin concentration is expressed as a percentage change from baseline (concentration before antibody administration is shown as 100%). ADA-positive animals were omitted from the analysis. The time-course serum osteocalcin concentrations measured by this method are shown in Figure 9. Mean values ​​(n=3-4) are shown along with error bars indicating the standard deviation. pH-dependent anti-sclerostin antibodies mabA_pH1-SG2 and mabA_pH2-SG2 showed higher osteocalcin levels compared to non-pH-dependent anti-sclerostin antibodies mabA_NpH1-SG2 and mabA_NpH2-SG2, respectively.

[0309] Example 8: In vivo efficacy of pH-dependent sclerostin antibody in normal rats In vivo study using normal rats The efficacy of pH-dependent anti-sclerostin antibodies was evaluated in normal rats. Conventional antibody mabA-hG2 at 10 mg / kg, non-pH-dependent antibodies mabA_NpH3-SG2 at 3 mg / kg and 10 mg / kg, and pH-dependent antibodies mabA_pH3-SG2 at 1 mg / kg, 3 mg / kg, and 10 mg / kg were administered intravenously (IV) once weekly for 4 weeks to 8-week-old female normal rats (Charles River Laboratories Japan, Inc.). Plasma samples were obtained by collecting blood from the jugular vein once weekly prior to antibody administration, and the plasma antibody levels were measured by ELISA. After 4 weeks of administration, the rats were sacrificed, and the lumbar vertebrae and right femur were obtained. Bone mineral density (BMD) of the lumbar vertebrae and right femur was evaluated by dual-energy X-ray absorptiometry (DXA) using DCS-600EX (Aloka). Data were expressed as mean + / - SE, and statistical significance was determined using JMP (Ver. 11.2.1, SAS Institute Inc.). Williams' test or Student's t-test was performed to detect significant differences in the antibody treatment group compared to the vehicle group or other antibody groups with the same dosage.

[0310] Measurement of anti-sclerostin antibody concentration in plasma by ELISA The concentration of anti-sclerostin antibody in rat plasma was measured by ELISA as described in Example 6. The rat plasma samples were diluted more than 100-fold. Figure 10 shows the time-course plasma anti-sclerostin antibody concentrations measured by this method. Plasma levels of the antibody increased in a dose-dependent manner.

[0311] The antibody mabA_pH3-SG2 significantly increased BMD of the lumbar spine and the entire femur at doses of 3 mg / kg and 10 mg / kg (Figures 11a and 11b). The antibody mabA_NpH3-SG2 increased lumbar spine BMD at 10 mg / kg and femoral BMD at 3 mg / kg and 10 mg / kg. The antibody mabA-hG2 significantly increased BMD of the lumbar spine and femur at 10 mg / kg. The BMD levels of the lumbar spine and the entire femur with 3 mg / kg of mabA_pH3-SG2 were significantly higher than those with 3 mg / kg of mabA_NpH3-SG2.

[0312] These results indicate that in normal rats, the anabolic effect of the pH-dependent antibody mabA_pH3-SG2 on BMD in the lumbar vertebrae and femur was higher than that of the non-pH-dependent antibody mabA_NpH3-SG2.

[0313] Example 9: In vivo efficacy of pH-dependent anti-sclerostin biparatopic antibody in SCID mice In vivo testing using SCID mice The efficacy of biparatopic anti-sclerostin sweeping antibodies was evaluated in SCID mice. The conventional antibody mabA-hG2, the pH-dependent antibody mabA_pH3-SG2, and the biparatopic sweeping antibody mabB / / mabA_pH3-BS01 were administered intravenously (IV) at doses of 2 mg / kg and 10 mg / kg to 8-week-old female SCID mice (Charles River Laboratories Japan, Inc.) once a week for 4 weeks. Plasma samples were obtained weekly by collecting blood from the jugular vein before antibody administration, and the plasma antibody levels were measured by ELISA. After 4 weeks of administration, the mice were sacrificed and the lumbar vertebrae were obtained. Bone mineral density (BMD) of the lumbar vertebrae was evaluated by dual-energy X-ray absorptiometry (DXA) using a DCS-600EX (Aloka). Data were expressed as mean + / - SE, and statistical significance was determined using JMP (Ver. 11.2.1, SAS Institute Inc.). Williams' test or Student's t-test was performed to detect significant differences in the antibody-treated group compared to the vehicle group or other antibody groups with the same dosage.

[0314] Measurement of anti-sclerostin antibody concentration in plasma by ELISA The concentration of anti-sclerostin antibody in mouse plasma was measured by ELISA as described in Example 6. The plasma mouse samples were diluted more than 100-fold. The time-course plasma anti-sclerostin antibody concentrations measured by this method are shown in Figure 12.

[0315] Measurement of total sclerostin concentration in plasma by electrochemiluminescence (ECL) The concentration of total sclerostin in mouse plasma was measured by ECL. For mouse plasma samples that did not contain anti-sclerostin antibody, anti-sclerostin immobilized plates were prepared by distributing the anti-sclerostin antibody SCL0122-SG110 onto an untreated MULTI-ARRAY 96-well plate (Meso Scale Discovery) and leaving it overnight at 4°C. Calibration curve samples and mouse plasma samples were prepared at a 10-fold dilution. Subsequently, the samples were added to the anti-sclerostin immobilized plates and incubated at 30°C for 1 hour. Next, SULFO TAG-labeled mabA-hG2 was added, and the plates were incubated at 30°C for 1 hour. Read Buffer T (×2) (Meso Scale Discovery) was immediately added to the plates, and the signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). For mouse plasma samples containing anti-sclerostin antibody, anti-sclerostin immobilized plates were prepared by distributing the anti-sclerostin antibody SCL0800-rbIgG onto an untreated MULTI-ARRAY 96-well plate (Meso Scale Discovery) and leaving it overnight at 4°C. Calibration curve samples and mouse plasma samples diluted 500-fold were prepared. The samples were mixed with the injected antibody solution (2 μg / mL) and incubated at 37°C for 1 hour to ensure that all sclerostin formed a complex with the antibody. Subsequently, the samples were added to the anti-sclerostin immobilized plates and incubated at 30°C for 1 hour. Next, SULFO TAG-labeled anti-human IgG antibody (SouthernBiotech) was added, and the plates were incubated at 30°C for 1 hour. Read Buffer T (×2) (Meso Scale Discovery) was immediately added to the plates, and the signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). Total sclerostin concentrations were calculated based on the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). The time-course plasma total sclerostin concentrations measured by this method are shown in Figure 13.

[0316] The antibody mabA-hG2 significantly increased lumbar spine BMD at 10 mg / kg (Figure 14). The antibody mabA_pH3-SG2 increased lumbar spine BMD at 2 mg / kg and 10 mg / kg. The antibody mabB / / mabA_pH3-BS01 significantly increased lumbar spine BMD at 2 mg / kg and 10 mg / kg. The level of lumbar spine BMD with 10 mg / kg of mabB / / mabA_pH3-BS01 was significantly higher than the levels with the same dosages of mabA-hG2 and mabA_pH3-SG2.

[0317] These results demonstrate that in SCID mice, the anabolic effect of the biparatopic anti-sclerostin sweeping antibody mabB / / mabA_pH3-BS01 against BMD is superior to that of the conventional antibody mabA-hG2 or the pH-dependent antibody mabA_pH3-SG2.

[0318] Example 10: Identification of the common light chain for mabA and mabB ("residue shuffling") For therapeutic drug development, it is necessary to optimize bispecific (biparatopic) antibodies through molecular engineering to enable large-scale production at clinical grade. A variety of molecular formats for bispecific antibodies have been studied, including single-chain diabodies, tandem scFv, IgG-scFv, DVD-Ig, CrossMab, double-binding Fa...

Claims

1. An isolated multispecific antibody that binds to sclerostin, comprising at least two different variable regions for binding to at least two different epitopes of sclerostin, and binding to sclerostin with higher affinity at neutral pH than at acidic pH.

2. Sclerostin forms an immune complex, The immune complex comprises at least two antibody molecules and at least two sclerostin molecules. The multispecific antibody according to claim 1.

3. The multispecific antibody according to claim 1 or 2, wherein at least one of two variable regions binds to the same epitope as the anti-sclerostin antibody comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO:

15.

4. A multispecific antibody according to any one of claims 1 to 3, wherein at least one of two variable regions binds to the same epitope as an anti-sclerostin antibody comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO:

27.

5. A multispecific antibody according to any one of claims 1 to 4, wherein at least one of two variable regions comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, and at least one amino acid is substituted at the following positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 60): positions 1, 3, 4, 5, 6, 7, and 8.

6. A multispecific antibody according to any one of claims 1 to 5, wherein at least one of two variable regions comprises VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, and at least one amino acid is substituted at the following positions: (a) in HVR-H1 (SEQ ID NO: 32): positions 1, 2, and 4; (b) in HVR-H2 (SEQ ID NO: 37): position 9; (c) in HVR-H3 (SEQ ID NO: 43): positions 2 and 9; (d) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 59): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 62): positions 1, 3, 4, 5, 6, 7, and 8.

7. A multispecific antibody according to any one of claims 1 to 6, having inhibitory activity against sclerostin.

8. A multispecific antibody according to any one of claims 1 to 7, wherein at least two different variable regions are included in a common light chain.

9. A bispecific antibody comprising a first variable region containing a VH sequence of SEQ ID NO: 101 or 102 and one VL sequence from among SEQ ID NOs: 98-100 and 105, and a second variable region containing a VH sequence of SEQ ID NO: 103 or 104 and one VL sequence from among SEQ ID NOs: 98-100 and 105.

10. The bispecific antibody according to claim 9, wherein the VL sequence of the first variable region and the VL sequence of the second variable region are identical.

11. A pharmaceutical formulation comprising the antibody according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. A method for treating an individual having a bone-related disease, comprising the step of administering an effective amount of an antibody described in any one of claims 1 to 10 to the individual.

13. A method for treating a disease or condition associated with an increased level of sclerostin in an individual, comprising the step of administering to the individual an effective amount of an antibody according to any one of claims 1 to 10.

14. A method for increasing bone formation and / or inhibiting bone resorption in an individual, comprising the step of administering an effective amount of an antibody according to any one of claims 1 to 10 to the individual in order to increase bone formation and / or inhibit bone resorption.

15. A method for increasing bone mineral density in an individual, comprising the step of administering an effective amount of an antibody described in any one of claims 1 to 10 to the individual in order to increase bone mineral density.

16. Isolated anti-sclerostin antibodies comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 15, wherein at least one amino acid is substituted at the following positions: (a) in HVR-H1 (SEQ ID NO: 31): position 1; (b) in HVR-H2 (SEQ ID NO: 34): positions 3, 5, 8, 9, 11, and 12; (c) in HVR-H3 (SEQ ID NO: 38): positions 2, 4, 5, 7, and 13; (d) in HVR-L1 (SEQ ID NO: 52): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 56): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 60): positions 1, 3, 4, 5, 6, 7, and 8.

17. Isolated anti-sclerostin antibodies comprising VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 27, wherein at least one amino acid is substituted at the following positions in the hypervariable region (HVR): (a) in HVR-H1 (SEQ ID NO: 32): positions 1, 2, and 4; (b) in HVR-H2 (SEQ ID NO: 37): position 9; (c) in HVR-H3 (SEQ ID NO: 43): positions 2 and 9; (d) in HVR-L1 (SEQ ID NO: 55): positions 1, 4, 5, 6, 7, 8, 9, 10, and 11; (e) in HVR-L2 (SEQ ID NO: 59): positions 1, 2, 4, 5, 6, and 7; and (f) in HVR-L3 (SEQ ID NO: 62): positions 1, 3, 4, 5, 6, 7, and 8.

18. The antibody according to claim 16 or 17, which binds to sclerostin with higher affinity at neutral pH than at acidic pH.

19. An antibody according to any one of claims 16 to 18, having inhibitory activity against sclerostin.

20. A pharmaceutical formulation comprising the antibody according to any one of claims 16 to 19 and a pharmaceutically acceptable carrier.

21. A method for treating an individual having a bone-related disease, comprising the step of administering an effective amount of an antibody according to any one of claims 16 to 19 to the individual.

22. A method for treating a disease or condition associated with an increased level of sclerostin in an individual, comprising the step of administering to the individual an effective amount of an antibody according to any one of claims 16 to 19.

23. A method for increasing bone formation and / or inhibiting bone resorption in an individual, comprising the step of administering an effective amount of an antibody according to any one of claims 16 to 19 to the individual in order to increase bone formation and / or inhibit bone resorption.

24. A method for increasing bone mineral density in an individual, comprising the step of administering an effective amount of an antibody according to any one of claims 16 to 19 to the individual in order to increase bone mineral density.

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