C-terminus antibody variants

Abstract

The present invention relates generally to anti-sclerostin antibodies having a C-terminal modification, and compositions comprising such antibodies.

Description

This application is a divisional, the application number of the parent application is 201980035260.3, the application date is March 29, 2019, and the name of the invention is “C-terminal antibody variant”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 650,762, filed on March 30, 2018, and U.S. Provisional Application No. 62 / 812,741, filed on March 1, 2019, the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0002] The present invention generally relates to anti-sclerostin antibodies having at least one C-terminal modification, and compositions comprising such antibodies. Incorporation by reference of electronically submitted material

[0003] Incorporated herein by reference in its entirety is a computer-readable Nucleotide / Amino Acid Sequence Listing filed concurrently herewith and defined as follows: ASCII (text) file name "52080_SeqListing.txt" created on March 21, 2019, 21,006 bytes. Incorporate by Reference

[0004] The following applications are incorporated herein by reference in their entireties: International Patent Publication No. PCT / US2012 / 049331, filed on August 2, 2012, which claims the benefit of U.S. Provisional Patent Application No. 61 / 515,191, filed on August 4, 2011; U.S. Patent Application No. 11 / 410,540, filed on April 25, 2006, which claims the benefit of 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 May 3, 2005; and U.S. Patent Application No. 11 / 411,003, filed April 25, 2006 (issued as U.S. Patent No. 7,592,429), which claims the benefit of 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 / 677,583, filed May 3, 2005. The following applications are also incorporated herein by reference: U.S. patent application Ser. No. 12 / 212,327, filed on Sep. 17, 2008, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60 / 973,024, filed on Sep. 17, 2007; and U.S. patent application Ser. No. 12 / 811,171, filed on Jun. 29, 2010, which is a U.S. national phase application under 35 U.S.C. §371 of International Patent Application Ser. No. PCT / US08 / 86864, filed on Dec. 15, 2008, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61 / 013,917, filed on Dec. 14, 2007. Background Art

[0005] The loss of bone mineral content can be caused by a variety of conditions, and may lead to serious medical problems. For example, osteoporosis is a debilitating disease in humans, and is characterized by a significant decrease in skeletal bone mass and mineral density, deterioration of bone structure (including the degeneration of bone microstructure and the corresponding increase of bone fragility (i.e., reduced bone strength)), and easy fractures in sick individuals. Although osteoporosis is considered to be due to an increased risk of fractures caused by a decrease in bone mass, currently available methods for treating bone disorders rarely increase the bone density of adults, and most current methods of treatment play a role mainly by inhibiting further bone resorption rather than stimulating new bone formation. Currently, estrogen prescriptions are implemented to delay bone loss. However, there is controversy about whether patients obtain any long-term benefits, and whether estrogen has any effect on patients over 75 years old. In addition, it is believed that the use of estrogen will increase the risk of breast cancer and endometrial cancer. Calcitonin is also recommended for postmenopausal women, with the addition of vitamin K or high-dose dietary calcium, with or without the addition of vitamin D osteocalcin. However, high doses of calcium often have undesirable gastrointestinal side effects, and serum and urine calcium levels 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 , olpadronate, neridronate, skelid, bonefos), parathyroid hormone, calcilytics, calcimimetic (e.g., cinacalcet), statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. However, these therapeutic agents 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 skeletal disease characterized by bone overgrowth and high density bones (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 by Brunkow et al. (supra) and disclosed herein as SEQ ID NO: 1. Sclerostin is a valuable target for mediating increased bone density. Summary of the invention

[0007] In one aspect, described herein are antibodies that specifically bind to sclerostin of SEQ ID NO: 1 and comprise the set of six CDRs set forth in SEQ ID NOs: 2-7, wherein the antibody comprises a heavy chain comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain. 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 the wild-type heavy chain amino acid sequence (i.e., lacking the C-terminal Pro-Ala-Arg-Gly). In some embodiments, the antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 12 and the heavy chain amino acid sequence shown 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 shown in SEQ ID NO: 12 and the heavy chain amino acid sequence shown in SEQ ID NO: 14.

[0008] In some embodiments, the C-terminus of one of the heavy chains of the antibody is amidated (ie, the antibody is monoamidated). In some embodiments, the C-termini of both heavy chains of the antibody are amidated (ie, the antibody is doubly amidated).

[0009] The present disclosure also provides a pharmaceutical composition comprising a population of antibodies described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a mixture of antibodies that specifically bind to sclerostin of SEQ ID NO: 1 and a pharmaceutically acceptable carrier, wherein the mixture of antibodies comprises a population of antibodies, the population of antibodies comprising a heavy chain having an amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain. In some embodiments, about 3%-5% of the antibodies in the composition are a population of antibodies comprising a heavy chain having an amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain. In some aspects, less than 70% of the population of antibodies is amidated on one or two heavy chains. In some aspects, all or part of the population of antibodies comprises a single heavy chain comprising a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, which is optionally amidated. In some aspects, all or part of the antibody population comprises a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence in two heavy chains, and the two heavy chains are optionally amidated. Optionally, less than about 35% of the antibody population is monoamidated, and / or less than about 35% of the antibody population is amidated on two heavy chains, and / or less than about 35% of the antibody population comprises unamidated heavy chains. In this regard, in many aspects, about 33% of the antibody population is not amidated, about 33% of the antibody population comprises an 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 acetate 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 methods of increasing bone mineral density in a subject in need thereof, the methods comprising administering to the subject a composition described herein in an amount effective to increase bone mineral density in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A nucleic acid sequence encoding a portion of the C-terminus of wild-type romosozumab is provided.

[0013] Figure 2A nucleic acid sequence encoding a portion of the C-terminus of a lomosozumab C-terminal variant (PARG variant) is provided.

[0014] Figure 3 is an overlay showing the magnified UV spectra of wild-type lomosozumab (dashed line) and lomosozumab PARG variant digested with Lys-C and analyzed by LC / MS peptide mapping (solid line).

[0015] Figure 4 is an overlay showing the cation exchange (CEX) spectra of the carboxypeptidase-treated lomosozumab PARG variant (dashed line) and the untreated lomosozumab PARG variant (solid line).

[0016] Figure 5 is a graph showing the percent recovery over time for the Scissor model of subcutaneous injection. For the simulated injection site, wild-type lomosozumab (circles) and PARG c-terminal variant lomosozumab diffuse at different rates.

[0017] Figure 6 is a graph showing that both wild-type lomosozumab and the PARG C-terminal variant lomosozumab bind to FcRn similarly, and that FcRn binding is not affected by PARG mutations.

[0018] Figure 7 is a graph showing that the relative binding of PARG C-terminal variant lomosozumab to FcγRIIa(131H) is much higher than that of wild-type lomosozumab. DETAILED DESCRIPTION

[0019] The present disclosure provides an antibody that specifically binds to sclerostin, wherein the antibody comprises a heavy chain comprising an amino acid sequence comprising Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain. In some embodiments, the antibody comprises a first heavy chain and a second heavy chain, the first heavy chain comprising an amino acid sequence comprising Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain, and the second heavy chain comprises a wild-type heavy chain amino acid sequence. In some embodiments, the antibody comprises 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 antibody (or a mixture of antibodies) and methods of using the antibody are also provided.

[0020] An "anti-sclerostin antibody" or "antibody that binds to sclerostin" is an antibody or portion thereof that binds to sclerostin of SEQ ID NO: 1. Recombinant human sclerostin / SOST is commercially available from, for example, R&D Systems (Minneapolis, MN, USA; 2006 Catalog No. 1406-ST-025). U.S. Patent Nos. 6,395,511 and 6,803,453, and U.S. Patent Publication Nos. 2004 / 0009535 and 2005 / 0106683 generally refer to anti-sclerostin antibodies. Examples of sclerostin antibodies suitable for use in the context of the present invention are also described in U.S. Patent Publication Nos. 2007 / 0110747 and 2007 / 0072797, which are incorporated herein by reference. Additional information regarding materials and methods for generating sclerostin antibodies can be found in U.S. Patent Publication No. 20040158045 (incorporated herein by reference).

[0021] The term "antibody" refers to intact immunoglobulin molecules (including polyclonal, monoclonal, chimeric, humanized, and / or human forms having full-length heavy and / or light chains).

[0022] As used herein, "specific binding" means that the antibody preferentially binds to the antigen rather than other proteins. In some embodiments, "specific binding" means that the antibody has a higher affinity for the antigen than for other proteins. An antibody that specifically binds to an antigen may have a binding affinity for the antigen that is less than or equal to 1 x 10 -7 M, less than or equal to 2x 10 -7 M, less than or equal to 3x 10 -7 M, less than or equal to 4x 10 -7 M, less than or equal to 5x 10 -7 M, less than or equal to 6x 10 -7 M, less than or equal to 7x 10 -7 M, less than or equal to 8x 10 -7 M, less than or equal to 9x 10 -7 M, less than or equal to 1x 10 -8 M, less than or equal to 2x 10 -8 M, less than or equal to 3x 10 -8 M, less than or equal to 4x 10 -8 M, less than or equal to 5x 10 -8 M, less than or equal to 6x10 -8 M, less than or equal to 7x 10 -8 M, less than or equal to 8x 10 -8 M, less than or equal to 9x 10 -8 M, less than or equal to 1x 10 -9 M, less than or equal to 2x 10 -9 M, less than or equal to 3x 10 -9 M, less than or equal to 4x 10 -9 M, less than or equal to 5x 10 -9 M, less than or equal to 6x 10 -9 M, less than or equal to 7x 10 -9 M, less than or equal to 8x 10 -9 M, less than or equal to 9x 10 -9 M, less than or equal to 1x 10 -10 M, less than or equal to 2x 10 -10 M, less than or equal to 3x 10 -10 M, less than or equal to 4x 10 -10 M, less than or equal to 5x10 -10 M, less than or equal to 6x 10 -10 M, less than or equal to 7x 10 -10 M, less than or equal to 8x 10 -10 M, less than or equal to 9x 10 -10 M, less than or equal to 1x 10 -11 M, less than or equal to 2x 10 -11 M, less than or equal to 3x 10 -11 M, less than or equal to 4x 10 -11 M, less than or equal to 5x 10 -11 M, less than or equal to 6x 10 -11 M, less than or equal to 7x 10 -11 M, less than or equal to 8x 10 -11 M, less than or equal to 9x 10 -11 M, less than or equal to 1x10 -12 M, less than or equal to 2x 10 -12 M, less than or equal to 3x 10 -12 M, less than or equal to 4x 10 -12 M, less than or equal to 5x 10 -12 M, less than or equal to 6x 10 -12 M, less than or equal to 7x 10 -12 M, less than or equal to 8x 10 -12 M, or less than or equal to 9x 10 -12 M.

[0023] In some or any embodiments, the antibody binds to sclerostin of SEQ ID NO: 1, or a naturally occurring variant thereof, at a concentration of less than or equal to 1 x 10 -7 M, less than or equal to 1x 10 -8M, less than or equal to 1x 10 -9 M, less than or equal to 1x 10 -10 M, less than or equal to 1x 10 -11 M, or less than or equal to 1x 10 -12 In some embodiments, the affinity of an antibody to sclerostin is determined by measuring the affinity (Kd) of the antibody to M. Affinity is determined using a variety of techniques, one example of which is 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 Publication No. 2007 / 0110747 (the disclosure of which is incorporated herein by reference) contains additional descriptions of affinity assays suitable for determining the affinity (Kd) of an antibody to sclerostin.

[0024] In some or any 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 binds 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 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.

[0025] In some or any embodiments, the anti-sclerostin antibodies described herein preferably modulate sclerostin function in a cell-based assay described in U.S. Patent Publication No. 2007 / 0110747 and / or in an in vivo assay described in U.S. Patent Publication No. 2007 / 0110747, and / or bind to one or more epitopes described in U.S. Patent Publication No. 2007 / 0110747, and / or cross-block binding of one of the antibodies described in U.S. Patent Publication No. 2007 / 0110747, and / or are cross-blocked by binding of one of the antibodies described in U.S. Patent Publication No. 2007 / 0110747 to sclerostin (incorporated herein in its entirety, along with descriptions of assays for characterizing anti-sclerostin antibodies).

[0026] "CDR" refers to the complementary determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy chain and the light chain, which are referred to as CDR1, CDR2 and CDR3. As used herein, the term "group of six CDRs" refers to a group of three CDRs that can bind to an antigen that appear in the light chain variable region and the heavy chain variable region. According to different systems, the exact boundaries of CDRs have been defined differently. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest [sequences of target immunoglobulins] National Institutes of Health [National Institutes of Health], Bethesda Software Company (Bethesda), Maryland (1987) and (1991)) not only provides an accurate residue numbering system applicable to any variable region of an antibody, but also provides accurate residue boundaries defining three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite having great diversity at the amino acid sequence level. These sub-portions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" represent the light chain region and the heavy chain region, respectively. These regions can be referred to as Chothia CDRs, which have borders that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are described by Padlan (FASEB J. [Journal of the Federation of American Societies for Experimental Biology] 9:133-139 (1995)) and MacCallum (J Mol Biol [Journal of Molecular Biology] 262(5):73245 (1996)). Other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, although they may be shortened or lengthened without significantly affecting antigen binding based on predictions or experimental results for particular residues or groups of residues, or even entire CDRs. The methods used herein can utilize CDRs defined according to any of these systems, but preferred embodiments use CDRs defined according to Kabat or Chothia.

[0027] The CDRs are obtained, for example, by constructing a polynucleotide encoding the CDR of interest. For example, such polynucleotides are prepared by synthesizing the variable region using mRNA from cells producing the antibody as a template using the polymerase chain reaction (see, e.g., 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.), p. 166, Cambridge University Press (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Clinical Applications, eds., p. 166, Cambridge University Press (1995); Monoclonal Antibodies: Principles and Applications, Birch et al. (Eds.), p. 137, Wiley-Liss, Inc. (1995).

[0028] In various aspects, 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, wherein CDR-H1 has the sequence given in SEQ ID NO: 2, CDR-H2 has the sequence given in SEQ ID NO: 3, CDR-H3 has the sequence given in SEQ ID NO: 4, CDR-L1 has the sequence given in SEQ ID NO: 5, CDR-L2 has the sequence given in SEQ ID NO: 6, and CDR-L3 has the sequence given in SEQ ID NO: 7. In various aspects, the anti-sclerostin antibody comprises two CDRs or six CDRs.

[0029] 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.

[0030] 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 shown 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 shown in SEQ ID NO: 10. In various aspects, the differences in sequence compared to SEQ ID NO: 9 or 10 are located outside the CDR regions in the corresponding sequences. In some or any embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence shown in SEQ ID NO: 9, and a heavy chain variable region comprising an amino acid sequence shown in SEQ ID NO: 10.

[0031] In some or any embodiments, the anti-sclerostin antibody comprises all or a portion of a heavy chain (e.g., two heavy chains) comprising an amino acid sequence that is at least 75% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identical) to the amino acid sequence shown in SEQ ID NO: 16, and all or a portion of a light chain (e.g., two light chains) comprising an amino acid sequence that is at least 75% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identical) to the amino acid sequence shown in SEQ ID NO: 12.

[0032] The antibody comprises a heavy chain comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain. In some embodiments, the C-termini of both heavy chains of the antibody comprise 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 the wild-type amino acid sequence. In various aspects, the antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 12 and the heavy chain amino acid sequence shown in SEQ ID NO: 13.

[0033] Alternatively, in some or any embodiments, the antibody comprises an amino acid sequence containing 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 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.

[0034] Examples of other anti-sclerostin antibodies include, but are not limited to, anti-sclerostin antibodies disclosed in International Patent Publication Nos. 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 herein by reference in its entirety).

[0035] Those skilled in the art will appreciate that some proteins (such as antibodies) can undergo a variety of post-translational modifications. The type and extent of these modifications generally depend on the host cell line used to express the protein and the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. Frequent modifications are due to the loss of carboxyl terminal basic residues (e.g., lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography [Journal of Chromatography] 705: 129-134, 1995).

[0036] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86

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

[0037] In some or any 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 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 embodiments, glycine is amidated. For example, amidation can occur as described 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 peptidyl glycine alpha-amidating monooxygenase (PAM) can catalyze the amidation of glycine. PAM has two active domains, peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL). PHM catalyzes the conversion of peptidylglycine (with ascorbate and oxygen) to peptidyl α-hydroxyglycine (with semidehydrogenascorbate and water). In turn, PAL catalyzes the conversion of peptidyl α-hydroxyglycine to amidated peptide (and glyoxylate).

[0038] The amidation of antibodies can be controlled by changing certain conditions during cell culture. For example, copper (e.g., in ammonium ferric citrate) and / or oxygen levels can be used to affect the level of amidation. It is expected that increasing copper concentration (e.g., in the culture medium) or oxygen availability (e.g., during culture) can increase amidation by affecting the activity of enzymes such as PHM.

[0039] Pharmaceutical composition

[0040] The present disclosure provides pharmaceutical compositions comprising a population of antibodies described herein together with a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0041] The present disclosure also provides a pharmaceutical composition comprising a mixture of antibodies that specifically bind to sclerostin of SEQ ID NO: 1 and a pharmaceutically acceptable carrier, wherein about 3%-5% of the antibodies in the composition are a population of antibodies described herein (e.g., antibodies comprising the set of six CDRs shown in SEQ ID NOs: 2-7 and having one heavy chain (or two heavy chains) comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of one or both heavy chains). The present disclosure also encompasses compositions comprising an alternative amount (e.g., 5%-10%, 1%-3%, 3%-15%, 2%-10%, 4%-20%, 1%-5%) of a population of antibodies described herein (e.g., antibodies comprising the set of six CDRs shown in SEQ ID NOs: 2-7 and having one heavy chain (or two heavy chains) comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of one or both heavy chains).

[0042] 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 about 35%, 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) comprises a heavy chain comprising a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, which 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 population of antibodies comprises a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence on both heavy chains, wherein both heavy chains are optionally amidated. It is also contemplated that both heavy chains comprise a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, but only one chain 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 a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence that is not amidated.In some embodiments, about 33% of the population of antibodies comprise a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence that is amidated, about 33% of the population of antibodies comprise a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence that is amidated on both heavy chains, and about 33% of the population of antibodies comprise one or both heavy chains having a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence (but which is not amidated).

[0043] In some embodiments, the pharmaceutical composition contains formulation materials used to modify, maintain, or preserve, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, 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 borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinyl pyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; 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 polyvinyl pyrrolidone ); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan, polysorbates (such as polysorbate 20, polysorbate), tritium, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity 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 ed. (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0044] The selection of specific formulation materials described herein can be driven by, for example, the intended route of administration, delivery form, 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. For example, suitable vehicles or carriers can be water for injection, saline solution, or artificial cerebrospinal fluid, possibly supplemented with other substances common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. In specific embodiments, the pharmaceutical composition contains a Tris buffer at a pH of about 7.0 - 8.5, or an acetate buffer at a pH of about 4.0 - 5.5, and can further include sorbitol or a suitable alternative. In certain embodiments, the composition can be prepared for storage by mixing the selected components of the desired purity with optional formulations (REMINGTON’S PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or an aqueous solution. Additionally, in some embodiments, an antibody or fragment can be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0045] The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Alternatively, the compositions can be selected for inhalation or delivery through 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 at concentrations acceptable at the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range from about 5 to about 8.

[0046] When considering parenteral administration, the therapeutic compositions for use in the present invention can be provided in a pyrogen-free, parenterally acceptable aqueous solution that contains 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 properly preserved sterile isotonic solution. In certain embodiments, an implantable drug delivery device can be used to introduce the desired antibody or fragment.

[0047] In some or any embodiments, the pharmaceutical composition described herein comprises 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 certain embodiments, the concentration of the calcium salt is 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 concentration of the calcium salt is not more than 11 mM, not more than 12 mM, not more than 13 mM, not more than 14 mM, not more than 15 mM, not more than 16 mM, not more than 17 mM, not more than 18 mM, not more than 19 mM, not more than 20 mM, not more than 21 mM, not more than 22 mM, not more than 23 mM, not more than 24 mM, or not more than 25 mM. Any range having a combination of the foregoing endpoints is contemplated including, but not limited to, from about 0.5 mM to about 10 mM, about 5 mM to about 10 mM, or about 5 mM to about 15 mM.

[0048] In some embodiments, the pharmaceutical composition comprises an acetate buffer (e.g., sodium acetate) having a concentration range 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 concentration of the acetate buffer is no greater than 10 mM, no greater than 15 mM, no greater than 20 mM, no greater than 25 mM, no greater than 30 mM, no greater than 35 mM, no greater than 40 mM, no greater than 45 mM, no greater than 50 mM, no greater than 55 mM, no greater than 60 mM, no greater than 65 mM, no greater than 70 mM, no greater than 75 mM, no greater than 80 mM, no greater than 85 mM, no greater than 90 mM, no greater than 95 mM, or no greater than 100 mM. Any range with a combination of the foregoing endpoints is contemplated, including but not limited to from about 5 mM to about 15 mM, or from about 5 mM to about 10 mM or from about 10 mM to about 25 mM. Preferably, the buffer is added to a concentration where the pH is maintained at about 5-6, or 5-5.5, or 4.5-5.5. In some embodiments, when the calcium salt of the formulation is calcium acetate, the total concentration of acetate is about 10 mM to about 55 mM, or about 20 mM to about 40 mM.

[0049] In some aspects, the pharmaceutical composition comprises acetate at a total concentration of at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, 45mM, or 50mM. In some embodiments, the concentration of acetate is no greater than about 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, or 90mM. Any range of combinations with the aforementioned endpoints is contemplated, including but not limited to: about 10mM to about 50mM, about 20mM to about 50mM, about 20mM to about 40mM, about 30mM to about 50mM, or about 30mM to about 75mM. In some embodiments, the calcium salt is calcium acetate, and the acetate buffer is sodium acetate. By way of 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 calcium cations), while a solution containing 10 mM sodium acetate will have 10 mM sodium cations and 10 mM acetate anions.

[0050] In some embodiments, the total concentration of ions (cations and anions) 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 concentration of ions is no greater than about 30 mM, no greater than about 35 mM, no greater than about 40 mM, no greater than about 45 mM, no greater than about 50 mM, no greater than about 55 mM, no greater than about 60 mM, no greater than about 65 mM, no greater than about 70 mM, no greater than about 75 mM, no greater than about 80 mM, no greater than about 85 mM, no greater than about 90 mM, no greater than about 95 mM, no greater than about 100 mM, no greater than about 110 mM, no greater than about 120 mM, no greater than about 130 mM, no greater than about 140 mM, no greater than about 150 mM, no greater than about 160 mM, no greater than about 170 mM, no greater than about 180 mM, no greater than about 190 mM, or no greater than about 200 mM. Any range having a combination of the foregoing endpoints is contemplated including, 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. By way of non-limiting example, a solution of 10 mM calcium acetate would have a total concentration of ions of 30 mM (10 mM cations and 20 mM anions).

[0051] In some or any embodiments, the pharmaceutical composition comprises a polyol. Polyol covers a class of excipients, which includes sugar (such as mannitol, sucrose, sorbitol) and other polyols (such as glycerol and propylene glycol). Exemplary polyols include but are not limited to propylene glycol, glycerol (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 etc. Higher sugars include but are not limited to dextran, propylene glycol or polyethylene glycol. Compared with non-reducing sugars, reducing sugars, such as fructose, maltose or galactose are more easily oxidized. Other examples of sugar alcohols are glucitol, maltitol, lactitol or isomaltulose. Other exemplary lyoprotectants include glycerol and gelatin, as well as 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 polyols selected from sugar alcohols and other linear polyols. Monosaccharide glycosides include compounds obtained by reducing disaccharides (e.g., lactose, maltose, lactulose and maltulose).

[0052] In some or any embodiments, the pharmaceutical composition comprises a polyol in a concentration range of from about 0% to about 40% w / v. In some or any embodiments, the composition comprises a polyol in 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 in a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9% to about 10% w / v. In some or any embodiments, the composition comprises a polyol in a concentration of about 2% to about 6% w / v. In some or any embodiments, the composition comprises a polyol in a concentration of about 4% w / v. In some or any embodiments, the composition comprises a polyol in about 6% w / v.

[0053] In some or any embodiments, the pharmaceutical composition comprises a surfactant. Exemplary surfactants include but are not limited to anionic surfactants, cationic surfactants, nonionic surfactants, zwitterionic surfactants and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include but are not limited to sodium lauryl sulfate, sodium dioctyl sulfonate and sodium dioctyl sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium lauryl sulfate, 1-octane sulfonic 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 cetyltrimethylammonium 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, the surfactant includes but is not limited to lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glyceryl monostearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80, soy 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.

[0054] Surfactant can be included in the composition alone or with a mixture of different proportions. In some or any embodiments, the composition comprises a concentration of about 0% 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 comprises a concentration of about 0.001% to about 0.5% w / v surfactant. In some or any embodiments, the composition comprises 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 comprises a surfactant at a concentration of about 0.01% to about 0.1% w / v.

[0055] In some or any embodiments, the pharmaceutical composition comprises 55 mM acetate, 13 mm calcium, 6.0% (w / v) sucrose, 0.006% (w / v) polysorbate 20, pH 5.2.

[0056] Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations that relate to antigen-binding proteins in a continuous or controlled delivery formulation. It is also known to those skilled in the art to prepare various other technologies (such as liposome carriers, bioerodible microparticles or porous beads and reservoir injection) that continue or control the delivery mode. Refer to, for example, International Patent Application No. PCT / US93 / 00829, which is incorporated herein by reference and describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. Sustained release formulations can include a semipermeable polymer matrix in the form of a molded article (e.g., film or microcapsule). Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 and European Patent Application Publication No. EP 058,481, each of which is incorporated herein by reference), copolymers of L-glutamic acid and γ-L-glutamic acid ethyl ester (Sidman et al., 1983, Biopolymers [Biopolymers] 2: 547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. [Journal of Biomedical Materials Research] 15: 167-277 and Langer, 1982, Chem. Tech. [Chemical Technology] 12: 98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D (-) -3-hydroxybutyric acid (European Patent Application Publication No. EP 133988). Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Application Nos. EP036676; EP088046 and EP143949, incorporated by reference.

[0057] Pharmaceutical compositions for in vivo administration are typically provided as sterile preparations. Sterilization can be accomplished by filtration through a sterile filter membrane. When the composition is freeze-dried, sterilization using the method can be performed before or after freeze-drying and reconstitution. Compositions for parenteral administration can be stored in a freeze-dried form or in a solution. Parenteral compositions are typically placed in a container with a sterile infusion port (e.g., an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle).

[0058] According to various embodiments of the present invention, free amino acids can be used in antibody or fragment formulations as bulking agents, stabilizers and antioxidants and other standard uses. Lysine, proline, serine and alanine can be used to stabilize the protein in the formulation. Glycine can be used for freeze drying to ensure the correct cake structure and characteristics. In both liquid formulations and freeze-dried formulations, arginine can be used to inhibit protein aggregation. Methionine can be used as an antioxidant.

[0059] Embodiments of antibody formulations may further comprise one or more antioxidants. By maintaining an appropriate level of ambient oxygen and temperature and avoiding exposure to light, harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent. Antioxidant excipients may also be used to prevent oxidative degradation of proteins. In this regard, useful antioxidants are reducing agents, oxygen / free radical scavengers and chelating agents. The antioxidants used in therapeutic protein formulations according to the present invention are preferably water soluble and maintain their activity over the shelf life of the whole product. In this regard, EDTA is a preferred antioxidant according to the present invention.

[0060] The formulations according to the invention may contain metal ions which are protein cofactors and are necessary for the formation of protein coordination complexes, such as zinc which is necessary for the formation of certain insulin suspensions. Metal ions may also inhibit some processes that degrade proteins. However, metal ions also catalyze physical and chemical processes that degrade proteins.

[0061] 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 DNAse. +2 , Mn +2 and Zn +2 Can destabilize recombinant human deoxyribonuclease (rhDNase). Similarly, Ca +2 and Sr +2 Can stabilize factor VIII, which can be absorbed by Mg +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 destabilized, and its aggregation can be caused by Al +3 Ions increase.

[0062] Embodiments of the antibody formulations may further comprise one or more preservatives.

[0063] Once the pharmaceutical composition is prepared, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal or as a dehydrated or lyophilized powder. Such preparations can be stored in a ready-to-use form or in a form (e.g., lyophilized form) to be reconstituted before administration. The present invention also provides a kit for producing a single-dose administration unit. The kit of the present invention can each contain a first container with dry protein and a second container with an aqueous preparation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0064] The therapeutically effective amount of the antibody-containing pharmaceutical composition to be used will depend, for example, on the therapeutic context and purpose. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will depend, in part, on the delivered molecule, one or more indications for which the antibody is 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.

[0065] stability

[0066] In the context of a composition comprising an antibody (or an antigen-binding fragment thereof), as used herein, the terms "stability" and "stable" refer to the resistance of the antibody (or an antigen-binding fragment thereof) in the composition to aggregation, degradation or fragmentation under given manufacturing, preparation, transportation and / or storage conditions. Antibody formulations comprising high stability exhibit enhanced reliability and safety and are therefore advantageous for clinical use.

[0067] The antibody stability in the composition is optionally assessed by checking the desired parameters (e.g., aggregation, degradation, chemical modification, etc.) of the antibody in the composition over time. In this regard, parameters are usually checked at the initial time point (T0) and the evaluation time point (T1), and the antibody is optionally exposed to any of a variety of environmental conditions at the same time, and compared. The initial time point can be, for example, the time when the antibody is first prepared in the composition or the quality is first checked (i.e., checking to determine whether the antibody composition meets the regulations or manufacturing specifications for aggregation or degradation). The initial time point can also be the time when the antibody is re-formulated in the composition (e.g., compared with the initial formulation, re-formulated with a higher or lower concentration). In various embodiments, the evaluation time point 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. The desired parameters (e.g., aggregation or degradation) of the antibodies or fragments thereof in the compositions can be evaluated under various storage conditions, such as temperature 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.), etc.

[0068] Exemplary methods for determining the degree, and / or type, and / or size of aggregation present in a composition comprising an antibody 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, endogenous 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 the molecules through a column filled with an appropriate resin, where larger molecules (e.g., aggregates) will elute before smaller molecules (e.g., monomers). Molecules are typically detected by UV absorbance at 280 nm, and molecules can be collected for further characterization. High pressure liquid chromatography columns are typically used for SEC analysis (HP-SEC). Alternatively, analytical ultracentrifugation (AUC) can be used. AUC is an orthogonal technique for determining the sedimentation coefficient of macromolecules in a liquid sample. Like SEC, AUC is capable of separating and detecting antibody fragments / aggregates from monomers, and further capable of providing information about molecular weight. Antibody aggregation in the composition can also be characterized by particle counter analysis using a Coulter counter, or by turbidity measurement using a nephelometer. 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. In addition, 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 the composition.

[0069] 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 on-line MS detection.

[0070] In various embodiments, less than 5% of the antibodies described herein in the composition are in aggregated form under the conditions of interest. For example, 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), less than 4%, or less than 3%, or less than 2%, or less than 1% of the antibodies in the composition are in aggregated form. In some embodiments, after storage at about 4°C for two weeks, less than 5% (or less than 4%, or less than 3%, or less than 2%, or less than 1%, or less) of the antibodies described herein in the composition are in aggregated form.

[0071] 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 antibodies in the composition are optionally present in a non-aggregated form (i.e., monomeric) after storage at -30°C, 4°C, 20°C, or 40°C for a period of 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 antibodies are present in a non-aggregated form in the composition after storage for two weeks at about 4°C. In some embodiments, at least 99% of the antibodies are present in a non-aggregated form in the composition after storage for two weeks at about 4°C for two weeks, and / or at least 95% of the antibodies are present in a non-aggregated form in the composition after storage for two weeks at 40°C.

[0072] In various embodiments, less than 5% of the antibody described herein in the composition is degraded.For example, less than 4% or less than 3% or less than 2% or less than 1% or less antibody in the composition is degraded under conditions of interest.For example, it is optionally stored in 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) of the composition of the time 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 is complete (that is, 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 antibodies are intact (i.e., non-degraded) after the composition is stored at about 4°C for a period of two weeks. In some embodiments, at least 99% of the antibodies remain intact when the composition is stored at about 4°C for two weeks, and / or at least 95% remain intact when the composition is stored at about 40°C for two weeks.

[0073] The functional or active stability of the antibody in the composition is also contemplated herein. Assays for detecting and / or quantifying, for example, the binding of the antibody to the target, or the neutralization of sclerostin are known in the art. Optionally, the antibody exhibits about 50%-100% activity under conditions of interest compared to the antibody activity at the initial time point. For example, the antibody maintains an activity level between about 60%-90% or 70%-80% compared to the activity at the initial time point. Therefore, the functional stability of the antibody includes at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% activity retention, and may include an activity measurement greater than 100%, such as 105%, 110%, 115%, 120%, 125% or 150% or more compared to the activity at the initial time point.

[0074] Viscosity

[0075] In some embodiments, the viscosity of a composition comprising one or more antibodies described herein is determined. As used herein, the term "viscosity" refers to "absolute viscosity". Absolute viscosity (sometimes referred to as dynamic or simple viscosity) is the product of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity x density). The dimension of kinematic viscosity is L. 2 / T, where L is the length and T is the time. Usually, kinematic viscosity is expressed in centistokes (cSt). The SI unit for 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.

[0076] The viscosity of the composition can be measured hours (e.g., 1-23 hours), days (e.g., 1-10 days), weeks (e.g., 1-5 weeks), months (e.g., 1-12 months), or years (e.g., 1-2 years, 1-3 years) after adding the antibody to the composition. Viscosity measurements can be performed at storage or administration temperatures (e.g., 2°C-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 1.5 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.

[0077] 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 these methods include, for example, using a capillary viscometer or a cone-plate rheometer. Any method can be used, provided that the same method is used to compare the test and reference formulations.

[0078] Treatment

[0079] The antibodies and pharmaceutical compositions described herein can be used to treat or prevent bone-related disorders, such as bone-related disorders associated with abnormal osteoblast or osteoclast activity. In some embodiments, the antibodies are administered to a subject suffering from a bone-related disease selected from the group consisting of: achondroplasia, clavicular dysplasia, enchondromatosis, fibrous dysplasia, Gaucher's Disease, hypophosphatemic rickets, Marfan syndrome, hereditary multiple exostoses, multiple neurofibromas, osteogenesis imperfecta, osteosclerosis, osteophytes, sclerotic lesions, pseudoarthrosis, suppurative osteomyelitis, periodontal disease, antiepileptic drug-induced bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism symptoms, weightlessness-induced bone loss, male osteoporosis, postmenopausal bone loss, osteoarthritis, osteoarthritis, bone infiltrative diseases, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease (juvenile Paget's disease, wax tear bone disease, metabolic bone disease, mastocytosis, sickle cell anemia / disease, bone loss due to organ transplantation, bone loss due to kidney transplantation, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile idiopathic arthritis, thalassemia, mucopolysaccharidosis, Fabry disease, Turner syndrome, Down syndrome, Klinefelter syndrome, leprosy, Perthe's disease, juvenile idiopathic scoliosis, infantile onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson's disease, ischemic bone diseases (such as Legg-Calve-Perthes disease) disease) and regional migration osteoporosis), depressed state, steroid-induced conditions, hormonal bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, nutritional disorders, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy-related bone loss, diabetes, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, genital dysfunction, immobilization or disusedisuse), reflex sympathetic dystrophy syndrome, localized osteoporosis, osteomalacia, bone loss associated with joint replacement, HIV-related bone loss, growth hormone-induced bone loss, cystic fibrosis-related bone loss, chemotherapy-related bone loss, tumor-induced bone loss, cancer-related bone loss, hormone-related bone loss, 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 skull bone loss, bone loss with age, facial bone loss with age, skull bone loss with age, jaw bone loss with age, skull bone loss with age, and bone loss associated with space travel.

[0080] In some embodiments, the antibodies described herein can be used to improve the results of plastic surgery, dental surgery, implant surgery, joint replacement, bone transplantation, bone plastic surgery and bone repair (such as fracture healing, nonunion healing, delayed union healing and facial reconstruction). Compositions comprising one or more antibodies can be administered before, during and / or after surgery, replacement, transplantation, surgical procedure or repair.

[0081] In some embodiments, the antibodies described herein can be used to treat any bone fracture that comprises a gap between two bone segments (e.g., a gap between two bone segments of at least about 1 mm). 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 about 5 mm to 1 cm, or up to 1 cm. The terms "bone gap defect" and "segmental bone defect" are used synonymously herein and refer to a gap between two bone segments (e.g., a gap of at least 1 mm).

[0082] Exemplary bone gap defects include, but are not limited to, comminuted fractures, non-union fractures, segmental bone defects, surgically created bone defects, surgically treated bone defects, and bone defects produced by traumatic injuries of bone or disease (including but not limited to arthritis, tumor removal (excision), or infection removal). In some or any embodiments, bone gap defects are produced by removing infected bone slices or removing cancer from bones due to bone cancer, including but not limited to osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma, and chordoma. In some or any embodiments, bone gap defects are developmental malformations, for example, due to genetic defects.

[0083] In some or any embodiments, the bone gap defect is created by removing a bone section 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, unilocular bone cyst, fibrous dysplasia of bone, and giant cell tumor of bone.

[0084] Administration of antibodies enhances or accelerates the healing of bone gap defects, thereby "treating" bone gap defects. "Enhancing" bone healing means mediating a bone healing level that exceeds (i.e., is greater than) the following: the bone healing level experienced in subjects (e.g., mammals, such as humans, i.e., control subjects) to which sclerostin inhibitors are not administered. Bone healing can be demonstrated by, for example, bridging status, improved bone mass, improved bone mineral content, and density within the fracture gap (i.e., the formation of bridging bone), mature callus, improved bone strength (optionally accompanied by a medically acceptable bone hardness level), or improved patient use of the affected area. "Improved" means an increase or decrease in a measured parameter (as needed). The increase can be a measured parameter that is returned in whole or in part to a baseline level (e.g., a level before a bone gap defect), returned to a value provided in a standard database used in the art, or returned to a contralateral functional level (e.g., returned in whole or in part to, for example, the functional performance of the contralateral limb). In some cases, the increase can be an improvement beyond the baseline level. If desired, measured parameters in patients administered one or more doses of the antibody can be compared to the same parameters in fracture patients (optionally age and sex matched) who were not administered the antibody to further analyze the efficacy of the methods described herein.

[0085] The formation of bridging bone, bone mineral content, and bone density, and / or mature callus at a bone defect site can be measured using radiography (e.g., radiographic absorptiometry), single-energy and / or dual-energy X-ray absorptiometry, quantitative computed tomography (QCT), ultrasonography, radiography (e.g., radiographic absorptiometry), and magnetic resonance imaging. In some embodiments, the antibody can be administered for a period of time at a dose effective to increase bridging bone formation, callus formation, or bone density (or volume) at a bone defect site by at least about 5% (about 6%, about 7%, about 8%, or about 9%). In some embodiments, bridging bone formation, callus formation, or bone density at a bone defect site increases 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 bone density of the bridging bone formation, callus formation, or bone defect site is increased by at least about 25% (e.g., at least about 26%, or at least about 28%) by a sclerostin inhibitor. In still other embodiments, the bone density of the bridging bone formation, callus formation, or bone defect site is increased 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 remodeling of bridging bone formation can be determined 1 week, 2 weeks, 3 weeks, or 4 weeks after the initial administration of the antibody. Alternatively, the bone density level 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). On the one hand, the method reduces the amount of time required to establish a desired level of bone formation, bone mass, callus or bone density (e.g., any percentage increase in bone formation, bone mineral density, callus or bone mass described herein) compared to an age and sex matched patient who did not receive the antibody, thereby reducing the recovery time of the subject. For example, in one embodiment, the antibody reduces the amount of time required to increase the bone density or volume of the defect 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%).

[0086] The antibody does not need to cure the subject's disorder or completely prevent the onset of a bone-related disorder to achieve a beneficial biological response. The antibody can be used prophylactically, meaning that all or part of the protection is from a bone-related disorder or its symptoms. The antibody can also be used therapeutically to improve a bone-related disorder or its symptoms in whole or in part, or to protect the further development of a bone-related disorder or its symptoms in whole or in part. In fact, the materials and methods of the present invention are particularly suitable for increasing bone mineral density, and optionally maintain increased bone mineral density over a period of time.

[0087] In some embodiments, one or more administrations of an antibody described herein are performed 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, one or more doses of an antibody described herein are administered to a subject for 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).

[0088] In addition, depending on the treatment regimen selected for a particular subject, it may be advantageous to give multiple doses of antibody or to space out the dosage. In certain embodiments, antibody or its fragment is regularly administered in a period of one 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, antibody or its fragment is administered to the human race once every about 3 days, or about 7 days, or 2 weeks, or 3 weeks, or 4 weeks, or 5 weeks, or 6 weeks, or 7 weeks, or 8 weeks, or 9 weeks, or 10 weeks, or 11 weeks, or 12 weeks, or 13 weeks, or 14 weeks, or 15 weeks, or 16 weeks, or 17 weeks, or 18 weeks, or 19 weeks, or 20 weeks, or 21 weeks, or 22 weeks, or 23 weeks, or 6 months, or 12 months.

[0089] In some embodiments, one or more doses of the antibody are administered in an amount and duration effective to increase bone mineral density or treat a bone disorder associated with reduced bone mineral density. In various embodiments, one or more doses of the antibody are administered weekly to a subject (e.g., a human subject) comprising from about 50 mg to about 1,000 mg. For example, a dose of the antibody can contain at least about 5 mg, 15 mg, 25 mg, 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, about 150 mg, about 200 mg, about 210 mg, about 240 mg, about 250 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 420 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or up to about 1,000 mg of the antibody. Also contemplated are ranges between any and all of these key points, such as 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, or about 150 mg to about 200 mg, or about 180 mg to about 270 mg, or about 280 to about 410 mg. Dosages are given at any interval, such as multiple times per week (e.g., twice or three times per week), once a week, once every two weeks, once every three weeks, or once every four weeks. In some or any embodiments, a dosage of about 120 mg to about 210 mg of an antibody is given twice per week. In some or any embodiments, a dosage of about 140 mg of an antibody is given twice per week. In various aspects, a dosage of about 210 mg of an antibody is given once a month.

[0090] In some embodiments, one or more doses of an antibody can include between about 0.1 to about 50 mg (e.g., between about 5 and about 50 mg), or about 1 to about 100 mg of antibody per kilogram of body weight (mg / kg). For example, a dose of an antibody can include at least about 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, about 30 mg / kg, about 31 mg / kg, about 32 mg / kg, about 33 mg / kg, about 34 mg / kg, about 35 mg / kg, about 36 mg / kg / kg, about 37mg / kg, about 38mg / kg, about 39mg / kg, about 40mg / kg, about 41mg / kg, about 42mg / kg, about 43mg / kg, about 44mg / kg, about 45mg / kg, about 46mg / kg, about 47mg / kg, about 48mg / kg, or about 49mg / kg, or about 50mg / kg, about 55mg / kg, about 60mg / kg, about 65mg / kg, about 70mg / kg, about 75mg / kg, about 80mg / kg, about 85mg / kg, about 90mg / kg, about 95mg / kg, or up to about 100mg / kg. Also contemplated are ranges between any and all of these endpoints, for example, 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.

[0091] Monitoring therapy

[0092] The antibody-mediated increase in bone mineral content or bone density can be measured using single-energy and dual-energy X-ray absorptiometry, ultrasound, computed tomography, radiography, and magnetic resonance imaging. The amount of bone mass can also be calculated by body weight or by using other methods (see, Guinness-Hey, Metab. Bone Dis. Relat. Res. [Metabolic bone disease and related research], 5: 177-181 (1984)). Animal models are used in the art to test the effects of pharmaceutical compositions and methods on parameters such as bone loss, bone resorption, bone formation, bone strength, or bone mineralization, which simulate the conditions of human diseases, such as osteoporosis and osteopenia. 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 described herein for measuring antibody activity can also be used to determine the efficacy of other sclerostin inhibitors.

[0093] In humans, bone mineral density can be determined clinically using, for example, dual X-ray absorptiometry (DXA) of the hip and spine. Other techniques include quantitative computed tomography (QCT), ultrasonography, single-energy X-ray absorptiometry (SXA), and radiographic absorptiometry. Common central skeletal sites for measurement include the spine and hip; peripheral sites include the forearm, fingers, wrist, and heel. In addition to ultrasonography, the American Medical Association states that BMD techniques typically involve the use of X-rays and are based on the principle that radiation attenuation depends on the thickness and composition of tissues in the radiation path. All techniques involve comparing the results to a normative database.

[0094] Alternatively, the physiological response to one or more anti-sclerostin antibodies can be measured by monitoring bone marker levels. Bone markers are products produced during bone remodeling and are released by bone, osteoblasts, and / or osteoclasts. Fluctuations in bone resorption and / or bone formation "marker" levels indicate changes in bone remodeling / modeling. The International Osteoporosis Foundation (IOF) recommends the use of bone markers to monitor bone density therapy (see, e.g., Delmas et al., Osteoporos Int. [International Osteoporosis], Suppl 6:S2-17 (2000), incorporated by reference). Markers indicating bone resorption (or osteoclast activity) include, for example, C-terminal peptides (e.g., C-terminal telopeptide (CTX) of type 1 collagen or serum cross-linked C-terminal peptides), N-terminal peptides (N-terminal telopeptide (NTX) of type 1 collagen), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline, galactosylhydroxylysine, and tartrate-resistant acid phosphatase (e.g., tartrate-resistant acid phosphatase isoform 5b). Bone formation / mineralization markers include, but are not limited to, bone-specific alkaline phosphatase (BSAP), peptides released from the N-terminal and C-terminal extensions of type I procollagen (P1NP, PICP), and osteocalcin (OstCa). Several kits are commercially available to detect and quantify markers in clinical samples (such as urine and blood).

[0095] Combination therapy

[0096] Treating pathology by combining two or more agents targeting the same pathogen or biochemical pathway or biological process sometimes results in greater efficacy and reduced side effects relative to each agent at a therapeutically relevant dose used alone. In some cases, the efficacy of the drug combination is additive (the efficacy of the combination is approximately equal to the sum of the effects of each drug alone), but in other cases, the effect is synergistic (the efficacy 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 agents are delivered in a simultaneous manner (e.g., simultaneously), or wherein one agent is first administered and then a second agent is administered, e.g., sequentially.

[0097] In some embodiments, the antibody is administered together with a standard care therapeutic agent for treating reduced bone mineral density (i.e., the antibody and the standard care therapeutic agent are part of the same treatment plan). As used herein, the term "standard care" refers to the treatment generally accepted by clinicians for a certain class of patients diagnosed with a certain disease. In some embodiments, the antibody is administered together with a second bone enhancer for treating reduced bone mineral density or bone defects. In some embodiments, the bone enhancer is selected from the group consisting of: antiresorptive agent, bone forming agent (i.e., anabolic agent), estrogen receptor modulator (including but not limited to raloxifene, bazedoxifene and lasofoxifene) and medicines with inhibitory effects on osteoclasts. In some embodiments, the second bone enhancer is selected from the group consisting of: bisphosphonates (including but not limited to alendronate sodium) Risedronate, ibandronate sodium and zoledronic acid ); estrogen or estrogen analogs; anti-RANK ligand (RANKL) inhibitors, such as anti-RANKL antibodies (e.g., denosumab, ); vitamin D, or vitamin D derivatives, or mimetics thereof; 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 enhancing agent includes, but is not limited to, parathyroid hormone (PTH) or a peptide fragment thereof, a PTH-related protein (PTHrp), a bone morphogenetic protein, an osteogenic agent, NaF, a PGE2 agonist, a statin, strontium ranelate, and a sclerostin inhibitor (e.g., an anti-sclerostin antibody described in U.S. Pat. Nos. 7,592,429 or 7,872,106). In some embodiments, the second bone enhancing agent is (teriparatide), In some embodiments, the second absorbent comprises a bone morphogenic 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).

[0098] In some embodiments, the combination therapy using antibodies described herein can be performed before or after giving one or more other therapeutic agents (e.g., a second bone strengthening agent) at intervals ranging from several minutes to several weeks to several months. For example, within about 24 hours of each other, for example, 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, a separate pattern is given. In some cases, it may be necessary to significantly extend the treatment time, wherein several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) fail between each administration of different patterns. It is particularly contemplated that repeated treatment using one or two agents / therapies of the combination therapy are used.

[0099] Maintenance treatment regimen

[0100] It is also contemplated to use a second bone enhancing agent and / or the antibodies described herein in a maintenance regimen, for example to prevent or slow the loss of bone mineral density. In this regard, the methods or uses described herein optionally include administering one or more amounts of a second bone enhancing agent that is effective to maintain bone mineral density for a maintenance period of about 1 week to about 5 years after the treatment period of the antibody ends. For example, in some embodiments, the methods or uses described herein comprise administering to the subject a second bone enhancing agent for about at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 4 months, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 5 months, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 6 months, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 7 months, about 29 weeks, about 30 weeks, about 31 weeks, or longer (e.g., about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 15 months ... In some embodiments, the maintenance period is about 18 months, about 2 years, about 3 years, about 4 years, about 5 years or more (e.g., over the lifetime of the subject). 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. In 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 bone mineral density parameters at similar levels experienced in subjects receiving antibody treatment.

[0101] Medicine Box

[0102] Pharmaceutical compositions comprising one or more antibodies described herein can be placed in a container (e.g., a vial or syringe) along with packaging materials that provide instructions for using such pharmaceutical compositions. Typically, such instructions will include a description of the antibody concentration and, in certain embodiments, a clear expression of the relative amounts of excipient ingredients or diluents (e.g., water, saline, or PBS) that may be necessary to reconstitute the pharmaceutical composition. Examples Example 1 - Analysis of Lomosozumab PARG C-terminal variants

[0103] Wild-type lomosozumab and the lomosozumab PARG C-terminal variant were digested by Lys-C and analyzed by LC / MS peptide mapping. The UV spectra of these two constructs were compared side by side ( Figure 3 ). It was determined that wild-type lomosozumab and lomosozumab PARG C-terminal variant had similar peaks eluting at 37.7 minutes, but it was determined that wild-type lomosozumab had a mass of 659.3 Da, while it was determined that lomosozumab PARG C-terminal variant had a mass of 886.7 Da. It is believed that most of the lysine (K) variants of lomosozumab (PGK) were removed from the process. The presence of a significant amount of the amidated form of the lomosozumab PARG C-terminal variant (828.6 Da peak) when compared to the wild-type lomosozumab PG sequence confirms that the amidation efficiency is sequence dependent.

[0104] The PARG C-terminal variant was then treated with carboxypeptidase (CP-B) and analyzed by CEX-HPLC method and compared with the PARG C-terminal variant control (which was not treated with CP-B). The peaks eluting at 17.5 min and 21 min were significantly shifted after treatment, but there was no shift for the peak at 24 min ( Figure 4 ). The 24 min peak is expected to be the doubly amidated form, which is protected from proteolytic degradation. Example 2 - C-terminal variant enrichment

[0105] The purification and enrichment of different lomosozumab species from a composition comprising wild-type lomosozumab and lomosozumab PARG C-terminal variants was achieved by cation exchange chromatography (CEX) classification. CEX separates proteins based on differences in surface charge. At a set pH, positively charged variants of wild-type lomosozumab are separated on a cation exchange column (e.g., Dionex Pro Pac WCX-10 analytical column, 2.0mm x250mm) and eluted using a salt gradient (e.g., mobile phase A: 10:90 (v / v) ACN, 19mM MES pH 6.2; mobile phase B: 10:90 (v / v) ACN, 19mM MES, 250mM NaCl, pH 6.2). Different C-terminal variants of lomosozumab are charged differently, and more positively charged variants are subsequently eluted in CEX. Thus, the elution order is: PG (wild type), P-amide (amidated wild type proline), PARG variant, and PAR-amide. The fraction collector can be programmed to collect CEX eluates containing different variants at different elution times. Example 3 - Analysis of aggregation of LOMOZOLZUMAB PARG C-terminal variants

[0106] Without being bound by any particular theory, it is expected that because the PARG C-terminal variants are highly charged, such forms will exclude the non-amidated forms in the composition, thereby reducing aggregation in the composition.

[0107] The lomosozumab PARG C-terminal variant Protein A pool was analyzed side by side with the wild-type lomosozumab Protein A pool using SEC-HPLC, a size exclusion HPLC method that separates proteins based on differences in their hydrodynamic volumes (Table 1).

[0108] Table 1 molecular %HMW AMG785ARG ProA Pool 3.4％ AMG785WT ProA pool 7.2％

[0109] The data showed that the LOMOZOZUMAB PARG C-terminal variant had less high molecular weight species compared to wild-type LOMOZOZUMAB. Example 4 - Viscosity analysis of lomosozumab PARGC terminal variants

[0110] Cone plate is used to analyze the antibody solution containing lomosozumab PARG C-terminal variant or wild-type lomosozumab. According to the approximate volume consumption, the solution is concentrated to 120mg / mL, and the protein absorbance at 280nm (finally in 0.1-1 absorbance unit (AU) after dilution) and protein-specific extinction coefficient are used to measure the final concentration (± 10%). Using CP-40 axis (spindle) and sample cup or ARES-G2 ammeter (thermal analysis instrument company (TAInstruments), Newcastle, Delaware, USA), TA Smart Swap 2 degree cone / plate axis is used to carry out viscosity analysis on BrookfieldLV-DVIII cone plate instrument (Brookfield Engineering, Boro, Massachusetts, USA). All measurements are carried out at 25 ℃ and controlled by a water bath attached to the sample cup. By increasing the RPM of the axis, multiple viscosity measurements are manually collected in the torque range (10%-90%) limited. The measurements were averaged so that one viscosity value was reported for each sample to simplify the resulting comparative charts. Example 5 - Solubility analysis of mosozumab PARG C-terminal variants

[0111] In order to determine the effect of subcutaneous (SC) injection of amino acid variation of lomosozumab PARG variants on solubility compared with wild-type lomosozumab, dialysis solubility assays were performed in parallel in wild-type and PARG-C terminal variant lomosozumab. This screening requires that samples of lomosozumab PARG C terminal variants and samples of wild-type lomosozumab be dialyzed into a solution of pH and ionic strength simulating SC space, and monitor the solubility and physical stability of the antibody under these conditions in a very short time. Samples are formulated into approximately 63mg / mL in formulation buffer (pH 5.2). Each sample is then injected into a dialysis cassette and dialyzed into PBS buffer to simulate SC space. Visual inspection was performed 24 hours after the initial dialysis. Wild-type lomosozumab usually shows precipitation after 24 hours.

[0112] The results showed that both molecules precipitated in this assay, but the PARG C-terminal variant precipitated less and at a slower rate. This suggests that the variant is more resistant to precipitation than the wild type, although the variant cannot completely eliminate precipitation. Example 6 - Diffusion analysis of lomosozumab PARGC terminal variants

[0113] To determine the effect of amino acid variations in the lomosozumab PARG C-terminal variants on diffusion from the subcutaneous (SC) space compared to wild-type lomosozumab, an assay was performed using Scissor (Pion Inc. (Pion Inc.), Billerica, MA). This assay requires the injection of approximately 70 mg / mL of sample (lomosozumab PARG C-terminal variant or wild-type lomosozumab) into a simulated SC space (composed of a collagen and hyaluronic acid matrix). The antibody is able to diffuse out of the matrix through a dialysis membrane into a reservoir of carbonate buffer at pH 7.4. Time points were collected for up to 3 days, and the protein concentration at each time point was determined by RP-HPLC. The resulting protein concentration versus time curve simulates the diffusion rate from the SC space. In addition, precipitation in the SC matrix was monitored by visual inspection.

[0114] As described above, both wild-type and PARG C-terminal variant lomosozumab were tested in Scissor. Figure 5 The results shown in indicate that wild-type lomosozumab diffuses from the simulated SC space at a much lower rate and retains more wild-type lomosozumab than the PARG C-terminal variant lomosozumab at the simulated injection site. Example 7 - FcRn binding

[0115] FcRn (neonatal Fc receptor) is an MHC class I-like heterodimer composed of a transmembrane α chain (homologous to MHC class I-like molecules) and a β2 microglobulin light chain. FcRn binds to the C chain of the IgG heavy chain in the Fc region of the IgG molecule under mildly acidic conditions (approximately pH 6). H 2 and C H The FcRn domains bind to the IgG3 domain interface and are released at neutral pH (approximately 7.4). Through this highly pH-dependent interaction, FcRn mediates IgG homeostasis in adults by maintaining serum IgG levels.

[0116] Using competitive binding assay ( The binding assay (PerkinElmer, San Jose, CA) evaluates the binding of the Fc domain of wild-type lomosozumab and the lomosozumab PARG C-terminal variant to FcRn. 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 thiophene 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 phthalocyanine, a photosensitizer, and streptavidin, which binds to biotinylated CHO-derived human Fc. When FcRn-His and biotinylated human Fc bind together, they bring the acceptor and donor beads into close proximity. When laser light is applied to this complex, ambient oxygen is converted to singlet oxygen by the donor beads. If the beads are in close proximity, energy transfer occurs to the acceptor bead, which results in the generation of light (luminescence), which is the result of the device Signal detection is measured in a plate reader.

[0117] When the antibody is present at a sufficient concentration to inhibit binding of FcRn-His to the biotinylated human Fc domain, a dose-dependent decrease in emission at 570 nm is observed. Test sample binding relative to the antibody reference standard is determined and reported as % relative binding and can be used to demonstrate the integrity of the Fc domain of the antibody. It is expected that compositions with PARG C-terminal variants will have a dose response curve similar to or better than the wild-type antibody.

[0118] The results are shown in Figure 6 It was observed that both wild-type lomosozumab and the PARG C-terminal variant lomosozumab bound FcRn similarly, and FcRn binding was not affected by the PARG mutation. Example 8 - FcγRIIa binding

[0119] 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, with two extracellular Ig-like domains and low binding affinity to monomeric IgG. There are two common allelic variants in humans, which are known to exist in FcγRIIa, expressing histidine or arginine at position 131 (131H and 131R, respectively).

[0120] A competitive binding assay was developed to evaluate the binding of wild-type lomosozumab and lomosozumab PARG C-terminal variants to FcγRIIa(131H). The FcγRIIa(131H) binding assay is a bead-based amplified luminescent proximity homogeneous assay ( Binding assay (PerkinElmer, San Jose, CA) that detects bimolecular interactions. The assay contains two bead types, acceptor beads and donor beads. The acceptor beads contain a fluorophore europium chelate and are coated with a hydrogel containing glutathione, which binds recombinant human FcγRIIa(131H)-glutathione-S-transferase (FcγRIIa(131H)-GST). The donor beads are coated with a hydrogel containing phthalocyanine, a photosensitizer, and streptavidin, which binds to biotinylated human IgG1. When FcγRIIa(131H)-GST and biotinylated human IgG1 bind together, they bring the acceptor and donor beads into proximity. When a laser is applied to this complex, ambient oxygen is converted to singlet oxygen by the donor beads. When the acceptor and donor beads are in proximity, singlet oxygen diffuses within the acceptor beads, which results in light generation (luminescence), which is measured in a plate reader equipped for luminescent signal detection.

[0121] When the antibody is present at a sufficient concentration to inhibit the binding of FcγRIIa(131H)-GST to biotinylated human IgG1, a dose-dependent decrease in emission at 570 nm is measured. Test sample binding relative to the antibody reference standard is determined and reported as % relative binding and can be used to demonstrate the integrity of the Fc domain of the antibody. The results are shown in Figure 7 It was observed that the relative binding of the PARG C-terminal variant lomosozumab to FcγRIIa(131H) was much higher than that of wild-type lomosozumab. Example 9 - Pharmacokinetic Study in Mice

[0122] To evaluate in vivo drug exposure and bioavailability, a single-dose pharmacokinetic study was performed in mice. The lomosozumab PARG C-terminal variant was injected intravenously (via the tail vein) or subcutaneously at a dose of 1 mg / kg. Nine animals were used in each group, and staggered sampling allowed data to be collected at a large number of time points without exceeding the maximum amount of blood that could be drawn from a single animal. At each time point, 0.05 ml of blood was drawn. Animals 1 to 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 the concentration of the test substance was determined by combining immunoassays such as ELISA (enzyme-linked immunosorbent assay). Changes in the concentration of the test article over time can be used to calculate pharmacokinetic parameters by two-compartment analysis. Parameters of interest include, but are not limited to, the area under the plasma concentration-time curve (AUC) and the half-life (t 1 / 2 ) and clearance (CL). Bioavailability can be determined as the ratio of the AUC of a subcutaneous dose to the AUC of an intravenous dose.

Claims

1. An antibody that specifically binds to sclerostin of SEQ ID NO: 1 and comprises a set of six CDRs shown in SEQ ID NO: 2-7, wherein the antibody comprises a heavy chain comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain.

2. The antibody of claim 1, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

10.

3. The antibody of claim 1 or claim 2, wherein the C-terminus of the heavy chain is amidated.

4. The antibody of any one of claims 1 to 3, wherein the C-termini of both heavy chains comprise the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8). The antibody of claim 4 , wherein the C-termini of both heavy chains are amidated.

6. The antibody according to any one of claims 1 to 5, 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:

13.

7. The antibody of claim 1 or claim 2, wherein the antibody comprises an amino acid sequence comprising Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the heavy chain.

8. The antibody according to claim 7, 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 a population of antibodies according to any one of claims 1 to 8, 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; Wherein about 3%-5% of the antibodies in the composition are the population of antibodies as described in any one of claims 1-8.

11. The pharmaceutical composition of claim 9 or claim 10, wherein all or part of the antibody population comprises a single heavy chain comprising a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence.

12. The pharmaceutical composition of claim 9 or claim 10, wherein all or part 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 of claim 12, wherein less than about 35% of the population of antibodies are monoamidated.

14. The pharmaceutical composition of claim 10, wherein all or part of the population of antibodies comprises a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence in both heavy chains.

15. The pharmaceutical composition of claim 14, wherein all or part of the antibody population comprises two heavy chains, both of which comprise an amidated C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence on both heavy chains.

16. The pharmaceutical composition of claim 15, wherein less than about 35% of the population of antibodies are amidated on both heavy chains.

17. The pharmaceutical composition of any one of claims 9-16, wherein less than about 35% of the population of antibodies comprises a heavy chain that is not amidated.

18. The pharmaceutical composition of any one of claims 9-17, wherein about 33% of the population of antibodies are not amidated, about 33% of the population of antibodies comprise one amidated heavy chain, and about 33% of the population of antibodies comprise two amidated heavy chains.

19. The pharmaceutical composition of any one of claims 9-18, further comprising a calcium salt, an acetate buffer, a polyol, and a surfactant.

20. The pharmaceutical composition of claim 19, wherein the calcium salt comprises calcium acetate.

21. The pharmaceutical composition of claim 19, wherein the acetate buffer comprises sodium acetate.

22. The pharmaceutical composition of claim 19, wherein the polyol comprises sucrose.

23. The pharmaceutical composition of claim 19, wherein the surfactant comprises polysorbate 20.

24. The pharmaceutical composition of any one of claims 9 to 23, further comprising 55 mM acetate, 13 mm calcium, 6.0% (w / v) sucrose, 0.006% (w / v) polysorbate 20, pH 5.

2.

25. A method of increasing bone mineral density in a subject in need thereof, the method comprising administering to the subject a composition of any one of claims 9-24 in an amount effective to increase bone mineral density in the subject.

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