C-terminal antibody variants

Abstract

To provide a pharmaceutical composition for use in methods that enhance bone mineral density.SOLUTION: An antibody specifically binds to sclerostin and comprises a specific sequence, wherein the antibody comprises a heavy chain comprising an amino acid sequence Pro-Ala-Arg-Gly at a C-terminus. There is also provided a pharmaceutical composition comprising the antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 650,762, filed March 30, 2018, and U.S. Provisional Patent Application No. 62 / 812,741, filed March 1, 2019, the disclosures of which are incorporated herein by reference in their entireties.

[0002] The present invention relates generally to anti-sclerostin antibodies having at least one C-terminal modification, and compositions comprising such antibodies.

[0003] Incorporation by reference of electronic submissions The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this specification is incorporated by reference in its entirety and is as follows: 21,006 byte ASCII (text) file with filename "52080_SeqListing.txt", created on March 21, 2019.

[0004] Incorporation by Reference The following applications are incorporated herein by reference in their entireties: International Patent Application No. PCT / US2012 / 049331, filed August 2, 2012 (claiming priority to U.S. Provisional Patent Application No. 61 / 515,191, filed August 4, 2011), U.S. Patent Application No. 11 / 410,540, filed April 25, 2006 (claiming priority to U.S. Provisional Patent Application No. 60 / 792,645, filed April 17, 2006, U.S. Provisional Patent Application No. 60 / 782,244, filed March 13, 2006, U.S. Provisional Patent Application No. 60 / 776,847, filed February 24, 2006, and U.S. Provisional Patent Application No. 60 / 776,847, filed May 3, 2005). No. 60 / 677,583, filed on April 25, 2006 (which claims priority to U.S. Provisional Patent Application No. 11 / 411,003, filed on April 25, 2006 (issued as U.S. Patent No. 7,592,429) (which claims priority to U.S. Provisional Patent Application No. 60 / 792,645, filed on April 17, 2006, U.S. Provisional Patent Application No. 60 / 782,244, filed on March 13, 2006, U.S. Provisional Patent Application No. 60 / 776,847, filed on February 24, 2006, and U.S. Provisional Patent Application No. 60 / 677,583, filed on May 3, 2005). The following applications are also incorporated by reference herein: U.S. Patent Application No. 12 / 212,327, filed September 17, 2008 (claiming priority to U.S. Provisional Patent Application No. 60 / 973,024, filed September 17, 2007), and U.S. Patent Application No. 12 / 811,171, filed June 29, 2010 (a U.S. national stage application under 35 U.S.C. 371 of International Patent Application No. PCT / US08 / 86864, filed December 15, 2008, claiming priority to U.S. Provisional Patent Application No. 61 / 013,917, filed December 14, 2007). [Background technology]

[0005] Loss of bone mineral density can be caused by a wide variety of pathologies and can result in serious medical problems. For example, osteoporosis is a debilitating human disease characterized by significant reductions in skeletal bone mass and bone mineral density, structural deterioration of bone, including deterioration of bone microarchitecture and corresponding increased bone fragility (i.e., decreased bone strength), and a higher susceptibility to fractures in affected individuals. Osteoporosis has been associated with increased fracture risk due to bone loss, but few currently available bone disorder treatments can increase bone mineral density in adults, and most currently available treatments function primarily by inhibiting further bone resorption rather than stimulating new bone formation. Estrogens are currently prescribed to slow bone loss. However, there is debate as to whether patients derive any long-term benefit and whether estrogen is effective in patients over 75 years of age. Furthermore, estrogen use is thought to increase the risk of breast and endometrial cancer. Calcitonin, osteocalcin containing vitamin K, or high-dose dietary calcium, with or without vitamin D, have also been suggested for postmenopausal women. However, high doses of calcium often have undesirable gastrointestinal side effects, and serum and urinary calcium concentrations must be continuously monitored (e.g., Khosla and Riggs, Mayo Clin. Proc. 70:978-982, 1995). Other current treatments for osteoporosis include bisphosphonates (e.g., Fosamax®). TM , Actonel TM , Bonviva TM , Zometa TM These include steroids, lanthanum and strontium salts, olpadronate, neridronate, skelid, bonefos), parathyroid hormone, calcilytics, calcium receptor agonists (e.g., cinacalcet), statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. However, such treatments are often associated with undesirable side effects (see Khosla and Riggs, supra).

[0006] Sclerostin, the product of the SOST gene, is absent in sclerosteosis, a bone disease characterized by bone overgrowth and high bone density (Brunkow et al., Am. J. Hum. Genet., 68:577-589, 2001; Balemans et al., Hum. Mol. Genet., 10:537-543, 2001). The amino acid sequence of human sclerostin is reported in Brunkow et al., supra, and is disclosed herein as SEQ ID NO: 1. Sclerostin is an important target for mediating increased bone density. Summary of the Invention [Means for solving the problem]

[0007] In one aspect, an antibody is described herein that specifically binds to the sclerostin of SEQ ID NO: 1 and comprises a set of six CDRs set forth in SEQ ID NOs: 2 to 7, the antibody comprising a heavy chain comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at its C-terminus. In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the antibody comprises the amino acid sequence Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the heavy chain. In some embodiments, the antibody comprises a first heavy chain comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the first heavy chain and a second heavy chain comprising a wild-type heavy chain amino acid sequence (i.e., lacking the C-terminal Pro-Ala-Arg-Gly). In some embodiments, the antibody comprises a light chain amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the antibody comprises the amino acid sequence Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the heavy chain. In some embodiments, the antibody comprises the light chain amino acid sequence set forth in SEQ ID NO: 12 and the heavy chain amino acid sequence set forth in SEQ ID NO: 14.

[0008] In some embodiments, the C-terminus of one of the antibody's heavy chains is amidated (i.e., the antibody is monoamidated), and in some embodiments, the C-terminus of both antibody heavy chains is amidated (i.e., the antibody is doubly amidated).

[0009] Pharmaceutical compositions comprising a population of antibodies described herein and a pharmaceutically acceptable carrier are also provided by the present disclosure. In some embodiments, the pharmaceutical composition comprises a mixture of antibodies that specifically bind to sclerostin of SEQ ID NO: 1, the mixture of antibodies comprising a population of antibodies comprising a heavy chain having the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at its C-terminus, and a pharmaceutically acceptable carrier. In some embodiments, approximately 3-5% of the antibodies in the composition are from the population of antibodies comprising a heavy chain having the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at its C-terminus. In some aspects, less than 70% of the antibody population are amidated on one or both heavy chains. In some aspects, all or a portion of the antibody population comprises one heavy chain comprising the C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, which is optionally amidated. In some aspects, all or a portion of the antibody population comprises both heavy chains comprising the C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, which is optionally amidated. Optionally, less than about 35% of the antibody population has one amidated heavy chain, and / or less than about 35% of the antibody population has both amidated heavy chains, and / or less than about 35% of the antibody population comprises a non-amidated heavy chain. In this regard, in various embodiments, about 33% of the antibody population is non-amidated, about 33% of the antibody population comprises one amidated heavy chain, and about 33% of the antibody population comprises two amidated heavy chains.

[0010] In some embodiments, the composition further comprises a calcium salt, an acetate buffer, a polyol, and a surfactant. In some embodiments, the calcium salt comprises calcium acetate, the acetate buffer comprises sodium acetate, the polyol comprises sucrose, and the surfactant comprises polysorbate 20. In some embodiments, the composition comprises 55 mM acetate, 13 mM calcium, 6.0% (w / v) sucrose, and 0.006% (w / v) polysorbate 20, pH 5.2.

[0011] The present disclosure also provides a method for increasing bone mineral density in a subject in need thereof, comprising administering to the subject a composition described herein in an amount effective to increase bone mineral density in the subject. The present invention also includes the following [1] to

[25] . [1] An antibody that specifically binds to sclerostin of SEQ ID NO: 1 and comprises a set of six CDRs shown in SEQ ID NOs: 2 to 7, and comprises a heavy chain containing the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain. [2] The antibody according to [1] above, comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10. [3] The antibody according to [1] or [2] above, wherein the C-terminus of the heavy chain is amidated. [4] The antibody according to any one of [1] to [3] above, wherein the C-terminus of both heavy chains comprises the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8). [5] The antibody according to [4] above, wherein the C-terminus of both heavy chains is amidated. [6] The antibody according to any one of [1] to [5] above, which comprises a light chain amino acid sequence shown in SEQ ID NO: 12 and a heavy chain amino acid sequence shown in SEQ ID NO: 13. [7] The antibody according to [1] or [2] above, which comprises an amino acid sequence containing Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the heavy chain. [8] The antibody according to [7] above, comprising the light chain amino acid sequence shown in SEQ ID NO: 12 and the heavy chain amino acid sequence shown in SEQ ID NO: 14. [9] A pharmaceutical composition comprising the population of antibodies according to any one of [1] to [8] above and a pharmaceutically acceptable carrier.

[10] A pharmaceutical composition comprising a mixture of antibodies that specifically bind to sclerostin of SEQ ID NO: 1 and a pharmaceutically acceptable carrier, A pharmaceutical composition in which approximately 3 to 5% of the antibodies in the composition are a population of antibodies according to any one of [1] to [8] above.

[11] The pharmaceutical composition according to [9] or

[10] above, wherein all or a portion of the antibody population comprises one heavy chain comprising the C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence.

[12] The pharmaceutical composition according to [9] or

[10] above, wherein all or a portion of the antibody population comprises a heavy chain comprising an amidated C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence.

[13] The pharmaceutical composition according to

[12] above, wherein less than about 35% of the antibody population has one amidated antibody.

[14] The pharmaceutical composition according to

[10] above, wherein all or a portion of the antibody population comprises a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence in both heavy chains.

[15] The pharmaceutical composition according to

[14] above, wherein all or a portion of the antibody population comprising two heavy chains each containing the C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence is amidated.

[16] The pharmaceutical composition according to

[15] above, wherein less than about 35% of the antibody population has both heavy chains amidated.

[17] The pharmaceutical composition according to any one of [9] to

[16] above, wherein less than about 35% of the antibody population comprises non-amidated heavy chains.

[18] The pharmaceutical composition according to any one of [9] to

[17] above, wherein approximately 33% of the antibody population is non-amidated, approximately 33% of the antibody population contains one amidated heavy chain, and approximately 33% of the antibody population contains two amidated heavy chains.

[19] The pharmaceutical composition according to any one of the above [9] to

[18] , further comprising a calcium salt, an acetate buffer solution, a polyol and a surfactant.

[20] The pharmaceutical composition according to

[19] above, wherein the calcium salt comprises calcium acetate.

[21] The pharmaceutical composition according to

[19] above, wherein the acetate buffer comprises sodium acetate.

[22] The pharmaceutical composition according to

[19] above, wherein the polyol comprises sucrose.

[23] The pharmaceutical composition according to

[19] above, wherein the surfactant comprises polysorbate 20.

[24] The pharmaceutical composition according to any one of [9] to

[23] above, further comprising 55 mM acetate, 13 mM calcium, 6.0% (w / v) sucrose, and 0.006% (w / v) polysorbate 20, pH 5.2.

[25] A method for increasing bone mineral content in a subject in need thereof, comprising administering to the subject an amount of the composition described in any of [9] to

[24] above in an amount effective to increase bone mineral content in the subject. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 provides the nucleic acid sequence encoding a portion of the C-terminus of wild-type romosozumab. [Figure 2] FIG. 2 provides the nucleic acid sequence encoding a portion of the C-terminus of the romosozumab C-terminal variant (PARG variant). [Figure 3] FIG. 3 is a graph showing the magnified UV profile of wild-type romosozumab (dotted line) overlaid with the romosozumab PARG variant (solid line) digested with Lys-C and analyzed by LC / MS peptide mapping. [Figure 4] FIG. 4 is a graph showing the cation exchange (CEX) profile of the carboxypeptidase-treated romosozumab PARG variant (dotted line) overlaid with the untreated romosozumab PARG variant (solid line). [Figure 5] Figure 5 is a graph showing percent recovery of the Scissor model of subcutaneous injection as a function of time. Wild-type romosozumab (circles) and PARG c-terminal variant romosozumab diffuse at different rates at the simulated injection site. [Figure 6] FIG. 6 is a graph showing that wild-type romosozumab and PARG C-terminally modified romosozumab both bind to FcRn in a similar manner, and that FcRn binding is not affected by PARG mutations. [Figure 7] FIG. 7 is a graph showing that the relative binding of PARG C-terminal variant romosozumab to FcγRIIa (131H) was much higher than that of wild-type romosozumab. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure provides antibodies that specifically bind to sclerostin, the antibodies comprising a heavy chain comprising an amino acid sequence comprising Pro-Ala-Arg-Gly (SEQ ID NO: 8) at its C-terminus. In some embodiments, the antibodies comprise a first heavy chain comprising an amino acid sequence comprising Pro-Ala-Arg-Gly (SEQ ID NO: 8) at its C-terminus and a second heavy chain comprising a wild-type heavy chain amino acid sequence. In some embodiments, the antibodies comprise an amino acid sequence comprising Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the heavy chain. Pharmaceutical compositions comprising the antibodies (or mixtures of antibodies) and methods of using the antibodies are also provided.

[0014] An "anti-sclerostin antibody" or "antibody that binds to sclerostin" is an antibody that binds to the sclerostin of SEQ ID NO: 1 or a portion thereof. Recombinant human sclerostin / SOST is commercially available, for example, from R&D Systems (Minneapolis, Minn., USA; 2006 Catalog No. 1406-ST-025). U.S. Patent Nos. 6,395,511 and 6,803,453, as well as U.S. Patent Application Publication Nos. 2004 / 0009535 and 2005 / 0106683, generally refer to anti-sclerostin antibodies. Examples of anti-sclerostin antibodies suitable for use in the context of the present invention are also described in U.S. Patent Application Publication Nos. 2007 / 0110747 and 2007 / 0072797, which are incorporated herein by reference. Further information regarding materials and methods for generating anti-sclerostin antibodies can be found in US Patent Application Publication No. 2004 / 0158045, which is incorporated herein by reference.

[0015] The term "antibody" refers to an intact immunoglobulin molecule (including polyclonal, monoclonal, chimeric, humanized, and / or human versions with full-length heavy and / or light chains).

[0016] As used herein, "specifically binds" means that an antibody binds to an antigen preferentially over other proteins. In some embodiments, "specifically binds" means that the antibody has a higher affinity for the antigen than for other proteins. An antibody that specifically binds to an antigen has a binding affinity for the antigen of 1×10 or greater. -7 M or less, 2×10 -7 M or less, 3×10 -7 M or less, 4×10 -7 M or less, 5×10 -7 M or less, 6×10 -7 M or less, 7×10 -7 M or less, 8×10 -7 M or less, 9×10 -7 M or less, 1×10 -8 M or less, 2×10 -8 M or less, 3×10 -8 M or less, 4×10 -8 M or less, 5×10 -8 M or less, 6×10 -8 M or less, 7×10 -8 M or less, 8×10 -8 M or less, 9×10 -8 M or less, 1×10 -9 M or less, 2×10 -9 M or less, 3×10 -9 M or less, 4×10 -9 M or less, 5×10 -9 M or less, 6×10 -9 M or less, 7×10 -9 M or less, 8×10 -9 M or less, 9×10 -9 M or less, 1×10 -10 M or less, 2×10 -10 M or less, 3×10 -10 M or less, 4×10 -10 M or less, 5×10 -10 M or less, 6×10 -10 M or less, 7×10 -10 M or less, 8×10 -10 M or less, 9×10 -10 M or less, 1×10 -11 M or less, 2×10 -11 M or less, 3×10 -11 M or less, 4×10 -11 M or less, 5×10 -11M or less, 6×10 -11 M or less, 7×10 -11 M or less, 8×10 -11 M or less, 9×10 -11 M or less, 1×10 -12 M or less, 2×10 -12 M or less, 3×10 -12 M or less, 4×10 -12 M or less, 5×10 -12 M or less, 6×10 -12 M or less, 7×10 -12 M or less, 8×10 -12 M or less, or 9 x 10 -12 It can be M or less.

[0017] In some or any embodiments, the antibody is present in a concentration of 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11 M or less, or 1 x 10 -12 The antibody binds to sclerostin of SEQ ID NO: 1 or a naturally occurring variant thereof with an affinity (Kd) of M or less. Affinity is determined using various techniques, one example being an affinity ELISA assay. In various embodiments, affinity is determined by a BIAcore assay. In various embodiments, affinity is determined by a kinetic method. In various embodiments, affinity is determined by an equilibrium / solution method. U.S. Patent Application Publication No. 2007 / 0110747, the disclosure of which is incorporated herein by reference, contains further description of affinity assays suitable for determining the affinity (Kd) of an antibody for sclerostin.

[0018] In some or any of the embodiments, the antibody (or antibody fragment thereof) binds to a sclerostin polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and to a region of sclerostin comprising the sequence of SEQ ID NO:5 (CGPARLLPNAIGRGKWWRPSGPDFRC; corresponding to amino acids 86-111 of SEQ ID NO:1). This region is also referred to herein as the "loop 2" region of sclerostin. The region of sclerostin outside the loop 2 region is defined herein as the "non-loop 2 region." Alternatively, or in addition, the anti-sclerostin antibody binds to a sclerostin polypeptide comprising amino acids 57-146 of SEQ ID NO:1. Alternatively, or in addition, the anti-sclerostin antibody binds to a sclerostin polypeptide comprising amino acids 89-103 of SEQ ID NO:1 and / or amino acids 137-151 of SEQ ID NO:1. In some or any of the embodiments, a sclerostin polypeptide that is a fragment of full-length sclerostin retains the tertiary structure of the corresponding polypeptide region of human sclerostin of SEQ ID NO:1.

[0019] In some or any embodiments, the anti-sclerostin antibodies described herein preferably modulate sclerostin function in the cell-based assays described in U.S. Patent Application Publication No. 2007 / 0110747 and / or in the in vivo assays described in U.S. Patent Application Publication No. 2007 / 0110747, and / or bind to one or more of the epitopes described in U.S. Patent Application Publication No. 2007 / 0110747, and / or cross-block the binding of one of the antibodies described in U.S. Patent Application Publication No. 2007 / 0110747, and / or are cross-blocked from sclerostin binding by one of the antibodies described in U.S. Patent Application Publication No. 2007 / 0110747 (which are incorporated by reference in their entireties and for purposes of describing assays for characterizing anti-sclerostin antibodies).

[0020] "CDR" refers to a complementarity-determining region within an antibody variable sequence. Each heavy and light chain variable region has three CDRs, designated CDR1, CDR2, and CDR3 in each variable region. The term "set of six CDRs," as used herein, refers to a group of three CDRs present in the light and heavy chain variable regions that are capable of antigen binding. The exact boundaries of the CDRs have been defined differently by different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) discovered that certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These subportions were designated L1, L2, and L3 or H1, H2, and H3 (where "L" and "H" refer to the light chain and heavy chain regions, respectively). These regions can be referred to as Chothia CDRs and have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J. Mol. Biol. 262(5):73245 (1996)). Still other definitions of CDR boundaries may not strictly follow one of the above systems, but still overlap with the Kabat CDRs, although they may be shortened or extended in light of predictions or experimental findings that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding.The methods used herein can utilize CDRs defined by either of these systems, although preferred embodiments use CDRs defined by Kabat or Chothia.

[0021] CDRs can be obtained, for example, by constructing a polynucleotide encoding the desired CDR. Such polynucleotides can be prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), page 137, Wiley-Liss, Inc. (1995)).

[0022] In various embodiments, the antibody comprises at least one CDR sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity) to a CDR selected from CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where CDR-H1 has the sequence set forth in SEQ ID NO:2, CDR-H2 has the sequence set forth in SEQ ID NO:3, CDR-H3 has the sequence set forth in SEQ ID NO:4, CDR-L1 has the sequence set forth in SEQ ID NO:5, CDR-L2 has the sequence set forth in SEQ ID NO:6, and CDR-L3 has the sequence set forth in SEQ ID NO:7. The anti-sclerostin antibody, in various aspects, comprises two CDRs or six CDRs.

[0023] In a preferred embodiment, the anti-sclerostin antibody comprises the following set of six CDRs: CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 5, CDR-L2 of SEQ ID NO: 6, and CDR-L3 of SEQ ID NO: 7.

[0024] In some or any embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity) to the amino acid sequence set forth in SEQ ID NO: 9, and a heavy chain variable region comprising an amino acid sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity) to the amino acid sequence set forth in SEQ ID NO: 10. In various aspects, the sequence differences compared to SEQ ID NO: 9 or 10 are outside of the CDR regions in the corresponding sequences. In some or any embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0025] In some or any embodiments, the anti-sclerostin antibody comprises all or a portion of a heavy chain comprising an amino acid sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity) to the amino acid sequence set forth in SEQ ID NO: 16, and all or a portion of a heavy chain comprising an amino acid sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity) to the amino acid sequence set forth in SEQ ID NO: 12.

[0026] The antibody comprises a heavy chain comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at its C-terminus. In some embodiments, the C-terminus of both heavy chains of the antibody comprises the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8). In some embodiments, the antibody comprises a first heavy chain comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) and a second heavy chain comprising a wild-type amino acid sequence. In various aspects, the antibody comprises a light chain amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain amino acid sequence set forth in SEQ ID NO: 13.

[0027] Alternatively, in some or any embodiments, the antibody comprises a sequence of amino acids comprising Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the heavy chain, optionally at the C-terminus of both heavy chains. In some embodiments, the antibody comprises a first heavy chain comprising the amino acid sequence Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) and a second heavy chain comprising the wild-type amino acid sequence (i.e., without the C-terminal Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11)). In various aspects, the antibody comprises a light chain amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain amino acid sequence set forth in SEQ ID NO: 14.

[0028] Other exemplary anti-sclerostin antibodies include, but are not limited to, the anti-sclerostin antibodies disclosed in International Patent Applications WO 2008 / 092894, WO 2008 / 115732, WO 2009 / 056634, WO 2009 / 047356, WO 2010 / 100200, WO 2010 / 100179, WO 2010 / 115932, and WO 2010 / 130830, each of which is incorporated by reference in its entirety.

[0029] Those skilled in the art will appreciate that proteins, such as antibodies, can undergo a variety of post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the protein. Such modifications can include variations in glycosylation, methionine oxidation, diketopiperidine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of a basic residue (e.g., lysine or arginine) at the carboxy terminus by the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995).

[0030] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (Tecreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86

[1983] (incorporated herein in its entirety)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0031] In some or any of the embodiments, the C-terminus of the heavy chain of the antibody comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) is amidated. In some or any of the embodiments, both heavy chains of the antibody comprise the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8), and both heavy chains are amidated. In some of the embodiments, glycine is amidated. Amidation can occur as described, for example, in Prigg, ST et al., "New insights into copper monooxygenases and peptide amidation: Structure, mechanism and function," Cell. Mol. Life Sci. 57 (2000) 1236-1259. The enzyme peptidylglycine α-amidating monooxygenase (PAM) can catalyze the amidation of glycine. PAM has two active domains: peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxylglycine α-amidating lyase (PAL). PHM catalyzes the conversion of peptidylglycine (together with ascorbate and oxygen) to peptidyl α-hydroxylglycine (together with semidehydrogenascorbate and water), and PAL then catalyzes the conversion of peptidyl α-hydroxylglycine to an amidated peptide (and glyoxylate).

[0032] Antibody amidation can be controlled by altering certain conditions during the cell culture process. For example, copper (e.g., in ferric ammonium citrate) and / or oxygen levels can be used to affect amidation levels. It is believed that increasing copper concentration (e.g., in the medium) or oxygen availability (e.g., during culture) can increase amidation by affecting the activity of enzymes such as PHM.

[0033] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising the populations of antibodies described herein in association with a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0034] The present disclosure also provides a pharmaceutical composition comprising a mixture of an antibody that specifically binds to sclerostin of SEQ ID NO: 1 and a pharmaceutically acceptable carrier. Approximately 3-5% of the antibodies in the composition are a population of antibodies described herein (e.g., antibodies comprising a set of six CDRs as set forth in SEQ ID NOs: 2-7 and having one heavy chain (or two heavy chains) containing the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain). The present disclosure also contemplates compositions containing alternative amounts (e.g., 5-10%, 1-3%, 3-15%, 2-10%, 4-20%, 1-5%) of the population of antibodies described herein (e.g., antibodies comprising a set of six CDRs as set forth in SEQ ID NOs: 2-7 and having one heavy chain (or two heavy chains) containing the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain).

[0035] In some embodiments, less than 70% (e.g., about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%, about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or less) comprise a heavy chain comprising a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, which heavy chain is optionally amidated. In some embodiments, less than 35% (e.g., about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or less) of the antibodies in the population comprise a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence on both heavy chains, and both heavy chains are optionally amidated. It is also contemplated that both heavy chains comprise the C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, but only one of the heavy chains is amidated. In some embodiments, less than 35% (e.g., about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or less) of the antibodies in the composition comprise the non-amidated C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence.In some embodiments, about 33% of the antibodies in the population comprise an amidated C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, about 33% of the antibodies in the population comprise an amidated C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence on both heavy chains, and about 33% of the antibodies in the population comprise a heavy chain with a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence but that is not amidated.

[0036] In some embodiments, pharmaceutical compositions contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); injectables; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt forms preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyloxapol); stabilization enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0037] The selection of specific formulation materials described herein can be driven, for example, by the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. The primary vehicle or carrier in a pharmaceutical composition can be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In specific embodiments, the pharmaceutical composition comprises Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, and may further comprise sorbitol or a suitable substitute thereof. In certain embodiments, the composition can be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing the selected composition having the desired purity with optional formulating agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Further, in some embodiments, the antibodies or fragments may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0038] The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Alternatively, the compositions can be selected for inhalation or for delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are preferably present in concentrations acceptable to the site of administration. In certain embodiments, a buffering agent is used to maintain the composition at physiological pH or a slightly lower pH, typically within the pH range of about 5 to about 8.

[0039] When parenteral administration is intended, therapeutic compositions for use in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired antibody or fragment in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the antibody or fragment is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, an implantable drug delivery device may be used to introduce the desired antibody or fragment construct.

[0040] In some or any embodiments, the pharmaceutical compositions described herein comprise a calcium salt, an acetate buffer, a polyol, and a surfactant. Exemplary calcium salts include, but are not limited to, calcium acetate, calcium carbonate, and calcium chloride. In some embodiments, the calcium salt is at a concentration of at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM. In certain embodiments, the calcium salt concentration is 11 mM or less, 12 mM or less, 13 mM or less, 14 mM or less, 15 mM or less, 16 mM or less, 17 mM or less, 18 mM or less, 19 mM or less, 20 mM or less, 21 mM or less, 22 mM or less, 23 mM or less, 24 mM, or 25 mM or less. Any range combining the above endpoints is contemplated, for example, but not limited to, about 0.5 mM to about 10 mM, about 5 mM to about 10 mM, or about 5 mM to about 15 mM.

[0041] In some embodiments, the pharmaceutical composition comprises an acetate buffer (e.g., sodium acetate) having a concentration ranging from about 0.1 mM to about 1000 mM (1 M). In some embodiments, the concentration of the acetate buffer is at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 200 mM, at least 500 mM, at least 700 mM, or at least 900 mM. In some embodiments, the acetate buffer concentration is 10 mM or less, 15 mM or less, 20 mM or less, 25 mM or less, 30 mM or less, 35 mM or less, 40 mM or less, 45 mM or less, 50 mM or less, 55 mM or less, 60 mM or less, 65 mM or less, 70 mM or less, 75 mM or less, 80 mM or less, 85 mM or less, 90 mM or less, 95 mM or less, or 100 mM or less. Any range combining the aforementioned endpoints is contemplated, such as, but not limited to, about 5 mM to about 15 mM, about 5 mM to about 10 mM, or about 10 mM to about 25 mM. The buffer is preferably added to a concentration that maintains a pH of about 5-6, 5-5.5, or 4.5-5.5. When the calcium salt in the formulation is calcium acetate, in some embodiments, the total concentration of acetate is from about 10 mM to about 55 mM, or from about 20 mM to about 40 mM.

[0042] In some aspects, the pharmaceutical composition comprises acetate at a total concentration of at least 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, or at least about 50 mM. In some embodiments, the acetate concentration is about 30 mM or less, about 35 mM or less, about 40 mM or less, about 45 mM or less, about 50 mM or less, about 55 mM or less, about 60 mM or less, about 65 mM or less, about 70 mM or less, about 75 mM or less, about 80 mM or less, about 85 mM or less, or about 90 mM or less. Any range combining the foregoing endpoints is contemplated, such as, but not limited to, about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 30 mM to about 50 mM, or about 30 mM to about 75 mM. In some embodiments, the calcium salt is calcium acetate and the acetate buffer is sodium acetate. As a non-limiting example, a solution containing 10 mM calcium acetate will have 20 mM acetate anions and 10 mM calcium cations due to the divalent nature of the calcium cation, while a solution containing 10 mM sodium acetate will have 10 mM sodium cations and 10 mM acetate anions.

[0043] In some embodiments, the total ion (cation and anion) concentration in the solution is at least 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, or at least about 85 mM. In some embodiments, the total ion concentration is about 30 mM or less, about 35 mM or less, about 40 mM or less, about 45 mM or less, about 50 mM or less, about 55 mM or less, about 60 mM or less, about 65 mM or less, about 70 mM or less, about 75 mM or less, about 80 mM or less, about 85 mM or less, about 90 mM or less, about 95 mM or less, about 100 mM or less, about 110 mM or less, about 120 mM or less, about 130 mM or less, about 140 mM or less, about 150 mM or less, about 160 mM or less, about 170 mM or less, about 180 mM or less, about 190 mM or less, or about 200 mM or less. Any range combining the foregoing endpoints is contemplated, for example, but not limited to, about 30 mM to about 60 mM, or about 30 mM to about 70 mM, or about 30 mM to about 80 mM, or about 40 mM to about 150 mM, or about 50 mM to about 150 mM. As a non-limiting example, a 10 mM calcium acetate solution has a total ion concentration of 30 mM (10 mM cations and 20 mM anions).

[0044] In some or any embodiments, the pharmaceutical composition includes a polyol. Polyols encompass a class of excipients including sugars (e.g., mannitol, sucrose, sorbitol) and other polyhydric alcohols (e.g., glycerol and propylene glycol). Exemplary polyols include, but are not limited to, propylene glycol, glycerol, threose, threitol, erythrose, erythritol, ribose, arabinose, arabitol, lyxose, maltitol, sorbitol, sorbose, glucose, mannose, mannitol, levulose, dextrose, maltose, trehalose, fructose, xylitol, inositol, galactose, xylose, fructose, sucrose, 1,2,6-hexanetriol, and the like. Higher sugars include, but are not limited to, dextran, propylene glycol, or polyethylene glycol. Reducing sugars, such as fructose, maltose, or galactose, oxidize more readily than non-reducing sugars. Further examples of sugar alcohols are glucitol, maltitol, lactitol, or isomaltulose. Further exemplary lyoprotectants include glycerin and gelatin, and the sugars melibiose, melezitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyalcohols. Monoglycosides include compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose, and maltulose.

[0045] In some or any embodiments, the pharmaceutical composition comprises a polyol at a concentration ranging from about 0% w / v to about 40% w / v, hi some or any embodiments, the composition comprises a polyol at a concentration of at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, or at least 40% w / v. In some or any embodiments, the composition comprises a polyol at a concentration of about 1 w / v%, about 2 w / v%, about 3 w / v%, about 4 w / v%, about 5 w / v%, about 6 w / v%, about 7 w / v%, about 8 w / v%, about 9 w / v% to about 10 w / v%. In some or any embodiments, the composition comprises a polyol at a concentration of about 2 w / v% to about 6 w / v%. In some or any embodiments, the composition comprises a polyol at a concentration of about 4 w / v%. In some or any embodiments, the composition comprises a polyol at a concentration of about 6 w / v%.

[0046] In some or any embodiment, the pharmaceutical composition includes a surfactant. Exemplary surfactants include anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, and glycodeoxycholic acid sodium salt. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80. In another embodiment, surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80, soybean lecithin and other phospholipids such as DOPC, DMPG, DMPC, and DOPG, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. In some or any embodiments, the surfactant is polysorbate 20.

[0047] The surfactants may be included in the composition individually or as a mixture in different ratios. In some or any embodiments, the composition comprises a surfactant at a concentration of about 0% w / v to about 5% w / v (e.g., about 0.001%, about 0.002%, about 0.005%, about 0.007%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, or about 4.5% w / v). In some or any embodiments, the composition includes a surfactant at a concentration of about 0.001% w / v to about 0.5% w / v. In some or any embodiments, the composition includes a surfactant at a concentration of about 0.004%, about 0.005%, about 0.007%, about 0.01%, about 0.05%, or about 0.1% w / v to about 0.2% w / v. In some or any embodiments, the composition includes a surfactant at a concentration of about 0.01% w / v to about 0.1% w / v.

[0048] In some or any embodiments, the pharmaceutical composition comprises 55 mM acetate, 13 mM calcium, 6.0% (w / v) sucrose, and 0.006% (w / v) polysorbate 20, pH 5.2.

[0049] Those skilled in the art will recognize that additional pharmaceutical compositions include formulations of antigen-binding proteins in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829 (incorporated by reference), which describes the controlled release of porous polymeric microparticles for delivering pharmaceutical compositions. Sustained-release formulations may include, for example, semipermeable polymer matrices in the form of shaped articles such as films or microcapsules. Sustained-release matrices can include polyesters, hydrogels, polylactides (disclosed in U.S. Pat. No. 3,773,919 and EP-A-058,481, each of which is incorporated by reference), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyrate (EP-A-133,988). Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art (see, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692, European Patent Applications 036676, 088046, and 143949, which are incorporated by reference).

[0050] Pharmaceutical compositions used for in vivo administration are generally provided as sterile preparations. Sterilization can be achieved by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or as a solution. Parenteral compositions are generally filled into a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0051] Free amino acids can be used in antibody or fragment formulations according to various embodiments of the present invention as bulking agents, stabilizers, and antioxidants, as well as other standard uses. Lysine, proline, serine, and alanine can be used to stabilize proteins in the formulation. Glycine is useful for ensuring proper cake structure and properties during lyophilization. Arginine can be useful for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0052] Embodiments of the antibody formulation may further comprise one or more antioxidants. Harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and by avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Particularly useful antioxidants in this regard are reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain activity throughout the product's shelf life. In this regard, EDTA is a preferred antioxidant according to the present invention.

[0053] The formulations according to the present invention may contain metal ions, which are protein cofactors and are required to form protein coordination complexes, such as zinc, which is required to form certain insulin suspensions. Metal ions can also inhibit some processes that degrade proteins. However, metal ions also catalyze the physical and chemical processes that degrade proteins.

[0054] Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartate to isoaspartate. +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. +2 , Mn +2 and Zn +2 can destabilize rhDNase. +2 and Sr +2 can stabilize factor VIII, which is +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 The aggregation can be destabilized by Al +3 It may be increased by ions.

[0055] Embodiments of the antibody formulation may further include one or more preservatives.

[0056] Once the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations can be stored in a form ready for use or to be reconstituted prior to administration (e.g., lyophilized). The present invention also provides kits for producing single-dose administration units. Each kit of the present invention includes both a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, kits are provided that include single- and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0057] The therapeutically effective amount of an antibody-containing pharmaceutical composition to be used will depend, for example, on the nature and purpose of the treatment. Those skilled in the art will recognize that appropriate dosage levels for treatment will vary in part depending on the molecule being delivered, the indication for which the antibody is being used, the route of administration, and the size (weight, body surface area, or organ size) and / or condition (age and general health) of the patient.

[0058] stability The terms "stability" and "stable," as used herein in reference to a composition comprising an antibody (or antigen-binding fragment thereof), refer to the resistance of the antibody (or antigen-binding fragment thereof) in the composition to aggregation, degradation, or fragmentation under given processing, preparation, shipping, and / or storage conditions. Antibody formulations that exhibit high stability exhibit high reliability and safety and are therefore advantageous for clinical use.

[0059] The stability of the antibody in the composition is optionally assessed by testing a desired parameter of the antibody in the composition over time (e.g., aggregation, heavy and / or light chain degradation, chemical modification, etc.). In this regard, the parameter is generally examined and compared at an initial time point (T0) and an evaluation time point (T1), optionally while exposing the antibody to any of several environmental conditions. The initial time point can be, for example, the time when the antibody is first formulated in the composition or first tested for quality (i.e., tested to determine whether the antibody composition meets regulatory or manufacturing specifications for aggregation or degradation). The initial time point can also be the time when the antibody is reformulated in the composition (e.g., reformulated at a higher or lower concentration compared to the initial formulation). The evaluation time point, in various embodiments, is about 1 week (or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 10 weeks, or about 3 months, or about 6 months, or about 1 year) after the initial time point. Desired parameters (e.g., aggregation or degradation) of the antibody or fragment thereof in the composition can be evaluated under various storage conditions, such as temperatures of -30°C, 4°C, 20°C, or 40°C, shaking, pH, storage in different container materials (e.g., glass vials, pre-filled syringes, etc.).

[0060] Exemplary methods for determining the degree of aggregation and / or type and / or size of aggregates present in an antibody-containing composition include, but are not limited to, size exclusion chromatography (SEC), high-performance size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and 1-anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques. Size exclusion chromatography (SEC) can be performed to separate molecules based on their size by passing them through a column packed with an appropriate resin, with larger molecules (e.g., aggregates) eluting before smaller molecules (e.g., monomers). Molecules are generally detected by UV absorbance at 280 nm and can be collected for further characterization. High-pressure liquid chromatography columns are often used for SEC analysis (HP-SEC). Alternatively, analytical ultracentrifugation (AUC) can be used. AUC is an organic technique for determining the sedimentation coefficient of macromolecules in a liquid sample. Similar to SEC, AUC can separate and detect antibody fragments / aggregates from monomers and can further provide information about molecular weight. Antibody aggregation in a composition can also be characterized by particle counter analysis using a Coulter counter or by turbidity measurement using a turbidimeter. Turbidity is a measure of the amount of light scattered by particles in a solution and can therefore be used as a general indicator of protein aggregation. Additionally, non-reducing polyacrylamide gel electrophoresis (PAGE) or capillary gel electrophoresis (CGE) can be used to characterize the aggregation and / or fragmentation state of antibodies or antibody fragments in a composition.

[0061] Exemplary methods for determining antibody degradation include, but are not limited to, size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and capillary electrophoresis with SDS (CE-SDS), and reverse-phase HPLC with in-line MS detection.

[0062] In various embodiments, less than 5% of the antibodies described herein in the composition are in aggregate form under the conditions of interest. For example, less than 4%, or less than 3%, or less than 2%, or less than 1% of the antibodies in the composition are in aggregate form after about 1 week (or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 10 weeks, or about 3 months, or about 6 months, or about 1 year) of storage at -30°C, 4°C, 20°C, or 40°C. In some embodiments, less than 5% (or less than 4%, or less than 3%, or less than 2%, or less than 1%) of the antibodies described herein in the composition are in aggregate form after 2 weeks of storage at about 4°C.

[0063] For example, at least 85% (or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) of the antibody in the composition is optionally present in unaggregated (i.e., monomeric) form after storage at -30°C, 4°C, 20°C, or 40°C for about 1 week (or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 10 weeks, or about 3 months, or about 6 months, or about 1 year). In some embodiments, at least 85% (or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or more) of the antibody is present in the composition in non-aggregated form after 2 weeks of storage at about 4° C. In some embodiments, at least 99% of the antibody is present in the composition in non-aggregated form after 2 weeks of storage at about 4° C., and / or at least 95% of the antibody present in the composition is in non-aggregated form after 2 weeks of storage at 40° C.

[0064] In various embodiments, less than 5% of the antibodies described herein in the composition are degraded. For example, less than 4%, or less than 3%, or less than 2%, or 1% or less of the antibodies in the composition are degraded under the conditions of interest. For example, optionally, at least 85% (or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) of the antibodies stored in the composition at about -30°C, about 4°C, about 20°C, or about 40°C for about 1 week (or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 10 weeks, or about 3 months, or about 6 months, or about 1 year) remain intact (i.e., not degraded). In some aspects, at least 85% (or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or more) of the antibody remains intact (i.e., not degraded) after storage in the composition for 2 weeks at about 4° C. In some embodiments, at least 99% of the antibody remains intact when stored in the composition for 2 weeks at about 4° C., and / or at least 95% remains intact when stored in the composition for 2 weeks at about 40° C.

[0065] Functional or activity stability of the antibody in the composition is also contemplated herein. For example, assays for detecting and / or quantifying antibody binding to a target or sclerostin neutralization are known in the art. Optionally, the antibody exhibits about 50-100% activity under the conditions of interest compared to the activity of the antibody at an initial time point. For example, the antibody retains about 60-90% or 70-80% activity level compared to the activity at the initial time point. Thus, functional stability of an antibody includes retention of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% activity, and can include activity measurements of greater than 100%, such as 105%, 110%, 115%, 120%, 125%, or 150% or more compared to the activity at the initial time point.

[0066] viscosity In some embodiments, the viscosity of a composition comprising one or more of the antibodies described herein is determined. The term "viscosity," as used herein, refers to "absolute viscosity." Absolute viscosity, sometimes called dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity x density). Kinematic viscosity has the dimension L 2 / T, where L is length and T is time. Kinematic viscosity is generally expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is 1 cSt. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is millipascal-second (mPa·s), where 1 cP = 1 mPa·s.

[0067] The viscosity of the composition can be measured several hours (e.g., 1-23 hours), several days (e.g., 1-10 days), several weeks (e.g., 1-5 weeks), several months (e.g., 1-12 months), or several years (e.g., 1-2 years, 1-3 years) after adding the antibody to the composition. Viscosity measurements can be performed at a storage or administration temperature, for example, 2-8°C or 25°C (room temperature). In some embodiments, the absolute viscosity of the liquid or reconstituted liquid composition at the storage and / or administration temperature is 15 cP or less, or 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 cP or less. In some embodiments, the absolute viscosity of the liquid or reconstituted liquid composition is 6 cP or less.

[0068] In some embodiments, the viscosity of the antibody composition is measured before and after the addition of the antibody. Methods for measuring viscosity are well known in the art and include, for example, the use of a capillary viscometer or a cone-plate rheometer. Any method can be used, provided that the same method is used to compare the test formulation and the reference formulation.

[0069] Treatment method The antibodies and pharmaceutical compositions described herein are useful for treating or preventing bone-related disorders, such as bone-related disorders associated with abnormal activity of osteoblasts or osteoclasts.In some embodiments, the antibody is directed against achondroplasia, cleidocranial dysplasia, enchondromatosis, fibrous dysplasia, Gaucher disease, hypophosphatemic rickets, Marfan syndrome, hereditary multiple exostoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, bone poikilosis, sclerotic lesions, pseudoarthrosis, pyogenic osteomyelitis, periodontal disease, antiepileptic drug-induced osteopenia, primary and secondary hyperparathyroidism, familial hyperparathyroidism syndrome, weightlessness-induced osteopenia, osteoporosis in men, postmenopausal osteopenia, osteoarthritis, renal osteodystrophy, infiltrative diseases of bone, oral osteopenia, osteonecrosis of the jaw, juvenile osteopenia, osteopenia of the jaw, osteoporosis ... -Jett's disease, melorheostosis, metabolic bone disease, mastocytosis, sickle cell anemia / disease, organ transplant-associated osteopenia, kidney transplant-associated osteopenia, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidosis, Fabry's disease, Turner's syndrome, Down's syndrome, Klinefelter's syndrome, Leye's disease, Perthes' disease, adolescent idiopathic scoliosis, infantile-onset multisystem inflammatory disease, Winchester's syndrome, Menkes' disease, Wilson's disease, ischemic bone disease (such as Legg-Calpe-Perthes disease and focal migratory osteoporosis), anemic states, steroid-induced diseases conditions, glucocorticoid-induced osteopenia, heparin-induced osteopenia, bone marrow disorders, scurvy, nutritional deficiencies, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory disease, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory bowel disease, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy-related osteopenia, diabetes mellitus, hyperthyroidism, thyroid disease, parathyroid disease, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, focal osteoporosis, osteomalacia, bone loss associated with joint replacement, HIV The present invention is administered to a subject suffering from a bone-related disorder selected from the group consisting of bone loss associated with growth hormone deficiency, bone loss associated with cystic fibrosis, bone loss associated with chemotherapy, bone loss due to tumors, cancer-related osteopenia, bone loss due to hormone deprivation, multiple myeloma, drug-induced bone loss, anorexia nervosa, disease-related facial bone loss, disease-related skull bone loss, disease-related jaw bone loss, disease-related cranial bone loss, age-related bone loss, age-related facial bone loss, age-related skull bone loss, age-related jaw bone loss, age-related skull bone loss, and bone loss associated with space travel.

[0070] In some embodiments, the antibodies described herein are useful for improving outcomes in orthopedic procedures, dental procedures, implant surgery, joint replacement, bone grafting, cosmetic bone surgery, and bone repair, such as fracture healing, non-union healing, delayed union healing, and facial reconstruction. Compositions comprising one or more antibodies can be administered before, during, and / or after the procedure, replacement, graft, surgery, or repair.

[0071] In some embodiments, the antibodies described herein are useful for treating any fracture involving a gap between two segments of bone (e.g., a gap of at least about 1 mm between two segments of bone). In some or any embodiments, the gap is at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, or at least about 1 cm or more. In some or any embodiments, the gap is between about 5 mm and 1 cm, or up to 1 cm. The terms "bone gap defect" and "partial skeletal defect" are used interchangeably herein and refer to a gap between two segments of bone (e.g., a gap of at least 1 mm).

[0072] Exemplary bone gap defects include, but are not limited to, comminuted fractures, non-union fractures, partial skeletal defects, surgically created bone defects, surgically treated bone defects, and bone defects created from trauma or disease to the bone (including, but not limited to, arthritis, tumor removal (resection), or infection removal). In some or any embodiments, the bone gap defect results from the removal of an infected portion of bone, or the removal of cancer from the bone due to bone cancer, such as, but not limited to, osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma, and chordoma. In some or any embodiments, the bone gap defect is a developmental deformity, for example, due to a genetic defect.

[0073] In some or any embodiments, the bone gap defect is created by removing a bone portion containing a benign tumor. Exemplary benign bone tumors include, but are not limited to, osteoma, osteoid osteoma, osteoblastoma, osteochondroma, enchondroma, chondromyxoid fibroma, aneurysmal bone cyst, unicameral bone cyst, fibrous dysplasia of bone, and giant cell tumor of bone.

[0074] Administration of the antibody enhances or promotes bone gap defect healing, thereby "treating" the bone gap defect. "Enhancing" bone healing means mediating a level of bone healing that exceeds (i.e., is greater than) the level of bone healing experienced in a subject (e.g., a mammal such as a human) not administered a sclerostin inhibitor (i.e., a control subject). Bone healing is manifested, for example, by improved bridging status, improved bone mass, improved bone mineral content and density within the fracture gap (i.e., formation of bridging bone), mature bony callus, improved bone strength (optionally with a medically acceptable level of bone stiffness), or improved use of the affected area by the patient. "Improved" means an increase or decrease (as desired) in the measured parameter. An increase can return the measured parameter, in whole or in part, to baseline levels (e.g., levels before the bone gap defect), to values ​​provided in standard databases used in the art, or to contralateral functional levels (e.g., return, in whole or in part, to the functional capacity of, for example, the contralateral limb). In some cases, an increase can be an improvement over baseline levels. If desired, the effectiveness of the methods described herein can be further analyzed by comparing the parameters measured in patients who have received one or more doses of the antibody with the same parameters in patients with fractures (optionally age- and sex-matched) who have not received the antibody.

[0075] Bridging bone formation, bone mineral content and bone density, and / or mature bony callus at the bone defect site can be measured using radiography (e.g., X-ray absorptiometry), single and / or dual-energy X-ray absorptiometry, quantitative computed tomography (QCT), ultrasonography, and magnetic resonance imaging. In some embodiments, the antibody can be administered at a dose and for a time effective to increase bridging bone formation, bony callus formation, or bone density (or bone mass) at the defect site by at least about 5% (e.g., about 6%, about 7%, about 8%, or about 9%). In some embodiments, bridging bone formation, bony callus formation, or bone density at the defect site is increased by at least about 10% (e.g., at least about 10%, at least about 12%, at least about 15%, at least about 18%, at least about 20%, or at least about 22%). In other embodiments, the sclerostin inhibitor increases bridging bone formation, bony callus formation, or bone density at the defect site by at least about 25% (e.g., at least about 26% or at least about 28%). In still other embodiments, the bridging bone formation, bony callus formation, or bone density at the defect site increases by at least about 30% (e.g., at least about 32%, at least about 35%, at least about 38%, or at least about 40%), or at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%). The increase or restoration of bridging bone formation can be determined 1 week, 2 weeks, 3 weeks, or 4 weeks after the initial administration of the antibody. Alternatively, bone density levels can be determined after the end of the treatment period (e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks after the end of the treatment period). In one aspect, the method reduces the amount of time required to establish a desired level of bone formation, bone mass, bony callus, or bone density (e.g., any percentage increase in bone formation, bone mineral content, bony callus, or bone mass described herein) compared to an age- and sex-matched patient who does not receive the antibody, thereby reducing the subject's recovery time.For example, in one embodiment, the antibody reduces the amount of time required to increase bone density or bone mass at the defect site by at least about 10% (e.g., at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%).

[0076] The antibody need not cure the subject of the disorder or completely protect against the development of a bone-related disorder to achieve a beneficial biological response. The antibody can be used prophylactically, meaning to protect, in whole or in part, against a bone-related disorder or its symptoms. The antibody can also be used therapeutically to ameliorate, in whole or in part, a bone-related disorder or its symptoms, or to protect, in whole or in part, against further progression of the bone-related disorder or its symptoms. Indeed, the materials and methods of the present invention are particularly useful for increasing bone mineral density, and optionally maintaining the increased bone mineral density over a period of time.

[0077] In some embodiments, one or more administrations of an antibody described herein are administered over a treatment period of, for example, about 1 week to about 18 months (e.g., about 1 month to about 12 months, about 1 month to about 9 months, or about 1 month to about 6 months, or about 1 month to about 3 months). In some embodiments, a subject receives one or more doses of an antibody described herein over a treatment period of, for example, about 1 month to about 12 months (52 weeks) (e.g., about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months).

[0078] Furthermore, depending on the treatment regimen selected for a particular subject, it may be advantageous to administer the antibody multiple times or space the administrations apart. In some embodiments, the antibody or fragment thereof is administered periodically over a period of 1 year (12 months, 52 weeks) or less (e.g., 9 months or less, 6 months or less, or 3 months or less). In this regard, the antibody or fragment thereof is administered to a human about every 3 days, or about every 7 days, or every 2 weeks, or every 3 weeks, or every 4 weeks, or every 5 weeks, or every 6 weeks, or every 7 weeks, or every 8 weeks, or every 9 weeks, or every 10 weeks, or every 11 weeks, or every 12 weeks, or every 13 weeks, or every 14 weeks, or every 15 weeks, or every 16 weeks, or every 17 weeks, or every 18 weeks, or every 19 weeks, or every 20 weeks, or every 21 weeks, or every 22 weeks, or every 23 weeks, or every 6 months, or every 12 months.

[0079] In some embodiments, the antibody is administered one or more times in an amount and for a time effective to increase bone mineral density or treat a bone disorder associated with bone mineral loss. In various embodiments, about 50 milligrams to about 1,000 milligrams of antibody are administered to a subject (e.g., a human subject) one or more times per week. For example, a single dose of antibody can contain at least about 5 mg, at least about 15 mg, at least about 25 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 120 mg, at least about 150 mg, at least about 200 mg, at least about 210 mg, at least about 240 mg, at least about 250 mg, at least about 280 mg, at least about 300 mg, at least about 350 mg, at least about 400 mg, at least about 420 mg, at least about 450 mg, at least about 500 mg, at least about 550 mg, at least about 600 mg, at least about 650 mg, at least about 700 mg, at least about 750 mg, at least about 800 mg, at least about 850 mg, at least about 900 mg, at least about 950 mg, or up to about 1,000 mg of antibody. Ranges between any and all of these endpoints are also contemplated, e.g., about 50 mg to about 80 mg, about 70 mg to about 140 mg, about 70 mg to about 270 mg, about 75 mg to about 100 mg, about 100 mg to about 150 mg, about 140 mg to about 210 mg, about 150 mg to about 200 mg, about 180 mg to about 270 mg, or about 280 mg to about 410 mg. Doses are administered at any interval, such as multiple times per week (e.g., two or three times per week), once per week, once every two weeks, once every three weeks, or once every four weeks. In some or any embodiments, an antibody dose in the range of about 120 mg to about 210 mg is administered twice per week. In some or any embodiments, an antibody dose of about 140 mg is administered twice per week. In various aspects, an antibody dose of about 210 mg is administered once per month.

[0080] In some embodiments, one or more doses of antibody can include about 0.1 to about 50 milligrams (e.g., about 5 to about 50 milligrams) of antibody per kg of body weight (mg / kg), or about 1 to about 100 milligrams of antibody per kg of body weight (mg / kg). For example, a single dose of antibody may be at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, at least about 25 mg / kg, at least about 26 mg / kg, at least about 27 mg / kg, at least about 28 mg / kg, at least about 29 mg / kg, at least about 30 mg / kg, at least about 31 mg / kg, at least about 32 mg / kg, at least about 33 mg / kg, at least about 34 mg / kg, at least about 35 mg / kg, at least about 36 mg / kg, at least about 37 mg / kg, at least about 38 mg / kg, at least about 39 mg / kg, at least about 40 mg / kg, at least about 41 mg / kg, at least about 42 mg / kg, at least about 43 mg / kg, at least about 44 mg / kg, at least about 45 mg / kg, at least about 46 mg / kg, at least about 47 mg / kg, at least about 48 mg / kg, at least about 49 mg / kg, at least about 50 mg / kg, at least about 51 mg / kg, at least about 52 mg / kg, at least about 53 mg / kg, at least about 54 mg / kg, at least about 55 mg / kg, at least about 56 mg / kg, at least about 57 mg / kg, at least about 58 mg / kg, at least about 59 mg / kg, at least about 60 mg / kg, at least about 61 mg / kg, at least about 62 mg / mg / kg, at least about 37 mg / kg, at least about 38 mg / kg, at least about 39 mg / kg, at least about 40 mg / kg, at least about 41 mg / kg, at least about 42 mg / kg, at least about 43 mg / kg, at least about 44 mg / kg, at least about 45 mg / kg, at least about 46 mg / kg, at least about 47 mg / kg, at least about 48 mg / kg, or at least about 49 mg / kg, or at least about 50 mg / kg, at least about 55 mg / kg, at least about 60 mg / kg, at least about 65 mg / kg, at least about 70 mg / kg, at least about 75 mg / kg, at least about 80 mg / kg, at least about 85 mg / kg, at least about 90 mg / kg, at least about 95 mg / kg, or up to about 100 mg / kg. Ranges between any and all of these endpoints are also contemplated, e.g., about 1 mg / kg to about 3 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 8 mg / kg, about 3 mg / kg to about 8 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 40 mg / kg, about 5 mg / kg to about 30 mg / kg, or about 5 mg / kg to about 20 mg / kg.

[0081] Treatment monitoring Antibody-mediated increases in bone mineral content or bone density can be measured using single- and dual-energy X-ray absorptiometry, ultrasound, computed tomography, radiography, and magnetic resonance imaging. Bone mass can also be calculated from body weight or using other methods (see Guinness-Hey, Metab. Bone Dis. Relat. Res., 5:177-181 (1984)). Animal models that mimic the pathology of human diseases, such as osteoporosis and osteopenia, are used in the art to test the effects of pharmaceutical compositions and methods on parameters of bone loss, bone resorption, bone formation, bone strength, or bone mineralization, for example. Examples of such models include the ovariectomized rat model (Kalu, Bone and Mineral, 15:175-192 (1991); Frost and Jee, Bone and Mineral, 18:227-236 (1992); and Jee and Yao, J. Musculoskel. Neuron. Interact., 1:193-207 (2001)). The methods for measuring antibody activity described herein can also be used to determine the efficacy of other sclerostin inhibitors.

[0082] In humans, bone mineral density can be determined clinically using, for example, dual-energy x-ray absorptiometry (DXA) of the hip and spine. Other techniques include quantitative computed tomography (QCT), ultrasonography, single-energy x-ray absorptiometry (SXA), and x-ray absorptiometry. Common central skeletal sites for measurement include the spine and hip, while peripheral sites include the forearm, finger, wrist, and heel. With the exception of ultrasonography, the American Medical Association notes that BMD techniques generally involve the use of x-rays and are based on the principle that radiation attenuation depends on the thickness and composition of tissue within the radiation path. All techniques involve comparing results to a normative database.

[0083] Alternatively, physiological responses to one or more anti-sclerostin antibodies can be measured by monitoring bone marker levels. Bone markers are products produced during the bone remodeling process and are released by bone, osteoblasts, and / or osteoclasts. Fluctuations in the levels of bone resorption and / or bone formation "markers" indicate changes in bone remodeling / modeling. The International Osteoporosis Foundation (IOF) recommends the use of bone markers to monitor bone density treatments (see, e.g., Delmas et al., Osteoporos Int., Suppl. 6:S2-17 (2000) (incorporated herein by reference)). 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 phosphatase (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- and C-terminal extensions of type I procollagen (P1NP, PICP), and osteocalcin (OstCa). Several kits for detecting and quantifying markers in clinical samples such as urine and blood are commercially available.

[0084] Combination therapy Treating a condition by combining two or more drugs that target the same pathogen or biochemical pathway or biological process sometimes results in greater efficacy and fewer side effects than the use of doses associated with treating each drug alone. In some cases, the effect of the drug combination is additive (the effect of the combination is approximately equal to the sum of the effects of each drug alone), while in other cases, the effect is synergistic (the effect of the combination is greater than the sum of the effects of each drug given alone). As used herein, the term "combination therapy" means that two or more drugs are delivered simultaneously (e.g., simultaneously, or in a manner in which one of the drugs is administered first, followed by the second drug, e.g., sequentially).

[0085] In some embodiments, the antibody is administered in conjunction with standard of care therapy for treating bone mineral loss (i.e., the antibody and standard of care therapy are part of the same treatment plan). As used herein, the term "standard of care" refers to treatment generally accepted by clinicians for certain patients diagnosed with certain diseases. In some embodiments, the antibody is administered in conjunction with a second bone-strengthening agent useful for treating bone mineral loss or bone defects. In some embodiments, the bone-strengthening agent is selected from the group consisting of anti-resorptive agents, bone-forming agents (i.e., anabolic agents), estrogen receptor modulators (including, but not limited to, raloxifene, bazedoxifene, and lasofoxifene), and drugs that have an inhibitory effect on osteoclasts. In some embodiments, the second bone-strengthening agent is selected from the group consisting of bisphosphonates (e.g., including but not limited to alendronate sodium (FOSAMAX®), risedronate, ibandronate sodium (BONIVA®), and zoledronic acid (RECLAST®)); estrogen or estrogen analogs; anti-RANK ligand (RANKL) inhibitors such as anti-RANKL antibodies (e.g., denosumab, PROLIA®); vitamin D or a vitamin D derivative or mimetic; a calcium source, a cathepsin-K (cat-K) inhibitor (e.g., odanacatib), tibolone, calcitonin, or calcitriol; and hormone replacement therapy. In some embodiments, the second bone-strengthening agent includes, but is not limited to, parathyroid hormone (PTH) or a peptide fragment thereof, PTH-related protein (PTHrp), a bone morphogenetic protein, osteogenin, NaF, a PGE2 agonist, a statin, strontium ranelate, and a sclerostin inhibitor (e.g., an anti-sclerostin antibody described in U.S. Pat. No. 7,592,429 or U.S. Pat. No. 7,872,106). In some embodiments, the second bone-strengthening agent is Forteo® (teriparatide), Preotact®, or Protelos®.In some embodiments, the second bone-enhancing agent comprises a bone morphogenetic protein (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).

[0086] In some embodiments, combination therapy using an antibody described herein may be preceded or followed by administration of an additional therapeutic agent (e.g., a second bone-strengthening agent) by intervals ranging from minutes to weeks to months. For example, separate methods may be administered within about 24 hours of each other, e.g., within about 6-12 hours of each other, or within about 1-2 hours of each other, or within about 10-30 minutes of each other. In some situations, it may be desirable to extend the treatment period significantly, such that several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between administration of each of the different methods. Repeated treatment with one or both agents / therapies of the combination therapy is specifically contemplated.

[0087] Maintaining a treatment plan Also contemplated is the use of a second bone-strengthening agent and / or antibody described herein in maintenance therapy, e.g., to prevent or slow bone mineral loss. In this regard, the methods or uses described herein optionally comprise administering one or more doses of a second bone-strengthening agent effective to maintain bone mineral density for a maintenance period of about one week to about five years after the antibody treatment period has ended. For example, in some embodiments, the methods or uses described herein comprise administering one or more doses of a second bone-strengthening agent effective to maintain bone mineral density for a maintenance period of at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about five weeks, at least about six weeks, at least about seven weeks, at least about eight weeks, at least about nine weeks, at least about ten weeks, at least about 11 weeks, at least about 12 weeks, at least about three months, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about four months, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about five months, at least about 21 weeks, or at least about 22 weeks. In some embodiments, the maintenance period is about 6-12 weeks. In some embodiments, the maintenance period is about 4-12 weeks or about 1-3 months. In some embodiments, the maintenance period is about 12-20 weeks or about 3-5 months. In some embodiments, the maintenance period is about 20-32 weeks or about 5-8 months. In some embodiments, the maintenance period is about 24-36 weeks or about 6-9 months, hi some embodiments, the maintenance period is about 1 year, about 2 years, about 3 years, about 4 years, about 5 years or more."Maintaining" bone mineral density includes maintaining a similar level of bone mineral density parameter experienced by subjects receiving antibody therapy.

[0088] kit Pharmaceutical compositions comprising one or more antibodies described herein can be placed in a container (e.g., a vial or syringe) along with packaging material providing instructions for use of such pharmaceutical compositions. Generally, such instructions will include specific wording describing the antibody concentration, as well as, in certain embodiments, the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) that may be required to reconstitute the pharmaceutical composition. [Example]

[0089] Example 1 - Analysis of romosozumab PARG C-terminal variants Wild-type romosozumab and the romosozumab PARG C-terminal variant were digested with Lys-C and analyzed by LC / MS peptide mapping. The UV profiles of these two constructs were compared side-by-side (Figure 3). While wild-type romosozumab and the romosozumab PARG C-terminal variant have similar peaks eluting at 37.7 minutes, wild-type romosozumab was determined to have a mass of 659.3 Da, and the romosozumab PARG C-terminal variant was determined to have a mass of 886.7 Da. The majority of the lysine (K) variant of romosozumab (PGK) was likely removed in the process. The presence of a significant amount of the amidated form of the romosozumab PARG C-terminal variant (828.6 Da peak) confirms that amidation efficiency is sequence-dependent when compared to the wild-type romosozumab PG sequence.

[0090] The PARG C-terminal variants were then treated with carboxypeptidase (CP-B) and analyzed by CEX-HPLC, and compared with a control PARG C-terminal variant that was not treated with CP-B. There was a significant shift after treatment in the peaks eluting at 17.5 and 21 minutes, but not in the peak at 24 minutes (Figure 4). The peak at 24 minutes is thought to be a doubly amidated form that is protected from proteolysis.

[0091] Example 2 - Enrichment of C-terminal variants Purification or enrichment of different romosozumab species from compositions containing wild-type romosozumab and romosozumab PARG C-terminal variants is achieved by cation exchange chromatography (CEX) fractionation. CEX separates proteins based on differences in their surface charge. Positively charged variants of wild-type romosozumab are separated on a cation exchange column (e.g., a Dionex Pro Pac WCX-10 analytical column, 2.0 mm x 250 mm) at a set pH and eluted using a salt gradient (e.g., mobile phase A: 10:90 (v / v) ACN, 19 mM MES, pH 6.2; mobile phase B: 10:90 (v / v) ACN, 19 mM MES, 250 mM NaCl, pH 6.2). Different C-terminal variants of romosozumab have different amounts of charge, and more positively charged variants elute later in CEX. The elution order is therefore as follows: PG (wild type), P-amide (amidated proline in wild type), PARG variants, and PAR-amide. The fraction collector can be programmed to collect CEX eluates containing different variants at different elution times.

[0092] Example 3 - Analysis of aggregation of romosozumab PARG C-terminal variants Without being bound by any particular theory, it is believed that because the PARG C-terminal variant is highly charged, such a form repels the non-amidated form in the composition, thus reducing aggregation in the composition.

[0093] The romosozumab PARG C-terminal variant protein A pool was analyzed alongside the wild-type romosozumab protein A pool using SEC-HPLC, a size-exclusion HPLC method that separates proteins based on differences in their hydrodynamic volumes (Table 1).

[0094] [Table 1]

[0095] The data demonstrated that the romosozumab PARG C-terminal variants had fewer high molecular weight species compared to wild-type romosozumab.

[0096] Example 4 - Viscosity analysis of romosozumab PARG C-terminal variants Antibody solutions containing romosozumab PARG C-terminal variants or wild-type romosozumab were measured using a cone and plate. These solutions were concentrated to 120 mg / mL according to the approximate volume reduction, and the final concentration was determined using the protein absorbance at 280 nm (after dilution to within 0.1–1 absorbance units (AU)) and the protein specific extinction coefficient (±10%). Viscosity analysis was performed using a Brookfield LV-DVIII cone and plate instrument (Brookfield Engineering, Middleboro, MA, USA) with a CP-40 spindle and sample cup, or an ARES-G2 rheometer (TA Instruments, New Castle, DE, USA) with a TA Smart Swap 2-degree cone / plate spindle. All measurements were performed at 25°C and controlled by a water bath attached to the sample cup. Multiple viscosity measurements were collected manually within the specified torque range (10–90%) by increasing the spindle RPM. For simplicity of the resulting comparison chart, measurements are averaged to report one viscosity value per sample.

[0097] Example 5 - Analysis of the solubility of romosozumab PARG C-terminal variants To determine the effect of amino acid modifications of the romosozumab PARG variants compared to wild-type romosozumab on their solubility upon subcutaneous (SC) injection, parallel dialysis solubility assays were performed on both wild-type and PARG C-terminal variant romosozumab. This screening involved dialyzing samples of the romosozumab PARG C-terminal variants and wild-type romosozumab into solutions simulating the pH and ionic strength of the SC space and monitoring the solubility and physical stability of the antibody under these conditions over a short period of time. Samples were formulated at approximately 63 mg / mL in formulation buffer (pH 5.2). Each sample was then injected into a dialysis cassette and dialyzed into PBS buffer to mimic the SC space. Visual observations were performed 24 hours after the initial dialysis. Wild-type romosozumab typically exhibited precipitation after 24 hours.

[0098] The results show that both molecules precipitate in this assay, but the PARG C-terminal variant precipitates less and at a slower rate, suggesting that the variant is more resistant to precipitation than the wild type, although it does not completely eliminate precipitation.

[0099] Example 6 - Diffusion analysis of romosozumab PARG C-terminal variants To determine the effect of amino acid modifications of the PARG C-terminal variant of romosozumab compared to wild-type romosozumab on diffusion from the subcutaneous (SC) space, an assay was performed using a Scissor (Pion Inc., Billerica, MA). The assay involved injecting samples (romosozumab PARG C-terminal variant or wild-type romosozumab) at approximately 70 mg / mL into a simulated SC space consisting of a collagen and hyaluronic acid matrix. The antibody was allowed to diffuse from this matrix through a dialysis membrane into a reservoir of carbonate buffer at pH 7.4. Time points were collected over a period of up to 3 days, and the protein concentration at each time point was measured by RP-HPLC. The generated protein concentration vs. time curve simulated the diffusion rate from the SC space. Furthermore, precipitation in the SC matrix was monitored by visual inspection.

[0100] Both wild-type and PARG C-terminal variant romosozumab were tested in the Scissor as described above. The results, shown in Figure 5, indicate that wild-type romosozumab diffused from the simulated SC space much more slowly than PARG C-terminal variant romosozumab, with more wild-type romosozumab remaining at the simulated injection site.

[0101] Example 7 - FcRn Binding FcRn, the fetal Fc receptor, is an MHC class I-like heterodimer consisting of a transmembrane α chain (homologous to MHC class I-like molecules) and a β2-microglobulin light chain. FcRn binds to the C of the IgG heavy chain in the Fc region of IgG molecules under slightly acidic conditions (approximately pH 6). H 2 Domain and C H It binds to the three-domain interface and releases it at neutral pH (approximately 7.4). Through this highly pH-dependent interaction, FcRn mediates IgG homeostasis in adult humans by maintaining serum IgG concentrations.

[0102] The binding of the Fc domains of wild-type romosozumab and romosozumab PARG C-terminal variants to FcRn was assessed using a competitive binding assay, the AlphaScreen® binding assay (PerkinElmer, San Jose, CA). The assay is a bead-based amplified luminescent proximity homogeneous assay ("Alpha") that detects bimolecular interactions. The assay contains two bead types: acceptor beads and donor beads. The acceptor beads are coated with a hydrogel containing a thioxene derivative and a nickel chelate that binds to the histidine domain of histidine-tagged FcRn (FcRn-His). The donor beads are coated with a hydrogel containing a phthalocyanine, a photosensitizer, and streptavidin that binds to biotinylated CHO-derived human Fc. Upon binding of FcRn-His and biotinylated human Fc, they bring the acceptor and donor beads into close proximity. When this complex is illuminated with laser light, ambient oxygen is converted to singlet oxygen by the donor bead. If the beads are in close proximity, energy transfer to the acceptor bead occurs, resulting in light production (luminescence), which is measured in a plate reader equipped for AlphaScreen® signal detection.

[0103] When the antibody is present at a concentration sufficient to inhibit binding of FcRn-His to the biotinylated human Fc domain, a dose-dependent decrease in emission at 570 nm is observed. Binding of the test sample relative to an antibody reference standard is determined, reported as % relative binding, and can be used to demonstrate the integrity of the antibody's Fc domain. Compositions with PARG C-terminal variants are expected to have dose-response curves similar to or better than those of wild-type antibodies.

[0104] The results are shown in Figure 6. It was observed that both wild-type romosozumab and PARG C-terminally modified romosozumab bound to FcRn in a similar manner, and that FcRn binding was not affected by the PARG mutation.

[0105] Example 8 - FcγRIIa Binding FcγRIIa is an activating Fc receptor expressed on monocytes, certain dendritic cells, neutrophils, B cells, platelets, and NK cells. FcγRIIa (CD32a) is the most widely distributed FcγR, possessing two extracellular Ig-like domains and low binding affinity for monomeric IgG. In humans, there are two common allelic variants known to exist in FcγRIIa, expressing either a histidine or an arginine at position 131 (131H and 131R, respectively).

[0106] A competitive binding assay was developed to evaluate the binding of wild-type romosozumab and romosozumab PARG C-terminal variants to FcγRIIa(131H). The FcγRIIa(131H) binding assay is a bead-based amplified luminescent proximity homogeneous assay (AlphaScreen® Binding Assay (PerkinElmer, San Jose, CA)) that detects bimolecular interactions. The assay contains two bead types: acceptor beads and donor beads. The acceptor beads are coated with a hydrogel containing a fluorophore europium chelate and glutathione that binds to recombinant human FcγRIIa(131H)-glutathione-S-transferase (FcγRIIa(131H)-GST). The donor beads are coated with a hydrogel containing phthalocyanine, a photosensitizer, and streptavidin that binds biotinylated human IgG1. When FcγRIIa(131H)-GST binds to biotinylated human IgG1, they bring the acceptor and donor beads into close proximity. When this complex is illuminated with a laser, ambient oxygen is converted to singlet oxygen by the donor beads. When the acceptor and donor beads are in close proximity, the singlet oxygen diffuses within the acceptor beads, producing light (luminescence), which is measured using a plate reader equipped for luminescence signal detection.

[0107] When the antibody is present at a concentration sufficient to inhibit binding of FcγRIIa(131H)-GST to biotinylated human IgG1, a dose-dependent decrease in emission at 570 nm is measured. Binding of the test sample relative to an antibody reference standard is determined, reported as % relative binding, and can be used to demonstrate the integrity of the antibody's Fc domain. The results are shown in Figure 7. It was observed that the relative binding of the PARG C-terminal variant romosozumab to FcγRIIa(131H) was much higher than that of wild-type romosozumab.

[0108] Example 9 - Mouse Pharmacokinetic Study To assess in vivo drug exposure and bioavailability, single-dose pharmacokinetic studies were conducted in mice. Romosozumab PARG C-terminal variants were injected intravenously (via the tail vein) or subcutaneously at a dose of 1 mg / kg. Staggered sampling, using nine animals per group, allowed for the collection of data at multiple time points without exceeding the maximum volume of blood that could be collected from an individual animal. At each time point, 0.05 ml of blood was collected. Animals 1-3 were sampled at 0.083, 24, 96, and 192 hours after administration. Animals 4-6 were sampled at 1, 48, 168, and 240 hours. Animals 7-9 were sampled at 6, 72, and 192 hours. Serum was collected from whole blood samples, and test substance concentrations were measured using a binding immunoassay such as ELISA (enzyme-linked immunosorbent assay). Changes in test substance concentration over time could be used to calculate pharmacokinetic parameters using a two-compartment analysis. The parameters of interest were the area under the plasma concentration-time curve (AUC) and half-life (t 1 / 2 Bioavailability can be determined as the ratio of the AUC of a subcutaneous dose to the AUC of an intravenous dose.

[0109] SEQUENCE LISTING <110> Amgen Inc. <120> C-Terminal Antibody Variants <150> US 62 / 650,762 <151> 2018-03-30 <150> US 62 / 812,741 <151> 2019-03-01 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 190 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Human Sclerostin <400> 1 Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu 1 5 10 15 Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr 20 25 30 Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu 35 40 45 Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg 50 55 60 Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu 65 70 75 80 Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile 85 90 95 Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile 100 105 110 Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly 115 120 125 Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys 130 135 140 Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly 145 150 155 160 Thr Glu Ala Ala Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala 165 170 175 Arg Ser Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr 180 185 190 <210> 2 <211> 5 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo HCDR1 <400> 2 Asp Tyr Asn Met His 1 5 <210> 3 <211> 17 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo HCDR2 <400> 3 Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 4 <211> 14 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo HCDR3 <400> 4 Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr Phe Asp Val 1 5 10 <210> 5 <211> 11 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romoLCDR1 <400> 5 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 6 <211> 7 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo LCDR2 <400> 6 Tyr Thr Ser Arg Leu Leu Ser 1 5 <210> 7 <211> 9 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo LCD3 <400> 7 Gln Gln Gly Asp Thr Leu Pro Tyr Thr 1 5 <210> 8 <211> 4 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-Terminal variant sequence <400> 8 Pro Ala Arg Gly 1 <210> 9 <211> 123 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo light chain variable region <400> 9 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 107 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo heavy chain variable region <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 5 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> C-terminal variant sequence <400> 11 Pro Ala Arg Gly Lys 1 5 <210> 12 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <220> <221> MISC_FEATURE <223> romo light chain <400> 12 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Asp Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 13 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <220> <221> MISC_FEATURE <223> romo heavy chain variant without lysine <400> 13 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr 115 120 125 Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 145 150 155 160 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr 210 215 220 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val 225 230 235 240 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 245 250 255 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Ala Arg Gly 465 <210> 14 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <220> <221> MISC_FEATURE <223> romo heavy chain variant with lysine <400> 14 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr 115 120 125 Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 145 150 155 160 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr 210 215 220 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val 225 230 235 240 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 245 250 255 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Ala Arg Gly Lys 465 470 <210> 15 <211> 26 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> amino acids 86-111 of SEQ ID NO: 1 <400> 15 Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp 1 5 10 15 Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys 20 25 <210> 16 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> Humanized Antibody sequence <220> <221> MISC_FEATURE <223> romo heavy chain wild type <400> 16 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr 115 120 125 Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 145 150 155 160 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr 210 215 220 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val 225 230 235 240 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 245 250 255 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465

Claims

1. A pharmaceutical composition comprising: (a) a population of antibodies that specifically bind to sclerostin of SEQ ID NO: 1; and (b) a pharmaceutically acceptable carrier, the population comprises a mixture of antibodies comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10, wherein the antibodies comprise two heavy chains, at least one of which comprises the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO:8) at its C-terminus, and the C-terminus of one or both of the two heavy chains is amidated; A pharmaceutical composition wherein the population of antibodies is administered in a dose of at least 90 mg.

2. 2. The pharmaceutical composition of claim 1, wherein the C-terminus of both heavy chains comprises the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8).

3. The pharmaceutical composition of claim 1 , wherein the C-termini of both heavy chains are amidated.

4. The pharmaceutical composition described in claim 1 or 2, wherein the antibody comprises a light chain amino acid sequence shown in SEQ ID NO: 12 and a heavy chain amino acid sequence shown in SEQ ID NO:

13.

5. The pharmaceutical composition of claim 1, wherein the antibody comprises an amino acid sequence comprising Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the first heavy chain, and an amino acid sequence comprising Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the second heavy chain.

6. The pharmaceutical composition described in claim 5, wherein the antibody comprises a light chain amino acid sequence shown in SEQ ID NO: 12 and a heavy chain amino acid sequence shown in SEQ ID NO:

14.

7. The pharmaceutical composition of claim 1 , wherein at least 1% but less than 35% of the antibodies in the population have both of their heavy chains amidated.

8. 8. The pharmaceutical composition of claim 7, wherein at least 1% but less than 35% of the antibodies in the population comprise non-amidated heavy chains.

9. 8. The pharmaceutical composition of claim 7, wherein about 33% of the antibodies in the population are non-amidated, about 33% of the antibodies in the population comprise one amidated heavy chain, and about 33% of the antibodies in the population comprise two amidated heavy chains.

10. 10. The pharmaceutical composition of claim 1, further comprising a calcium salt, an acetate buffer, a polyol, and a surfactant.

11. 11. The pharmaceutical composition of claim 10, wherein the calcium salt comprises calcium acetate.

12. 11. The pharmaceutical composition of claim 10, wherein the acetate buffer comprises sodium acetate.

13. 11. The pharmaceutical composition of claim 10, wherein the polyol comprises sucrose.

14. 11. The pharmaceutical composition of claim 10, wherein the surfactant comprises polysorbate 20.

15. A pharmaceutical composition described in any one of claims 1 to 14, further comprising a solution of pH 5.2 having the following composition: 55 mM acetate, 13 mM calcium, 6.0% (w / v) sucrose, and 0.006% (w / v) polysorbate 20.

16. The pharmaceutical composition according to any one of claims 1 to 15 for increasing bone mineral density in a subject in need thereof.

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