BRPI0817879B1 - ISOLATED ANTIBODIES AGAINST SCLEROSTIN, THEIR USES, AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

ANTIBODY AGAINST SCLEROSTIN OR FUNCTIONAL PROTEIN, USE AND PHARMACEUTICAL COMPOSITION COMPRISING THEM, POLYNUCLEOTIDE SEQUENCE, CLONING OR EXPRESSION VECTOR COMPRISING SAID POLYNUCLEOTIDE SEQUENCE, RECOMBINANT HOST CELL COMPRISING SAID CLONING OR EXPRESSION VECTOR, PROCESS FOR PRODUCING AN ANTIBODY OR FUNCTIONAL PROTEIN, DIAGNOSTIC KIT AND METHOD FOR IDENTIFYING A CELL OR TISSUE EXPRESSING SCLEROSTIN IN VITRO. The present invention relates to antibodies against sclerostin and compositions and methods of use for said antibodies for the treatment of diseases related to bone abnormalities such as, for example, osteoporosis.

Description

TECHNICAL FIELD

[001] The present invention relates to antibodies against sclerostin and compositions and methods of use for said antibodies for the treatment of a pathological disorder that is mediated by sclerostin or disease related to bone abnormalities such as, for example, osteoporosis.

FUNDAMENTALS OF THE INVENTION

[002] The SOST gene encodes the sclerostin protein, which is a secreted glycoprotein of 213 amino acids. Sclerostin is a member of the cystine knot-containing factor superfamily. Sclerostin is related to the DAN/Cerberus protein family, which directly interferes with BMP signaling by inhibiting BMP binding to receptors and thus the BMP signaling cascade (Avsian-Kretchmer, Mol. Endocrinol. 2004, 18(1): 1-12).

[003] Sclerostin mRNA expression is detected in adult humans predominantly in bone and kidney. Sclerostin protein is detectable predominantly in bone. Within bone, its expression is restricted to mature, terminally differentiated bone-forming cells, osteocytes.

[004] Sclerostin is a potent negative regulator of bone formation in man and mice. Lack of SOST expression gives rise to sclerosteosis (Balemans et al. Hum. Mol. Genet., 2001, 10(5): 537-43; Brunkow et al. Am. J. Hum. Genet., 2001, 68(3): 577-89). Patients suffer from lifelong excessive bone growth, resulting in increased bone mineral density and strength. They exhibit no other endocrinological abnormalities - all complications they experience during their lifetime are related to abnormal bone accrual. Heterozygous carriers for this recessive disorder also exhibit increased bone mass (Gardner et al. J. Clin. Endocrinol. Metab., 2005, 90(12): 6,392-5). The phenotype can be recapitulated in SOST-deficient mice, and its overexpression results in osteopenia. Furthermore, Van Buchem disease [MIM 239100]—a phenotypic copy of sclerosteosis—is caused by dysregulation of SOST due to genomic deletion of a broad-spectrum bone enhancer (Balemans et al., J. Med. Gene, 2002, 39(2): 91-7; Loots et al., Genome Res., 2005, 15(7): 928-35). Finally, SOST is downregulated by parathyroid hormone (PTH)—a clinically validated bone-forming principle—during bone formation, suggesting that part of the anabolic action of PTH may be mediated through SOST (Keller and Kneissel Bone, 2005, 37(2): 148-58).

[005] Sclerostin binds to BMPs (bone morphogenic proteins) and can act as a BMP antagonist in vitro (Winkler et al. EMBO J., 2003, 22(23): 6267-76). Sclerostin also acts as a negative regulator of canonical Wnt signaling, either directly by binding to LRP5/LRP6 (Li et al. J. Biol. Chem., 2005, 20; 280(20); Semenov, J. Biol. Chem. 2006 Oct. 19; van Bezooijen et al. J. Bone Miner. Res., 2006 Oct. 10), or indirectly (Winkler et al. J. Biol. Chem., 2005, 28; 280(4): 2498502).

[006] Absence of sclerostin expression results in increased bone formation, while bone resorption is not affected (Sclerosteosis, Van Buchem disease) (Balemans et al. 2001; Brunkow et al. Am. J. Hum. Genet., 2001, 68(3): 577-89, Balemans et al. 2006; Loots et al., Genome Res., 2005, 15(7): 928-35).

[007] Few of the currently available treatments for skeletal disorders can increase bone density in adults, and most currently available treatments work primarily by inhibiting further bone resorption rather than by stimulating new bone formation.

[008] An example of a drug used for the treatment of bone loss is estrogen. However, it is unclear whether or not estrogen has any long-term beneficial effects. In addition, estrogen may carry the risk of increased prevalence of several types of tumors, such as breast and endometrial cancer. Other current therapeutic approaches for osteoporosis include bisphosphonates (e.g., FosamaxTM, ActonelTM, BonvivaTM, ZometaTM, olpadronate, neridronate, skelid, bonefos), parathyroid hormone, calcilytics, calcimimetics (e.g., cinacalcet), statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. These therapeutic substances, however, are often associated with undesirable side effects.

SUMMARY OF THE INVENTION

[009] One embodiment of the present invention provides an antibody or a functional protein comprising an antigen binding portion of said antibody for a target on the sclerostin polypeptide (SEQ ID NO: 155), characterized in that the antibody or functional protein specifically binds to the sclerostin polypeptide and can increase at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality, and bone strength in a mammal.

[0010] In one embodiment, the antibodies according to the invention have the ability to reverse sclerostin inhibition of bone mineralization in vitro. In a related embodiment, they have the ability to reverse sclerostin inhibition of the wnt-1-mediated signaling pathway. In another related embodiment, they disrupt sclerostin LRP6 binding and can block the inhibitory effect that sclerostin has at high doses on BMP-induced Smad1 phosphorylation. In another embodiment, antibodies of the invention bind to a region of sclerostin between amino acids 112 and 126, inclusive (i.e., said region consists of amino acids 112 to 126 of SEQ ID NO: 155) of SEQ ID NO: 155, and/or the region between amino acids 160-174, inclusive (i.e., said region consists of amino acids 160 to 174 of SEQ ID NO: 155) of SEQ ID NO: 155, and more specifically, bind to a region comprising both ARLLPNAIGRGKWWR (SEQ ID NO: 156) and RLVASCKCKRLTRFH (SEQ ID NO: 157).

[0011] Sclerostin inhibits wnt1-mediated activation of STF (Supertopflash, a reporter readout for canonical Wnt signaling) in HEK293 cells. In some embodiments, antibodies of the invention restore the reporter readout of Wnt signaling in a highly reproducible manner.

[0012] It has been demonstrated that the observed inhibitory effect of the antibodies according to the invention on the action of sclerostin in the Wnt signaling reporter assay in non-osteoblastic cells translates into induction of bone formation responses as a function of sclerostin inhibition in vivo. Indeed, in vivo experiments on aged rodents show that the antibodies according to the invention promote strong bone anabolism. The increase in bone mass reached the level of the effect of intermittent daily treatment with extremely high anabolic doses of parathyroid hormone (which was used as a positive control).

[0013] Therefore, according to another preferred embodiment, the antibodies according to the invention have affinities for sclerostin in the reduced pM range and inhibit the impact of sclerostin on wnt signaling with an IC50 of around 10 nM.

[0014] More preferably, in another preferred embodiment, the antibodies according to the invention bind to a region of sclerostin composed between amino acids 112 and 126, inclusive (i.e. said region consists of amino acids 112 to 126 of SEQ ID NO: 155) and between amino acids 160 and 174, inclusive (i.e. said region consists of amino acids 160 to 174 of SEQ ID NO: 155) of SEQ ID NO: 155 and, more specifically, a region that overlaps at least with the following peptides ARLLPNAIGRGKWWR (SEQ ID NO: 156) and RLVASCKCKRLTRFH (SEQ ID NO: 157), respectively, and have affinities for sclerostin in the low pM range and inhibit the impact of sclerostin on wnt signaling with an IC50 of around 10 nM. These antibodies have the ability to increase bone mass in the axial and appendicular skeleton of a mouse animal model at the level of the effect of daily subcutaneous treatment with an extremely high anabolic dose of parathyroid hormone (positive control) and are therefore useful in the treatment of diseases related to bone abnormalities such as osteoporosis.

[0015] Additional embodiments include compositions comprising the antibodies of the invention in combination with alternative therapies for the treatment of osteoporosis such as, for example, bisphosphonates, parathyroid hormone, parathyroid hormone releasing agents (calcilytics), LRP4 neutralizing antibodies, and DKK-1 neutralizing antibodies.

BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1: Effect of MOR05813_IgG2lambda on the dewnt-1 assay.

[0017] Figure 2: MOR05813_IgG2lambda in BMP-2-induced mineralization in MC3T3-1b cells.

[0018] Figure 3: Effect of MOR05813_IgG2lambda on LRP6-SOST ELISA.

[0019] Figure 4: Effect of MOR05813_IgG2lambda on the Phospho-Smad1 assay.

[0020] Figure 5: A - Effect of LRP4 knockdown (siRNA) on the inhibitory action of SOST in the wnt-1 assay in Hek293 cells (Black numbers: relative to STF activities in the absence of SOST, Bold black numbers: proportion of STF activities in the presence/absence of SOST); B - Specificity of the effect of LRP4 overexpression on the IC50 of SOST and IC50 of Dkk1 in the wnt-1 assay in Hek293 cells; C - Specificity of the effect of LRP4 overexpression on the inhibitory action of SOST and Dkk1 in the wnt-1 assay in C28a2 cells; D - Specificity of the effect of LRP4 knockdown (siRNA) on the inhibitory action of SOST and Dkk1 in the wnt-1 assay in Hek293 cells; E - Modulation of MOR05813 activity by LRP4.

[0021] Figure 6: Mouse study, in vivo pQCT - 2.5-week treatment with MOR05813 increases total bone mineral content in the proximal tibial metaphysis.

[0022] Figure 7: Mouse study, in vivo pQCT - 2.5-week treatment with MOR05813 increases total bone mineral density in the proximal tibial metaphysis.

[0023] Figure 8: Mouse study, in vivo pQCT - 2.5-week treatment with MOR05813 increases cortical thickness in the proximal tibial metaphysis.

[0024] Figure 9: Mouse study, in vivo uQCT - 2.5-week treatment with MOR05813 increases cancellous bone volume in the proximal tibial metaphysis.

[0025] Figure 10: Mouse study, in vivo uQCT - 2.5-week treatment with MOR05813 increases trabecular thickness in the proximal tibial metaphysis.

[0026] Figure 11: Mouse study, in vivo pQCT - 5-week treatment with MOR05813 increases total bone mineral density further in the proximal tibial metaphysis.

[0027] Figure 12: Mouse study, ex vivo DEXA - 5-week treatment with MOR05813 increases bone mineral density further in the tibia.

[0028] Figure 13: Mouse study, ex vivo DEXA - 5-week treatment with MOR05813 increases bone mineral density further in the femur.

[0029] Figure 14: Mouse study, ex vivo DEXA - 5-week treatment with MOR05813 increases bone mineral density further in the spine.

[0030] Figure 15: Mouse study, ex vivo histomorphometry - 2.5-week treatment with MOR05813 increases bone formation rates in the appendicular skeleton (distal femur metaphysis).

[0031] Figure 16: Mouse study, ex vivo histomorphometry - 2.5-week treatment with MOR05813 increases the rate of mineral apposition in the appendicular skeleton (distal femoral metaphysis).

[0032] Figure 17: Mouse study, ex vivo histomorphometry - 2.5-week treatment with MOR05813 increases the mineralization surface in the appendicular skeleton (distal femoral metaphysis).

[0033] Figure 18: Mouse study, ex vivo histomorphometry - 2.5-week treatment with MOR05813 increases bone formation rates in the axial skeleton (lumbar vertebra).

[0034] Figure 19: Mouse study, ex vivo histomorphometry - 2.5-week treatment with MOR05813 does not affect bone resorption in the appendicular skeleton (distal femoral metaphysis), assessed by osteoclast surface.

[0035] Figure 20: ELISA showing the effect of MOR05813_IgG2lambda on SOST binding of LRP6. 0.9 nM SOST was used in each case.

[0036] Figure 21: Mouse study, in vivo pQCT after co-treatment with MOR05813 and zoledronic acid, (A) Total bone mineral density, (B) Total bone mineral content, (C) Cortical thickness, and (D) Cancellous bone mineral density.

[0037] Figure 22: Mouse study, in vivo pQCT: MOR05813 treatment after alendronate (alen) pretreatment, (A) Total bone mineral density, (B) Total bone mineral content, (C) Cortical thickness, and (D) Cancellous bone mineral density.

[0038] Figure 23: Mouse study, in vivo pQCT after anabolic co-treatment with MOR05813 and (i) anti-DKK1 or (ii) PTH, (A) Total bone mineral density, (B) Total bone mineral content, (C) Cortical thickness, and (D) Bone mineral density of cancellous bone.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention relates to isolated antibodies, particularly human antibodies, that specifically bind to sclerostin and that inhibit functional properties of sclerostin. In certain embodiments, the antibodies of the invention are derived from particular heavy and light chain sequences and/or comprise particular structural features such as, for example, CDR regions comprising particular amino acid sequences. The invention provides isolated antibodies, methods of producing such antibodies, immunoconjugates and multivalent or multispecific molecules comprising such antibodies, and pharmaceutical compositions containing the antibodies, immunoconjugates or bispecific molecules of the invention. The invention also relates to methods of using the antibodies to inhibit a disorder or condition associated with the presence of sclerostin expression, for example, in the treatment of a pathological disorder that is mediated by sclerostin or that is associated with an increased level of sclerostin; for example, a bone-related disease such as, for example, osteoporosis.

[0040] In order that the present invention may be more easily understood, some terms will first be defined. Additional definitions are presented throughout the detailed description.

[0041] The term "comprising" encompasses "including" as well as "consisting of"; for example, a composition "comprising" X may consist exclusively of X or may include something else, for example, X + Y.

[0042] The term "about" in relation to a numerical value x means, for example, x ± 10%.

[0043] The word "substantially" does not exclude "completely"; for example, a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

[0044] The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by the above cells or by the liver (including antibodies, cytokines and complement) that result in selective damage, destruction or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancer cells or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0045] The term "sclerostin" refers to human sclerostin as defined in SEQ ID NO: 155. Recombinant human sclerostin can be obtained from R&D Systems (Minneapolis, MN, USA; 2006 Catalog No. 1406-ST-025). Additionally, recombinant mouse sclerostin/SOST is commercially available from R&D Systems (Minneapolis, MN, USA; 2006 Catalog No. 1589-ST-025). U.S. Patent Nos. 6,395,511 and 6,803,453, and U.S. Patent Publications Nos. 20040009535 and 20050106683, refer to anti-sclerostin antibodies in general.

[0046] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (herein abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0047] The term "antigen-binding portion" of an antibody (or simply "antigen portion"), as used herein, refers to a full-length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., sclerostin). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; an F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341: 544-546), consisting of a VH domain; and an isolated complementarity determining region (CDR).

[0048] Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods, by a synthetic linker that allows them to be made as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also encompassed within the term "antigen-binding region" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for utility in the same manner as intact antibodies.

[0049] The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies that have different antigenic specificities (e.g., an isolated antibody that specifically binds to sclerostin is substantially free of antibodies that specifically bind to antigens other than sclerostin). An isolated antibody that specifically binds to sclerostin may, however, have cross-reactivity to other antigens, e.g., sclerostin molecules from other species. In addition, an isolated antibody may be substantially free of other cellular materials and/or chemicals.

[0050] The terms "monoclonal antibody" or "monoclonal antibody composition", as used herein, refer to a preparation of antibody molecules of unique molecular composition. A monoclonal antibody composition exhibits unique specificity and binding affinity for a particular epitope.

[0051] The term "human antibody" as used herein is intended to include antibodies that have variable regions in which both the framework regions and the CDR regions are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences, or antibodies containing consensus framework sequences derived from analysis of human framework sequences, as described in Knappik, et al. (2000, J. Mol. Biol. 296, 57-86).

[0052] Human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., mouse, have been grafted onto human framework sequences.

[0053] The term "human monoclonal antibody" refers to antibodies exhibiting a single binding specificity that have variable regions in which both the framework regions and the CDR regions are derived from human sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a non-human transgenic animal, e.g., a transgenic mouse, that has a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0054] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, raised or isolated by recombinant means, e.g., antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a combinatorial library of recombinant human antibodies, and antibodies prepared, expressed, raised or isolated by any other means that involves splicing all or a portion of a human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human germline repertoire of antibodies in vivo.

[0055] As used herein, the term "isotype" refers to the class of antibodies (e.g., IgM, IgE, IgG such as IgG1 or IgG2) that is contributed by heavy chain constant region genes.

[0056] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0057] As used herein, an antibody that "specifically binds to sclerostin polypeptide" is intended to refer to an antibody that binds to sclerostin polypeptide with a KD of 1 x 10-8 M or less, 1 x 10-9 M or less, or 1 x 10-10 M or less. An antibody that "cross-reacts with an antigen other than sclerostin" is intended to refer to an antibody that binds to that antigen with a KD of 0.5 x 10-8 M or less, 5 x 10-9 M or less, or 2 x 10-9 M or less. An antibody that "does not cross-react with a particular antigen" is intended to refer to an antibody that binds to that antigen with a KD of 1.5 x 10-8 M or greater, or a KD of between 5 x 10-8 M and 10 x 10-8 M or 1 x 10-7 M or greater. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding against those proteins in standardized binding assays.

[0058] As used herein, an antibody that "blocks the inhibitory effect of sclerostin in a cellular assay of Wnt signaling" is intended to refer to an antibody that restores Wnt-induced signaling in the presence of sclerostin in a super top flash (STF) cell-based assay with an IC50 of less than 1 mM, 100 nM, 20 nM, 10 nM or less. Such an STF assay is described in more detail in the examples below.

[0059] As used herein, an antibody that "blocks the inhibitory effect of sclerostin in a cellular mineralization assay" is intended to refer to an antibody that restores BMP2-induced mineralization in the presence of sclerostin in a cell-based assay with an IC50 of less than 1 mM, 500 nM, 100 nM, 10 nM, 1 nM or less. Such an assay is described in more detail in the examples below.

[0060] As used herein, an antibody that "blocks the inhibitory effect of sclerostin in a Smad1 phosphorylation assay" is intended to refer to an antibody that restores BMP6-induced Smad1 phosphorylation in the presence of sclerostin in a cell-based assay with an IC50 of less than 1 mM, 500 nM, 100 nM, 10 nM, 1 nM or less. Such an assay is described in more detail in the examples below.

[0061] As used herein, an antibody that "inhibits the binding of sclerostin to LRP-6" refers to an antibody that inhibits the binding of sclerostin to LRP-6 with an IC50 of 1 mM, 500 nM, 100 nM, 10 nM, 5 nM, 3 nM, 1 nM or less. This assay is described in more detail in the examples below.

[0062] As used herein, an antibody that "increases bone formation, mass and density" refers to an antibody that is capable of bringing bone formation, mass and density to the level of intermittent daily treatment with a high anabolic dose of PTH, as shown in Example 10.

[0063] The term "Kassoc" or "Ka," as used herein, is intended to refer to the association rate of a particular antigen-antibody interaction, while the term "Kdis" or "KD," as used herein, is intended to refer to the dissociation rate of a particular antigen-antibody interaction. The term "KD," as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of KD to Ka (i.e., KD/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. One method for determining the KD of an antibody is by using surface plasmon resonance, or by using a biosensor system such as a Biacore® system.

[0064] As used herein, the term "affinity" refers to the strength of the interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region "arm" of the antibody interacts through weak non-covalent forces with antigen at multiple sites; the more interactions, the stronger the affinity.

[0065] As used herein, the term "avidity" refers to an informative measure of the overall stability or potency of the antigen-antibody complex. It is controlled by three main factors: the epitope affinity of the antibody; the valence of both the antigen and the antibody; and the structural arrangement of the interacting moieties. Ultimately, these factors define the specificity of the antibody, that is, the probability that that particular antibody will bind to a precise epitope of the antigen.

[0066] In order to obtain a probe with higher avidity, a dimeric conjugate can be constructed, thereby producing low affinity interactions (e.g., with germline antibody) more readily detected by FACS. Additionally, another means of increasing antigen binding avidity involves generating dimers, trimers, or multimers of any of the anti-sclerostin antibody constructs described herein. Such multimers can be generated by covalent linkage between individual modules, e.g., by mimicking the natural C-terminal to N-terminal linkage or by mimicking antibody dimers that are held together by their constant regions. The linkages created at the Fc/Fc interface can be covalent or non-covalent. Additionally, dimerization or multimerization partners other than Fc can be used in sclerostin hybrids to create these higher order structures. For example, it is possible to use multimerization domains such as the trimerization domain described in Borean (WO 2004039841) or the pentamerization domain described in published Patent Application WO98/18943.

[0067] As used herein, the term "cross-reactivity" refers to an antibody or population of antibodies that bind to epitopes on other antigens. This may be caused by low antibody avidity or specificity or by multiple distinct antigens that have identical or very similar epitopes. Cross-reactivity is sometimes desirable when general binding to a related group of antigens is desired or when cross-species labeling is attempted when the antigen epitope sequence is not highly evolutionarily conserved.

[0068] As used herein, the term "high affinity" for an IgG antibody refers to an antibody that has a KD of 10-8 M or less, 10-9 M or less, or 10-10 M or less for a target antigen. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody that has a KD of 10-7 M or less or 10-8 M or less.

[0069] As used herein, the term "individual" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals, and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0070] As used herein, the term "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the producing cell or organism, usually a eukaryotic cell, e.g., a Pichia or Trichoderma cell, the Chinese hamster ovary (CHO) cell, or a human cell. The optimized nucleotide sequence is designed to retain completely or as closely as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence. The optimized sequences presented herein have been genetically engineered to have codons that are preferred in mammalian cells; however, optimized expression of these sequences in other eukaryotic cells is also envisioned herein. Amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.

[0071] Various aspects of the invention will be described in more detail in the following subsections.

[0072] Standardized assays for evaluating the binding ability of antibodies to sclerostin from various species are known in the art, including, for example, ELISAs, Western blots, and RIAs. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of antibodies can also be evaluated by standardized assays known in the art, such as, for example, by Biacore analysis. Assays for evaluating the effects of antibodies on functional properties of sclerostin (e.g., receptor binding, prevention or attenuation of osteolysis) will be described in more detail in the Examples.

[0073] Accordingly, it will be understood that an antibody that "inhibits" one or more of these functional properties of sclerostin (e.g., biochemical, immunochemical, cellular, physiological or other biological activities, or the like), as determined according to methodologies known in the art and described herein, is related to a statistically significant decrease in the particular activity relative to that observed in the absence of the antibody (or when a control antibody of irrelevant specificity is present). An antibody that inhibits sclerostin activity effects such a statistically significant decrease by at least 10% of the measured parameter, by at least 50%, 80% or 90%, and in certain embodiments, an antibody of the invention can inhibit greater than 95%, 98% or 99% of the functional activity of sclerostin.

[0074] The terms "cross-block", "cross-blocked" and "cross-blocking" are used interchangeably herein to mean the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to sclerostin in a standardized competitive binding assay.

[0075] The ability or extent to which an antibody or other binding agent is able to interfere with the binding of another antibody or binding molecule to sclerostin, and therefore whether it can be considered to be cross-blocking in accordance with the invention, can be determined using standardized binding competition assays. One suitable assay involves the use of Biacore technology (e.g., using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-blocking uses an ELISA-based approach.

[0076] Additional details on both methods will be presented in the examples.

[0077] According to the invention, a cross-blocking antibody or other binding agent according to the invention binds to sclerostin in the described Biacore cross-blocking assay, such that the recorded binding of the combination (mixture) of the antibodies or binding agents is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to 4%), and more specifically between 65% and 0.1% (e.g. 65% to 4%) of the maximum theoretical binding (as defined above) of the two antibodies or binding agents in combination.

[0078] An antibody is defined as having cross-blocking in the ELISA assay as described in the examples if the solution phase anti-sclerostin antibody is capable of causing a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the sclerostin detection signal (i.e. the amount of sclerostin bound by the coated antibody), when compared to the sclerostin detection signal obtained in the absence of the solution phase anti-sclerostin antibody (i.e. the positive control wells).

Monoclonal antibodies

[0079] Antibodies of the invention include human monoclonal antibodies isolated as described in the examples. The VH amino acid sequences of isolated antibodies of the invention are set forth in SEQ ID NOs: 69-77. The VL amino acid sequences of isolated antibodies of the invention are set forth in SEQ ID NOs: 80-88, respectively. The corresponding preferred full-length heavy chain amino acid sequences of antibodies of the invention are set forth in SEQ ID NOs: 113-121. The corresponding preferred full-length light chain amino acid sequences of antibodies of the invention are set forth in SEQ ID NOs: 124-132, respectively. Other antibodies of the invention include amino acids that have been mutated, yet have at least 60, 70, 80, 90, or 95 percent or more identity in the CDR regions to the CDR regions disclosed in the sequences described above. In some embodiments, the invention includes mutant amino acid sequences in which at most 1, 2, 3, 4, or 5 amino acids have been mutated in the CDR regions, when compared to the CDR regions disclosed in the sequence described above.

[0080] Additionally, variable heavy chain parental nucleotide sequences are set forth in SEQ ID NOs: 89-90. Variable heavy chain parental nucleotide sequences are set forth in SEQ ID NOs: 100-101. Full-length light chain nucleotide sequences optimized for expression in a mammalian cell are set forth in SEQ ID NOs: 146-154. Full-length heavy chain nucleotide sequences optimized for expression in a mammalian cell are set forth in SEQ ID NOs: 135-143. Full-length light chain amino acid sequences encoded by optimized light chain nucleotide sequences are set forth in SEQ ID NOs: 124-132. Full-length heavy chain amino acid sequences encoded by optimized heavy chain nucleotide sequences are set forth in SEQ ID NOs: 113-121. Other antibodies of the invention include amino acids or nucleic acids that have been mutated, yet possess at least 60, 70, 80, 90, or 95 percent or more identity to the sequences described above. In some embodiments, the invention includes mutant amino acid sequences in which at most 1, 2, 3, 4, or 5 amino acids have been mutated in the variable regions, as compared to the variable regions disclosed in the sequence described above, yet retain substantially the same therapeutic activity.

[0081] To the extent that each of these antibodies can bind to sclerostin, the full-length light chain sequences and the full-length heavy chain sequences, VH, VL (nucleotide sequences and amino acid sequences), can be "mixed and matched" to create other anti-sclerostin binding molecules of the invention. The binding to sclerostin of these "mixed and matched" antibodies can be tested using the binding assays described above and in the examples (e.g., ELISAs). When these chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence. Likewise, a full-length heavy chain sequence from a particular full-length heavy chain/full-length light chain pairing should be replaced with a structurally similar full-length heavy chain sequence. Likewise, a VL sequence of a particular VH/VL pairing should be replaced with a structurally similar VL sequence. Likewise, a full-length light chain sequence of a particular full-length heavy chain/full-length light chain pairing should be replaced with a structurally similar full-length light chain sequence. Accordingly, in one aspect, the invention provides an isolated monoclonal antibody or an antigen-binding region thereof having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 69-77; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 80-88; wherein the antibody specifically binds to sclerostin.

[0082] In another aspect, the invention provides:

[0083] an isolated monoclonal antibody having: a full-length heavy chain comprising an amino acid sequence that has been optimized for expression in a mammalian cell selected from the group consisting of SEQ ID NOs: 113-121; and a full-length light chain comprising an amino acid sequence that has been optimized for expression in a mammalian cell selected from the group consisting of SEQ ID NOs: 124-132; or

[0084] a functional protein comprising an antigen-binding portion thereof.

[0085] In another aspect, the invention provides:

[0086] an isolated monoclonal antibody having: a full-length heavy chain comprising a nucleotide sequence that has been optimized for expression in a mammalian cell selected from the group consisting of SEQ ID NOs: 135-143; and a full-length light chain comprising a nucleotide sequence that has been optimized for expression in a mammalian cell selected from the group consisting of SEQ ID NOs: 146-154; or,

[0087] a functional protein comprising an antigen-binding portion thereof.

[0088] In yet another aspect, the invention provides antibodies comprising the CDR1s, CDR2s, and CDR3s of the heavy chain and light chain of the antibodies, or combinations thereof. The amino acid sequences of the VH CDR1s of the antibodies are set forth in SEQ ID NOs: 1-11. The amino acid sequences of the VH CDR2s of the antibodies are set forth in SEQ ID NOs: 12-22. The amino acid sequences of the VH CDR3s of the antibodies are set forth in SEQ ID NOs: 23-33. The amino acid sequences of the VL CDR1s of the antibodies are set forth in SEQ ID NOs: 34-44. The amino acid sequences of the VL CDR2s of the antibodies are set forth in SEQ ID NOs: 45-55. The amino acid sequences of the VL CDR3s of the antibodies are set forth in SEQ ID NOs: 56-66. CDR regions are delineated using the Kabat system (Kabat, E.A., et al., 1991 "Sequences of Proteins of Immunological Interest", Fifth Edition, "U.S. Department of Health and Human Services", NIH Publication No. 91-3242).

[0089] Considering that each of these antibodies can bind to sclerostin and that antigen binding specificity is provided primarily by the CDR1, 2, and 3 regions, the VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched), although each antibody must contain a VH CDR1, 2, and 3 and a VL CDR1, 2, and 3 to create other anti-sclerostin binding molecules of the invention. The binding to sclerostin of these "mixed and matched" antibodies can be tested using the binding assays described above and in the examples (e.g., ELISAs). When VH CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence of a particular VH sequence should be replaced with structurally similar CDR sequence(s). Likewise, when VL CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence of a particular VL sequence should be replaced with structurally similar CDR sequence(s). It will be apparent to those skilled in the art that new VH and VL sequences can be created by replacing one or more sequences of the VH and/or VL CDR region with structurally similar sequences of the CDR sequences disclosed herein for monoclonal antibodies of the present invention.

[0090] An isolated monoclonal antibody, or antigen-binding region thereof, has: a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11; a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1222; a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-33; a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-44; a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-55; and a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-66; in which the antibody specifically binds to sclerostin.

[0091] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 3; a heavy chain variable region CDR2 of SEQ ID NO: 14; a heavy chain variable region CDR3 of SEQ ID NO: 25; a light chain variable region CDR1 of SEQ ID NO: 36; a light chain variable region CDR2 of SEQ ID NO: 47; and a light chain variable region CDR3 of SEQ ID NO: 58.

[0092] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 4; a heavy chain variable region CDR2 of SEQ ID NO: 15; a heavy chain variable region CDR3 of SEQ ID NO: 26; a light chain variable region CDR1 of SEQ ID NO: 37; a light chain variable region CDR2 of SEQ ID NO: 48; and a light chain variable region CDR3 of SEQ ID NO: 59.

[0093] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 5; a heavy chain variable region CDR2 of SEQ ID NO: 16; a heavy chain variable region CDR3 of SEQ ID NO: 27; a light chain variable region CDR1 of SEQ ID NO: 38; a light chain variable region CDR2 of SEQ ID NO: 49; and a light chain variable region CDR3 of SEQ ID NO: 60.

[0094] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 6; a heavy chain variable region CDR2 of SEQ ID NO: 17; a heavy chain variable region CDR3 of SEQ ID NO: 28; a light chain variable region CDR1 of SEQ ID NO: 39; a light chain variable region CDR2 of SEQ ID NO: 50; and a light chain variable region CDR3 of SEQ ID NO: 61.

[0095] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 7; a heavy chain variable region CDR2 of SEQ ID NO: 18; a heavy chain variable region CDR3 of SEQ ID NO: 29; a light chain variable region CDR1 of SEQ ID NO: 40; a light chain variable region CDR2 of SEQ ID NO: 51; and a light chain variable region CDR3 of SEQ ID NO: 62.

[0096] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 8; a heavy chain variable region CDR2 of SEQ ID NO: 19; a heavy chain variable region CDR3 of SEQ ID NO: 30; a light chain variable region CDR1 of SEQ ID NO: 41; a light chain variable region CDR2 of SEQ ID NO: 52; and a light chain variable region CDR3 of SEQ ID NO: 63.

[0097] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 9; a heavy chain variable region CDR2 of SEQ ID NO: 20; a heavy chain variable region CDR3 of SEQ ID NO: 31; a light chain variable region CDR1 of SEQ ID NO: 42; a light chain variable region CDR2 of SEQ ID NO: 53; and a light chain variable region CDR3 of SEQ ID NO: 64.

[0098] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 10; a heavy chain variable region CDR2 of SEQ ID NO: 21; a heavy chain variable region CDR3 of SEQ ID NO: 32; a light chain variable region CDR1 of SEQ ID NO: 43; a light chain variable region CDR2 of SEQ ID NO: 54; and a light chain variable region CDR3 of SEQ ID NO: 65.

[0099] In certain embodiment, the antibody comprises: a heavy chain variable region CDR1 of SEQ ID NO: 11; a heavy chain variable region CDR2 of SEQ ID NO: 22; a heavy chain variable region CDR3 of SEQ ID NO: 33; a light chain variable region CDR1 of SEQ ID NO: 44; a light chain variable region CDR2 of SEQ ID NO: 55; and a light chain variable region CDR3 of SEQ ID NO: 66.

[00100] As used herein, a human antibody comprises heavy or light chain variable regions or full-length heavy or light chains that are "the product of" or "derived from" a germline sequence, particularly if the variable regions or full-length chains of the antibody are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a phage-displayed human immunoglobulin gene library with the antigen of interest. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence closest in sequence (i.e., highest % identity) to the human antibody sequence. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences when compared to the germline sequence, due to, for example, naturally occurring somatic mutations or the intentional introduction of a site-directed mutation. However, a selected human antibody typically is at least 90% identical in amino acid sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human when compared to the amino acid sequences of germline immunoglobulins from other species (e.g., murine germline sequences). In certain cases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95% or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will exhibit at most 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may exhibit at most 5, or even at most 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[00101] In some embodiments, germline immunoglobulin amino acid sequences are selected from those comprising the variable heavy chain sequences consisting of SEQ ID NO: 67-68, respectively, and the variable light chain sequences consisting of SEQ ID NO: 7879, respectively.

Homologous antibodies

[00102] In yet another embodiment, an antibody of the invention has full-length heavy and light chain amino acid sequences; full-length heavy and light chain nucleotide sequences, variable region heavy and light chain nucleotide sequences, or variable region heavy and light chain amino acid sequences that are homologous to the amino acid and nucleotide sequences of the antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-sclerostin antibodies of the invention.

[00103] For example, the invention provides an isolated monoclonal antibody (or a functional protein comprising an antigen-binding portion thereof) comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6777; the light chain variable region comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7888; the antibody specifically binds to sclerostin, and the antibody exhibits at least one of the following functional properties: the antibody blocks the inhibitory effect of sclerostin in a cellular Wnt signaling assay, the antibody blocks the inhibitory effect of sclerostin in a cellular mineralization assay, or the antibody blocks the inhibitory effect of sclerostin in a Smad1 phosphorylation assay, or the antibody inhibits the binding of sclerostin to LRP-6, or the antibody increases bone formation, bone mass, and bone density.

[00104] In a further example, the invention provides an isolated monoclonal antibody (or a functional protein comprising an antigen-binding portion thereof) comprising a full-length heavy chain and a full-length light chain, wherein: the full-length heavy chain comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 111-121; the full-length light chain comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 122-132; the antibody specifically binds to sclerostin, and the antibody exhibits at least one of the following functional properties: the antibody blocks the inhibitory effect of sclerostin in a cellular Wnt signaling assay, the antibody blocks the inhibitory effect of sclerostin in a cellular mineralization assay, or the antibody blocks the inhibitory effect of sclerostin in a Smad1 phosphorylation assay, or the antibody inhibits the binding of sclerostin to LRP-6, or the antibody increases bone formation, bone mass, and bone density.

[00105] In another example, the invention provides an isolated monoclonal antibody (or a functional protein comprising an antigen-binding portion thereof), comprising a full-length heavy chain and a full-length light chain, wherein: the full-length heavy chain is encoded by a nucleotide sequence that is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 133-143; the full-length light chain comprises a nucleotide sequence that is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 144-154; the antibody specifically binds to sclerostin, and the antibody exhibits at least one of the following functional properties: the antibody blocks the inhibitory effect of sclerostin in a cellular Wnt signaling assay, the antibody blocks the inhibitory effect of sclerostin in a cellular mineralization assay, or the antibody blocks the inhibitory effect of sclerostin in a Smad1 phosphorylation assay, or the antibody inhibits the binding of sclerostin to LRP-6, or the antibody increases bone formation, bone mass, and bone density.

[00106] In various embodiments, the antibody can exhibit one or more, two or more, or three of the functional properties discussed above. The antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[00107] In other embodiments, the amino acid sequences of VH and/or VL can be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth above. In other embodiments, the amino acid sequences of VH and/or VL can be identical except for an amino acid substitution at most 1, 2, 3, 4, or 5 amino acid positions. An antibody having VH and VL regions with high (i.e., 80% or greater) identity to the VH and VL regions of SEQ ID NOs: 67-77 and SEQ ID NOs: 78-88, respectively, can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs: 89-99 and 100-110, respectively, followed by testing the encoded altered antibody for retained function (i.e., the functions set forth above) using the functional assays described herein.

[00108] In other embodiments, the amino acid sequences of the full-length heavy chain and/or the full-length light chain can be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth above. An antibody having a full-length heavy chain and full-length light chain that have high (i.e., 80% or greater) identity to the full-length heavy chains of any of SEQ ID NOs: 111-121 and full-length light chains of any of SEQ ID NOs: 122-132, respectively, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs: 133-143 and SEQ ID NOs: 144-154, respectively, followed by testing the encoded altered antibody for retained function (i.e., the functions set forth above) using the functional assays described herein.

[00109] In other embodiments, the nucleotide sequences of the full-length heavy chain and/or the full-length light chain can be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth above.

[00110] In other embodiments, the variable regions of the heavy chain and/or light chain nucleotide sequences can be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth above.

[00111] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # identical positions/# total positions x 100), taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of the percent identity between two sequences may be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[00112] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17, 1988) that was incorporated into the ALIGN program (version 2.0), using a PAM120 residue weighting table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453, 1970) that was incorporated into the GAP program in the GCG suite of programs (available at http://www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix, and a gap weighting of 16, 14, 12, 10, 8, 6, or 4, and a in length of 1, 2, 3, 4, 5 or 6.

[00113] Additionally or alternatively, the protein sequences of the present invention may further be used as an "interrogate sequence" for performing a search against public databases for, for example, the identification of related sequences. Such searches may be performed using the XBLAST program (version 2.0) of Altschul, et al., 1990 J. Mol. Biol. 215: 403-10. Protein BLAST searches may be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be used as described in Altschul et al., 1997 Nucleic Acids Res. 25(17): 3,389-3,402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http:www.ncbi.nhn.nih.gov.

Antibodies with conservative modifications

[00114] In certain embodiments, an antibody of the invention has a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences has specified amino acid sequences based on the antibodies described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-sclerostin antibodies of the invention. Accordingly, the invention provides an isolated monoclonal antibody, or a functional protein comprising an antigen-binding portion thereof, consisting of a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: the amino acid sequences of the CDR1 of the heavy chain variable region are selected from the group consisting of SEQ ID NOs: 1-11, and conservative modifications thereof; the amino acid sequences of the CDR2 of the heavy chain variable region are selected from the group consisting of SEQ ID NOs: 12-22, and conservative modifications thereof; the amino acid sequences of the CDR3 of the heavy chain variable region are selected from the group consisting of SEQ ID NOs: 23-33, and conservative modifications thereof; the amino acid sequences of the CDR1 of the light chain variable regions are selected from the group consisting of SEQ ID NOs: 34-44, and conservative modifications thereof; the amino acid sequences of the CDR2 of the light chain variable regions are selected from the group consisting of SEQ ID NOs: 45-55, and conservative modifications thereof; the amino acid sequences of the CDR3 of the light chain variable regions are selected from the group consisting of SEQ ID NOs: 56-66, and conservative modifications thereof; the antibody specifically binds to sclerostin, and the antibody exhibits at least one of the following functional properties: the antibody blocks the inhibitory effect of sclerostin in a cellular Wnt signaling assay, the antibody blocks the inhibitory effect of sclerostin in a cellular mineralization assay, or the antibody blocks the inhibitory effect of sclerostin in a Smad1 phosphorylation assay, or the antibody inhibits the binding of sclerostin to LRP-6, or the antibody increases bone formation, bone mass, and bone density.

[00115] In various embodiments, the antibody can exhibit one or more, two or more, or three or more of the listed functional properties discussed above. Such antibodies can be, for example, human antibodies, humanized antibodies, or chimeric antibodies.

[00116] In other embodiments, an antibody of the invention optimized for expression in a mammalian cell has a full-length heavy chain sequence and a full-length light chain sequence, wherein one or more of these sequences have specified amino acid sequences based on the antibodies described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-sclerostin antibodies of the invention. Accordingly, the invention provides an isolated monoclonal antibody optimized for expression in a mammalian cell consisting of a full-length heavy chain and a full-length light chain, wherein: the full-length heavy chain has amino acid sequences selected from the group of SEQ ID NOs: 111-121, and conservative modifications thereof; and the full-length light chain has amino acid sequences selected from the group of SEQ ID NOs: 122-132, and conservative modifications thereof; the antibody specifically binds to sclerostin; and the antibody exhibits at least one of the following functional properties: the antibody blocks the inhibitory effect of sclerostin in a cellular Wnt signaling assay, the antibody blocks the inhibitory effect of sclerostin in a cellular mineralization assay, or the antibody blocks the inhibitory effect of sclerostin in a Smad1 phosphorylation assay, or the antibody inhibits the binding of sclerostin to LRP-6, or the antibody increases bone formation, bone mass, and bone density.

[00117] In various embodiments, the antibody can exhibit one or more, two or more, or three or more of the listed functional properties discussed above. Such antibodies can be, for example, human antibodies, humanized antibodies, or chimeric antibodies.

[00118] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications may be introduced into an antibody of the invention by standard techniques known in the art, such as, for example, site-directed and PCR-mediated mutagenesis.

[00119] Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for retained function using the functional assays described herein.

Antibodies that bind to the same epitope as the anti-sclerostin antibodies of the invention

[00120] In another embodiment, the invention provides antibodies that bind to the same epitope as the various specific anti-sclerostin antibodies of the invention described herein. It has surprisingly been found that all of the antibodies described in the examples are capable of:

[00121] block the inhibitory effect of sclerostin in a cellular Wnt signaling assay;

[00122] blocking the inhibitory effect in a cellular demineralization assay;

[00123] inhibit the binding of sclerostin to LRP-6; and,

[00124] increase bone formation, mass and density,

[00125] bind to the same epitope on sclerostin with high affinity, said epitope being a conformational epitope that includes amino acids from both SEQ ID NO: 156 and SEQ ID NO: 157. Without being bound to any specific model, it is proposed herein that the amino acid sequences of SEQ ID NO: 156 and SEQ ID NO: 157 delineate a region of the conformational epitope on the sclerostin polypeptide that is recognized by the antibodies of the invention.

[00126] Therefore, additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit binding, in a statistically significant manner) with other antibodies of the invention in standardized sclerostin binding assays. The ability of a test antibody to inhibit the binding of antibodies of the present invention to human sclerostin demonstrates that the test antibody can compete with that antibody for binding to human sclerostin; such an antibody can, according to a non-limiting theory, bind to the same or a related epitope (e.g., a structurally similar or spatially proximate epitope) on human sclerostin as the antibody with which it competes. In one embodiment, the antibody that binds to the same epitope on human sclerostin as the antibodies of the present invention is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described in the examples.

Genetically engineered and modified antibodies

[00127] An antibody of the invention may further be prepared using an antibody having one or more of the VH and/or VL sequences shown herein as starting material to genetically create a modified antibody, which modified antibody may have altered properties relative to the starting antibody. An antibody may be genetically created by modifying one or more residues within one or both of the variable regions (i.e., VH and/or VL), e.g., within one or more CDR and/or within one or more framework regions. Additionally or alternatively, an antibody may be genetically created by modifying residues within the constant region(s), e.g., to alter the effector function(s) of the antibody.

[00128] One type of variable region genetic engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse among individual antibodies than sequences outside the CDRs. Because CDR sequences are responsible for most antigen-antibody interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. et al., 1989 Proc. Natl. Acad. Sci. U.S.A. 86:10,029-10,033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patents 5,530,101, 5,585,089, 5,693,762 and 6,180,370 for Queen et al.).

[00129] Accordingly, another embodiment of the invention pertains to an isolated monoclonal antibody, or a functional protein comprising an antigen-binding portion thereof, comprising a heavy chain variable region comprising CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 111; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1222; CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2333, respectively; and a light chain variable region having CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-44; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-55; and CDR3 sequences consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5666, respectively. Thus, these antibodies contain the VH and VL CDR sequences of monoclonal antibodies, although they may still contain different framework sequences from those antibodies.

[00130] Such framework sequences can be obtained from public DNA databases or from published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the human germline sequence database "VBase" (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al., 1991, "Sequences of Proteins of Immunological Interest", Fifth Edition, "U.S. Department of Health and Human Services", NIH Publication No. 91-3242; Tomlinson, I. M., et al., 1992, J. Mol. Biol. 227: 776-798; and Cox, J. P. L. et al., 1994, Eur. J. Immunol. 24: 827-836; wherein the contents of each such reference are expressly incorporated herein by reference.

[00131] An example of framework sequences for use in antibodies of the invention are those that are structurally similar to the framework sequences used by selected antibodies of the invention, e.g., consensus sequences and/or framework sequences used by monoclonal antibodies of the invention. The VH CDR1, 2, and 3 sequences, and the VL CDR1, 2, and 3 sequences may be grafted into framework regions that have sequence identical to that found in the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences may be grafted into framework regions that contain one or more mutations when compared to the germline sequences. For example, it has been found that in certain cases it is beneficial to mutate residues within framework regions to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[00132] Another type of variable region modification is the mutation of amino acid residues within the CDR1, CDR2, and/or CDR3 regions of VH and/or VL to thereby increase one or more binding properties (e.g., affinity) of the antibody of interest, a process known as "affinity maturation." Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on antibody binding, or on another functional property of interest, can be assessed in in vitro or in vivo assays as described herein and provided in the Examples.

[00133] Conservative modifications may be introduced (as discussed above). Mutations may be amino acid substitutions, additions, or deletions. Furthermore, typically at most one, two, three, four, or five residues within a CDR region are altered.

[00134] Accordingly, in another embodiment, the invention provides isolated anti-sclerostin monoclonal antibodies, or a functional protein comprising an antigen-binding portion thereof, consisting of a heavy chain variable region having: a VH CDR1 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions, as compared to SEQ ID NOs: 1-11; a CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-22, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions, as compared to SEQ ID NOs: 12-22; a VH CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-33, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions, when compared to SEQ ID NOs: 23-33; a VL CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-44, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions, when compared to SEQ ID NOs: 34-44; a VL CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-55, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions, when compared to SEQ ID NOs: 45-55; and a VL CDR3 region that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5666, or an amino acid sequence that has one, two, three, four, or five amino acid substitutions, deletions, or additions, when compared to SEQ ID NOs: 56-66.

Grafting of antigen-binding domains onto alternative frameworks or scaffolds

[00135] A wide variety of antibody/immunoglobulin frameworks or scaffolds may be employed, provided that the resulting polypeptide includes at least one binding region that specifically binds to sclerostin. Such frameworks or scaffolds include the 5 major idiotypes of human immunoglobulins, or fragments thereof (such as those disclosed elsewhere herein), and include immunoglobulins from other animal species, preferably those that possess humanized aspects. Single heavy chain antibodies, such as those identified in camelids, are of particular interest in this regard. New frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

[00136] In one aspect, the invention pertains to the generation of non-immunoglobulin-based antibodies using non-immunoglobulin frameworks onto which the CDRs of the invention can be grafted. Known or future non-immunoglobulin frameworks and scaffolds may be employed, provided that they comprise a specific binding region for the target protein of SEQ ID NO: 155. Such compounds are known herein as "polypeptides comprising a target-specific binding region". Examples of non-immunoglobulin frameworks are further described in the sections below (camelid antibodies and non-antibody scaffold).

Camelid antibodies

[00137] Antibody proteins obtained from members of the camel and dromedary family (Camelus bactrianus and Camelus dromaderius) including new worldwide members such as llama species (Lama paccos, Lama glama and Lama vicugna), have been characterized with respect to size, structural complexity and antigenicity to human subjects. Certain IgG antibodies of this mammalian family as found in nature are devoid of light chains and are thus structurally distinct from the typical four-chain quaternary structure having two heavy chains and two light chains for antibodies of other animals. See PCT/EP93/02214 (WO 94/04678 published March 3, 1994).

[00138] A region of the camelid antibody that is the small unique variable domain identified as VHH can be genetically engineered to generate a small protein that has high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "camelid nanobody". See U.S. Patent No. 5,759,808 issued June 2, 1998; see also Stijlemans, B. et al., 2004 J. Biol. Chem. 279:1256-1261; Dumoulin, M. et al., 2003 Nature 424:783-788; Pleschberger, M. et al. 2003 Bioconjugate. Chem. 14:440-448; Cortez-Retamozo, V. et al. 2002 Int. J. Cancer. 89: 456–62; and Lauwereys, M. et al. 1998 EMBO J. 17: 3512–3520. Genetically engineered libraries of camelid antibodies and antibody fragments are commercially available, e.g., from Ablynx, Ghent, Belgium. As with other antibodies of nonhuman origin, an amino acid sequence of a camelid antibody can be recombinantly altered to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be “humanized.” In this way, the low natural antigenicity of camelid antibodies for humans can be further reduced.

[00139] The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. A consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents for the detection of antigens that are otherwise cryptic using classical immunological techniques, and as potential therapeutic agents. Thus, yet another consequence of the small size is that a camelid nanobody may inhibit by binding to a specific site in a narrow groove or cleft of a target protein, and thus may serve in a capacity that more closely resembles a function of a classical low molecular weight drug than that of a classical antibody.

[00140] The low molecular weight and compact size further result in the fact that camelid nanobodies are extremely thermostable, stable up to extreme pH and proteolytic digestion, and weakly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and may treat disorders affecting nervous tissue. Nanobodies may further facilitate drug transport across the blood-brain barrier. See U.S. Patent Application 20040161738 published August 19, 2004. These characteristics, combined with low antigenicity to humans, indicate great therapeutic potential. Furthermore, these molecules can be fully expressed in prokaryotic cells such as E. coli, and are expressed as bacteriophage fusion proteins and are functional.

[00141] Accordingly, a feature of the present invention is a camelid antibody or nanobody that has high affinity for sclerostin. In certain embodiments presented herein, the camelid antibody or nanobody is naturally produced in the camelid animal, i.e., it is produced by the camelid after immunization with sclerostin or a peptide fragment thereof, using techniques described herein for other antibodies. Alternatively, the camelid anti-sclerostin nanobody is genetically created, i.e., produced by selection, for example, from a phage library that presents suitably mutagenized camelid nanobody proteins using panning procedures with sclerostin as a target, as described in the examples presented herein. Genetically created nanobodies can further be customized by genetic engineering to have a half-life in a recipient individual of 45 minutes to two weeks. In a specific embodiment, the camelid antibody or nanobody is obtained by grafting the CDR sequences of the heavy or light chain of the human antibodies of the invention into nanobody or single domain antibody framework sequences, as described, for example, in PCT/EP93/02214 (WO94/04678).

Non-antibody framework

[00142] Known non-immunoglobulin frameworks or scaffolds include, without limitation, Adnectins (fibronectin) (Compound Therapeutic Substances, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc. (Mountain View, CA)), Protein A (Affibody AG, Sweden) and aphyllin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

Adnectins - Compound therapeutic substances

[00143] Adnectin scaffolds are based on the fibronectin type III domain (e.g., the tenth module of fibronectin type III (domain 10 Fn3)). The fibronectin type III domain has 7 or 8 beta strands that are distributed between two beta sheets, which pack against each other to form the core of the protein, and which further contain loops (analogous to CDRs) that connect the beta strands together and are exposed to solvent. There are at least three such loops on each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (U.S. 6,818,418).

[00144] These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that in the smallest functional antibody fragment, the heavy chain variable region, which comprises the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in an in vitro loop randomization and shuffling strategy that is similar to the in vivo antibody affinity maturation process. These fibronectin-based molecules can be used as scaffolds, wherein the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.

Ankyrin - Molecular partners

[00145] The technology is based on the use of proteins with ankyrin-derived repeat modules as scaffolds to house variable regions that can be used for binding to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two antiparallel α-helices and a β-turn. Binding of the variable regions is optimized mainly by the use of ribosome presentation.

Maxybodies/Avimeros - Avidia

[00146] Avimers are derived from natural proteins that contain A domains, such as LRP-1. These domains are used by nature for protein-protein interactions and in humans in over 250 proteins that are structurally based on A domains. Avimers consist of several different "A domain" monomers (2-10) linked via amino acid linkers. Avimers that bind to target antigen can be created using the methodology described, for example, in 20040175756, 20050053973, 20050048512, and 20060008844.

Protein A - Affibody

[00147] Affibody® affinity ligands are small, simple proteins composed of a three-helix bundle based on the framework of one of the IgG-binding domains of Protein A. Protein A is a surface protein of the bacterium Staphylococcus aureus. This framework domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants (see, e.g., U.S. 5,831,012). Affibody® molecules mimic antibodies, and have a molecular weight of 6 kDa, compared with the molecular weight of antibodies, which is 150 kDa. Despite their small size, the binding site of Affibody® molecules is similar to that of an antibody.

Anticalins - Pieris

[00148] Anticalins® are products developed by Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small, robust proteins that are typically involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human body tissues or fluids.

[00149] The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain of 160 to 180 amino acid residues, being only slightly larger than a single immunoglobulin domain.

[00150] The set of four loops, which constitute the binding pocket, exhibits pronounced structural plasticity and tolerates a variety of side chains. The binding site can thus be remodeled in a proprietary process in order to recognize differently shaped prescribed target molecules with high affinity and specificity.

[00151] A protein of the lipocalin family, the bilin-binding protein (BBP) from Pieris brassicae, has been used for the development of anticalins by mutagenesis of the four-loop set. An example of a patent application describing "anticalins" is PCT WO199916873.

Afilin Proteins - Scil

[00152] Affilin™ molecules are small non-immunoglobulin proteins that are engineered for specific affinities against proteins and small molecules. New Affilin™ molecules can be selected very rapidly from two libraries, each based on a different human-derived scaffold protein.

[00153] Affilin™ molecules do not exhibit any structural homology to immunoglobulin proteins. Scil proteins employ two Affilin™ scaffolds, one of which is gamma crystallin, a structural protein of the human eye lens, and the other is formed by proteins of the "ubiquitin" superfamily. Both human scaffolds are very small, exhibit stability at elevated temperatures and are nearly resistant to pH changes and denaturing agents. This high stability is mainly caused by the expanded beta sheet structure of the proteins. Examples of gamma crystallin-derived proteins are described in WO 200104144 and examples of "ubiquitin-like" proteins are described in WO 2004106368.

Protein epitope mimetics (PEM)

[00154] PEM are medium-sized, cyclic peptide-like molecules (molecular weight of 1-2 kDa), which mimic beta-hairpin secondary structures of proteins, the main secondary structure involved in protein-protein interactions. More generally, any polypeptide that mimics the 3D structure of the epitope of the disclosed antibodies is part of the present invention. Preferred embodiments are polypeptides of 30-100 amino acids comprising at least the following polypeptides E1-L-E2, wherein E1 is SEQ ID NO: 156 and E2 is SEQ ID NO: 157 and L is a polypeptide linker that allows E1 and E2 to reproduce the 3D structure of the region recognized by the antibodies of the invention. According to a preferred embodiment, L is a linker consisting of 10-20 amino acids selected from the amino acids glycine or serine. Preferably, the linker L comprises the peptide GGGSGGGGSGGGG (SEQ ID NO: X/SEQ ID NO: 158) or GGGGSGGGGSGGGGSGGGG (SEQ ID NO: Y/SEQ ID NO: 159); more preferably, the linker L consists essentially of SEQ ID NO: X or SEQ ID NO: Y.

[00155] These polypeptides should retain high affinity for the antibodies of the invention. These polypeptides may also be used advantageously as immunogens to raise antibodies against sclerostin.

[00156] These polypeptides can also be used as sclerostin antagonist or agonist and therefore have applications similar to those described for the antibodies of the present invention.

[00157] Polypeptides with one or more amino acid substitutions or deletions, preferably not less than 1, 2 or 3 amino acid substitutions or deletions in the E1 and/or E2 sequence, are also part of the invention. Such polypeptides can further be genetically engineered to increase half-life or improve solubility. In particular, fusion constructs of such polypeptides with serum proteins, e.g. Fc fragments of human IgG or serum albumin, can be generated to increase half-life, similarly to the genetically engineered Fc described in the following paragraph for fragments of antibody molecules of the invention.

Framework or Fc genetic creation

[00158] The genetically engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and/or VL, for example, to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues to the corresponding germline sequence. More specifically, a somatically mutated antibody may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequences of the antibody to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "back mutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "back mutated" antibodies are also encompassed by the invention.

[00159] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also termed "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043 to Carr et al.

[00160] In addition to or as an alternative to modifications made within the framework or CDR regions, antibodies of the invention may also be genetically engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody such as, for example, serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments will be described in detail below. The residue numbering in the Fc region is that of the Kabat EU index.

[00161] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered, e.g., to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[00162] In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired binding to staphylococcal protein A (SpA) relative to native binding of SpA to the Fc-hinge domain. This approach is described in more detail in U.S. Patent No. 6,165,745 to Ward et al.

[00163] In another embodiment, the antibody is modified to increase its biological half-life. Possible approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a wild-type receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patents Nos. 5,869,046 and 6,121,022 to Presta et al.

[00164] In still other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue, such that the antibody has an altered affinity for an effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand for which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both to Winter et al.

[00165] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue, such that the antibody has altered C1q binding and/or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 to Idusogie et al.

[00166] In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT Publication WO 94/29351 by Bodmer et al.

[00167] In yet another embodiment, the Fc region is modified to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fcy receptor by modifying one or more amino acids. This approach is further described in PCT Publication WO 00/42072 by Presta. In addition, the binding sites on human IgG1 for FcyRI, FcyRII, FcyRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chem. 276:6591-6604).

[00168] In yet another embodiment, the glycosylation of an antibody is modified. For example, an antibody can be made aglycosylated (i.e., the antibody is devoid of glycosylation). The glycosylation can be altered, for example, by increasing the affinity of the antibody for the "antigen." Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more framework glycosylation sites of the variable region, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. One such approach is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[00169] Additionally or alternatively, an antibody may be made that has an altered type of glycosylation, for example, a hypofucosylated antibody that has reduced amounts of fucosyl residues or an antibody that has increased bisected GlcNac structures. Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such carbohydrate modifications may be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and may be used as the host cells in which to express the recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hang et al describes a cell line with a functionally disrupted FUT8 gene, encoding a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 to Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to carbohydrates linked by Asn(297), which also results in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 to Umana et al. describes cell lines genetically engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the genetically engineered cell lines exhibit increased bisected GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17: 176–180).

[00170] Another modification of antibodies that is contemplated by the invention is pegylation. An antibody may be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, is typically reacted with polyethylene glycol (PEG), e.g., a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Pegylation may be accomplished by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous water-soluble reactive polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the invention. See, for example, EP 0,154,316 by Nishimura et al. and EP 0,401,384 by Ishikawa et al.

[00171] Another modification of antibodies that is contemplated by the invention is a protein conjugate or fusion of at least the antigen-binding region of the antibody of the invention to serum protein, e.g., human serum albumin or a fragment thereof, to increase the half-life of the resulting molecule. Such an approach is described, for example, in Ballance et al. EP 0322094.

[00172] Another possibility is a fusion of at least the antigen-binding region of the antibody of the invention to proteins capable of binding to serum proteins, e.g. human serum albumin, to increase the half-life of the resulting molecule. Such an approach is described, e.g., in Nygren et al., EP 0,486,525.

Methods for the genetic creation of altered antibodies

[00173] As discussed above, anti-sclerostin antibodies having VH and VL sequences or full-length heavy and light chain sequences set forth herein can be used to create novel anti-sclerostin antibodies by modifying full-length heavy and/or light chain sequences, VH and/or VL sequences, or the constant region(s) appended thereto. Thus, in another aspect of the invention, structural features of an anti-sclerostin antibody of the invention are used to create structurally related anti-sclerostin antibodies that retain at least one functional property of the antibodies of the invention, e.g., binding to human sclerostin, and that also inhibit one or more functional properties of sclerostin (e.g., receptor binding, prevention or attenuation of osteolysis).

[00174] For example, one or more CDR regions of the antibodies of the present invention, or mutations thereof, may be recombinantly combined with framework regions and/or other known CDRs to create additional recombinantly engineered anti-sclerostin antibodies of the invention, as discussed above. Other types of modifications include those described in the previous section. The starting material for the genetic engineering method is one or more of the VH and/or VL sequences provided herein, or one or more CDR regions thereof. To create the genetically engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) an antibody that possesses one or more of the VH and/or VL sequences provided herein, or one or more CDR regions thereof. Instead, the information contained in the sequence(s) is used as the starting material to create a "second generation" sequence(s) derived from the original sequence(s), and then the "second generation" sequence(s) is prepared and expressed as a protein.

[00175] Accordingly, in another embodiment, the invention provides a method for preparing an anti-sclerostin antibody consisting of: a heavy chain variable region antibody sequence having a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-11, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 12-22, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 23-33; and a light chain variable region antibody sequence having a CDR1 sequence selected from the group consisting of SEQ ID NOs: 34-44, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 45-55, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 56-66; altering at least one amino acid residue within the antibody heavy chain variable region sequence and/or the antibody light chain variable region sequence to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein.

[00176] Accordingly, in another embodiment, the invention provides a method for preparing an anti-sclerostin antibody optimized for expression in a mammalian cell consisting of: a full-length heavy chain antibody sequence having a sequence selected from the group of SEQ ID NOs: 111-121; and a full-length light chain antibody sequence having a sequence selected from the group of 122-132; altering at least one amino acid residue within the full-length heavy chain antibody sequence and/or the full-length light chain antibody sequence to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein.

[00177] The altered antibody sequence may also be prepared by screening antibody libraries having fixed CDR3 sequences selected from the group consisting of SEQ ID NO: 23-33 or minimal essential binding determinants as described in US20050255552 and diversity in CDR1 and CDR2 sequences. The screening may be performed according to any screening technology suitable for screening antibodies from antibody libraries, such as phage display technology.

[00178] Standard molecular biology techniques can be used for the preparation and expression of the altered antibody sequence. The antibody encoded by the altered antibody sequence(s) is one that retains one, some, or all of the functional properties of the anti-sclerostin antibodies described herein, which functional properties include, without limitation, specifically binding to human sclerostin; and the antibody exhibits at least one of the following functional properties: the antibody blocks the inhibitory effect of sclerostin in a cellular Wnt signaling assay, the antibody blocks the inhibitory effect of sclerostin in a cellular mineralization assay, or blocks the inhibitory effect of sclerostin in a Smad1 phosphorylation assay, or the antibody inhibits the binding of sclerostin to LRP-6, or the antibody increases bone formation and bone mass and density.

[00179] The altered antibody may exhibit one or more, two or more, or three or more of the functional properties discussed above.

[00180] The functional properties of the altered antibodies can be assessed using standardized assays available in the art and/or described herein, such as those set forth in the examples (e.g., ELISAs).

[00181] In certain embodiments of the antibody genetic engineering methods of the invention, mutations can be introduced randomly or selectively throughout all or part of an anti-sclerostin antibody coding sequence, and the resulting modified anti-sclerostin antibodies can be evaluated for binding activity and/or other functional properties, as described herein. Mutational methods have been described in the art. For example, PCT Publication WO 02/092780 to Short describes methods for creating and evaluating antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication WO 03/074679 to Lazar et al describes methods of using computational evaluation methods to optimize the physicochemical properties of antibodies.

Nucleic acid molecules encoding antibodies of the invention

[00182] Another aspect of the invention pertains to nucleic acid molecules encoding the antibodies of the invention. Examples of parental full-length light chain nucleotide sequences are shown in SEQ ID NOs: 144-145. Examples of parental full-length heavy chain nucleotide sequences are shown in SEQ ID NOs: 133-134. Examples of full-length light chain nucleotide sequences optimized for expression in a mammalian cell are shown in SEQ ID NOs: 146-154. Examples of full-length heavy chain nucleotide sequences optimized for expression in a mammalian cell are shown in SEQ ID NOs: 135-143.

[00183] Nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified from other cellular components or other contaminants, e.g., other nucleic acids or cellular proteins, by standard techniques, including alkaline/SDS treatment, CsCl ligation, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. 1987 "Current Protocols in Molecular Biology", Greene Publishing and Wiley Interscience, New York. A nucleic acid of the invention may be, e.g., DNA or RNA, and may or may not contain intronic sequences. In one embodiment, the nucleic acid is a cDNA molecule. The nucleic acid may be present in a vector, such as a phage display vector, or in a recombinant plasmid vector.

[00184] The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas made by transgenic mice carrying human immunoglobulin genes, as further described below), cDNAs encoding the antibody light and heavy chains made by the hybridoma can be obtained by standardized PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), the nucleic acid encoding the antibody can be recovered from multiple phage clones that are members of the library.

[00185] After the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or an scFv gene. In such manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA molecule, or to a fragment encoding another protein, for example, an antibody constant region or a flexible linker. The term "operatively linked" as used in this context means that the two DNA fragments are joined in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such that the protein is expressed under the control of a desired promoter.

[00186] Isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2, and CH3). Human heavy chain constant region gene sequences are known in the art (see, e.g., Kabat, E. A., et al., 1991 "Sequences of Proteins of Immunological Interest", Fifth Edition, "U.S. Department of Health and Human Services", NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standardized PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. Preferably, the heavy chain constant region is selected from IgG2 isotypes. For a Fab fragment heavy chain gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only the CH1 heavy chain constant region.

[00187] Isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the DNA encoding VL to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, E. A., et al., 1991 "Sequences of Proteins of Immunological Interest", Fifth Edition, "U.S. Department of Health and Human Services", NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standardized PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[00188] To create an scFv gene, the DNA fragments encoding VH- and VL are operably ligated to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, e.g., Bird et al., 1988 Science 242: 423-426; Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., 1990 Nature 348: 552-554).

Generation of monoclonal antibodies of the invention

[00189] Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standardized somatic cell hybridization technique of Kohler and Milstein, 1975 Nature 256: 495. Many techniques can be employed for monoclonal antibody production, e.g., viral or oncogenic transformation of B lymphocytes.

[00190] One animal system for the preparation of hybridomas is the murine system. Hybridoma production in the mouse is a well-established procedure. Immunization protocols and techniques for the isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[00191] The chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and genetically engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, murine CDR regions can be inserted into a human framework using methods known in the art. See, for example, U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[00192] In one embodiment, the antibodies of the invention are human monoclonal antibodies. Such human monoclonal antibodies directed against sclerostin may be generated using transgenic or transchromosomal mice that carry parts of the human immune system, rather than the mouse system. Such transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

[00193] The HuMAb® mouse (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human immunoglobulin heavy chain (μ and Y) and K light chain sequences, along with targeted mutations that inactivate the endogenous μ and K chain loci (see, e.g., Lonberg, et al., 1994 Nature 368 (6474): 856-859). Consequently, mice exhibit reduced expression of mouse IgM or k, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgGk monoclonal antibody (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N., 1994 "Handbook of Experimental Pharmacology," 113: 49-101; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol. 13: 65-93 and Harding, F. and Lonberg, N., 1995 Ann. N. Y. Acad. Sci. 764: 536-546). The preparation and use of HuMAb mice, and the genomic modifications carried out by these mice, are further described in Taylor, L. et al., 1992 Nucleic Acids Research 20: 6287-6295; Chen, J. et al., 1993 International Immunology 5: 647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94: 3720-3724; Choi et al., 1993 Nature Genetics 4: 117-123; Chen, J. et al., 1993 EMBO J. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152: 2912-2920; Taylor, L. et al., 1994 International Immunology 579-591; and Fishwild, D. et al., 1996 Nature Biotechnology 14: 845-851, the contents of which are specifically incorporated herein by reference in their entirety. See also U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, all to Lonberg and Kay; U.S. Patent No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92/103918, WO 93/12227, WO 94/25585, WO 97/113852, WO 98/24884, and WO 99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 to Korman et al.

[00194] In another embodiment, human antibodies of the invention can be raised using a mouse that carries human immunoglobulin sequences in transgenes and transchromosomes, such as a mouse carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice," are described in detail in PCT Publication WO 02/43478 to Ishida et al.

[00195] In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise the anti-sclerostin antibodies of the invention. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

[00196] In addition, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to raise the anti-sclerostin antibodies of the invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, termed "TC mice," can be used; such mice are described in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA 97: 722-727. In addition, cows carrying both human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., 2002 Nature Biotechnology 20: 889-894), and can be used to raise the anti-sclerostin antibodies of the invention.

[00197] Human monoclonal antibodies of the invention can also be prepared using phage display methods to screen human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art or described in the examples below. See, for example: U.S. Patent Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[00198] The human monoclonal antibodies of the invention may also be prepared using SCID mice in which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

Generation of hybridomas that produce human monoclonal antibodies

[00199] For the generation of hybridomas producing human monoclonal antibodies of the invention, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, e.g., a mouse myeloma cell line. The resulting hybridomas can be evaluated for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to one-sixth the number of non-secretory mouse myeloma cells P3X63-Ag8.653 (ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2 x 145 in flat-bottomed microtiter plates, followed by a two-week incubation in selective medium containing 20% fetal clone serum, 18% "653" conditioned media, 5% Origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamicin, and 1X HAT (Sigma; HAT is added 24 hours after fusion). After approximately two weeks, cells can be cultured in medium in which HAT is replaced with HT. Individual wells can then be assayed by ELISA for human monoclonal antibodies IgM and IgG. After intense hybridoma growth has occurred, normal medium can be observed after 10-14 days. Antibody-secreting hybridomas can be replaced, re-evaluated, and, if still positive for human IgG, monoclonal antibodies can be subcloned at least twice by limiting dilution. Stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

[00200] To purify human monoclonal antibodies, selected hybridomas can be grown in two-liter spinner bottles for monoclonal antibody purification. Supernatants can be filtered and concentrated prior to affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at -80°C.

Generation of transfectomas that produce monoclonal antibodies

[00201] Antibodies of the invention may also be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods, as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202).

[00202] For example, for the expression of antibodies, or antibody fragments thereof, DNAs encoding partial or full-length heavy and light chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNAs can be inserted into expression vectors in such a way that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" means that an antibody gene is ligated into a vector in such a way that the transcriptional and translational control sequences within the vector perform their desired function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites in the antibody gene fragment and vector, or blunt-end ligation if no restriction site is present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors that already encode heavy chain constant regions and light chain constant regions of the desired isotype, such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain by a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[00203] In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel ("Gene Expression Technology. Methods in Enzymology" 185, Academic Press, San Diego, CA 1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as, for example, the choice of host cell to be transformed, the level of protein expression desired, etc. Regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, e.g., promoters and/or enhancers derived from cytomegalovirus (CMV), Simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, nonviral regulatory sequences such as the ubiquitin promoter or the β-globin promoter can be used. In addition, regulatory elements composed of sequences from different sources can be used, e.g., the SRa promoter system, which contains sequences from the SV40 early promoter and the human T-cell leukemia virus type 1 long terminal repeat (Takebe, Y. et al., 1988 Mol. Cell. Biol. 8: 466–472).

[00204] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, e.g., sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all to Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, e.g., G418, hygromycin, or methotrexate, in a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and the neo gene (for selection with G418).

[00205] For expression of the heavy and light chains, the expression vector (or expression vectors) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for introducing exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, and the like. It is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells. Expression of antibodies in eukaryotic cells, in particular mammalian host cells, is discussed because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic antibody expression genes have been reported to be ineffective for producing high yields of active antibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today 6: 12-13).

[00206] Mammalian host cells for the expression of the recombinant antibodies of the invention include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, described by Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R.J. Kaufman and P.A. Sharp, 1982 Mol. Biol. 159:601-621, NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036, and EP 338,841. When recombinant expression vectors encoding DHFR genes are used, the recombinant expression vectors encoding DHFR genes are used. antibodies are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Bispecific molecules

[00207] In another aspect, the present invention features bispecific or multispecific molecules comprising an anti-sclerostin antibody, or a fragment thereof, of the invention. An antibody of the invention, or antigen binding regions thereof, may be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the invention may, in fact, be derivatized or linked to more than one functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule results.

[00208] Accordingly, the present invention includes bispecific molecules comprising at least a first binding specificity for sclerostin and a second binding specificity for a second target epitope. For example, the second target epitope is another sclerostin epitope different from the first target epitope. Another example is a bispecific molecule comprising at least a first binding specificity for sclerostin and a second binding specificity for an epitope within Dkk-1. Another example is a bispecific molecule comprising at least a first binding specificity for sclerostin and a second binding specificity for an epitope within LRP4.

[00209] Additionally, for an invention in which the bispecific molecule is multispecific, the molecule may further include a third binding specificity in addition to the first and second target epitopes.

[00210] In one embodiment, the bispecific molecules of the invention comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, for example, a Fab, Fab', F(ab')2, Fv, or a single chain Fv. The antibody can also be a light chain or heavy chain dimer, or any minimal fragment thereof such as, for example, an Fv or a single chain construct, as described in Ladner et al. in U.S. Patent No. 4,946,778, the contents of which are expressly incorporated by reference.

[00211] Other antibodies that can be used in the bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.

[00212] The bispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated together. When the binding specificities are proteins or peptides, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, M. A. et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229: 81-83, and Glennie et al., 1987 J. Immunol. 139: 2367-2375. Conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[00213] When the binding specificities are antibodies, they may be conjugated by sulfhydryl bridges of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, e.g., one, prior to conjugation.

[00214] Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, or linker x Fab fusion protein. A bispecific molecule of the invention may be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Patent No. 5,013,653, U.S. Patent No. 5,258,498 and U.S. Patent No. 5,482,858.

[00215] Binding of the bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

Multivalent antibodies

[00216] In another aspect, the present invention provides multivalent compounds comprising at least two identical or different antigen-binding portions of the sclerostin-binding antibodies of the invention. Preferably, considering the trimeric nature of sclerostin, the compounds of the invention provide at least three or four antigen-binding portions of the antibodies. The antigen-binding portions may be linked together via protein fusion or covalent or non-covalent linkage. Alternatively, linkage methods for bispecific molecules have been described. Tetravalent compounds may be obtained, for example, by cross-linking the antibodies of the invention with an antibody that binds to the constant regions of the antibodies of the invention, for example, the Fc or hinge region.

[00217] Trimerization domains are described, for example, in Borean patent EP 1,012,280 B1. Pentamerization modules are described, for example, in PCT/EP97/05897.

Pharmaceutical compositions

[00218] In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, that contains one or a combination of monoclonal antibodies, or antigen-binding region(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies that bind to different epitopes on the target antigen or that have complementary activities.

[00219] The pharmaceutical compositions of the invention may also be administered in combination therapy, that is, combined with other agents. For example, the combination therapy may include an anti-sclerostin antibody of the present invention combined with at least one other anti-inflammatory or anti-osteoporotic agent. Examples of therapeutic agents that may be used in combination therapy are described in more detail below in the section on uses of the antibodies of the invention. The compositions are preferably formulated at physiological pH.

[00220] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. The carrier must be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[00221] The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects (see, e.g., Berge, S.M., et al., 1977 J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorus, and the like, and non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[00222] A pharmaceutical composition of the invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[00223] Examples of suitable aqueous and non-aqueous vehicles that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as, for example, glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as, for example, olive oil, and injectable organic esters such as, for example, ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials such as, for example, lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[00224] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, e.g., paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable dosage form may be produced by the inclusion of agents that delay absorption, e.g., aluminum monostearate and gelatin.

[00225] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except where any media or agents are incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplemental active compounds may also be incorporated into the compositions.

[00226] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Adequate fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, may be included in the composition. Prolonged absorption of injectable compositions may be produced by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[00227] Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the ingredients enumerated above, as required, followed by sterilization by microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying (lyophilization) which generate a powder of the active ingredient plus any additional desired ingredient from a solution thereof previously sterilized by filtration.

[00228] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the individual treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent total, this amount will range from about 0.01 percent to about ninety-nine percent active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent active ingredient in combination with a pharmaceutically acceptable carrier.

[00229] Dosage regimens are adjusted to provide the desired optimum response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dosage may be reduced or increased proportionately as dictated by the demands of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form to facilitate administration and to standardize dosage. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the invention are dictated by and depend directly on the unique characteristics of the active compound and the particular therapeutic effect to be obtained, and on the inherent limitations of the art of producing such an active compound for the treatment of sensitivity in subjects.

[00230] For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more typically 0.01 to 5 mg/kg, of the host's body weight. For example, dosages may be 0.3 mg/kg of body weight, 1 mg/kg of body weight, 3 mg/kg of body weight, 5 mg/kg of body weight, or 10 mg/kg of body weight, or within the range of 1-10 mg/kg. An exemplary treatment regimen consists of administration once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every 3 months, or once every three to six months. Dosage regimens for an anti-sclerostin antibody of the invention include 1 mg/kg of body weight or 3 mg/kg of body weight by intravenous administration, with the antibody being given using one of the following dosage regimens: every four weeks for six doses, then every three months; every three weeks; 3 mg/kg of body weight once, followed by 1 mg/kg of body weight every three weeks.

[00231] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously or sequentially, whereupon the dosage of each antibody administered falls within the indicated ranges. The antibody is typically administered on multiple occasions. The intervals between single dosages may be, for example, weekly, monthly, every three months, or yearly. The intervals may also be irregular, as indicated by measurement of blood levels of antibody to the target antigen in the patient. In some methods, the dosage is adjusted to obtain a plasma antibody concentration of about 1-1,000 μg/ml, and in some methods, about 25-300 μg/ml.

[00232] Alternatively, the antibody may be administered as a sustained-release formulation, at which point less frequent administration is required. Dosage and frequency may vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until disease progression is slowed or terminated or until the patient shows partial or complete improvement in disease symptoms. The patient may then be placed on a prophylactic regimen.

[00233] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied in order to obtain an amount of the active ingredient that is effective in obtaining the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions of the present invention employed, or the ester, salt, or amide thereof, the route of administration, the timing of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health, and previous medical history of the patient treated, and similar factors well known in the medical art.

[00234] A "therapeutically effective dosage" of an anti-sclerostin antibody of the invention may result in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease.

[00235] A composition of the present invention may be administered by one or more routes of administration using one or more of several methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired results. Routes of administration for the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[00236] Alternatively, an antibody of the invention may be administered by a non-parenteral route, for example, by topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[00237] The active compounds may be prepared with carriers that will protect the compound against rapid release, e.g., a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biocompatible and biodegradable polymers may be used, e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are proprietary or generally known to those skilled in the art. See, e.g., "Sustained and Controlled Release Drug Delivery Systems," J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[00238] The therapeutic compositions can be administered with medical devices known in the art. For example, in one embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, for example, the devices shown in U.S. Patent Nos. 5,399,163 , 5,383,851 , 5,312,335 , 5,064,413 , 4,941,880 , 4,790,824 or 4,596,556 . Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603 , which shows an implantable microinfusion pump for delivering medication at a controlled rate; U.S. Patent No. 4,486,194 , which shows a therapeutic device for administering medication through the skin; U.S. Patent No. 4,447,233, which teaches a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which teaches a variable-flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which teaches an osmotic drug delivery system having multi-chambered compartments; and U.S. Patent No. 4,475,196, which teaches an osmotic drug delivery system. These patents are incorporated herein by reference. Many other implants, delivery systems, and modules of this type are known to those skilled in the art.

[00239] In certain embodiments, human monoclonal antibodies of the invention can be formulated to ensure proper delivery in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, into liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents 4,522,811 , 5,374,548 , and 5,399,331 . Liposomes can comprise one or more moieties that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, V.V. Ranade, 1989 J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al.); mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun. 153:1,038); antibodies (P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M. Owais et al., 1995 Antimicrob. Agents Chernother. 39:180); surfactant protein A receptor (Briscoe et al., 1995 Am. J. Physiol. 1,233:134); p120 (Schreier et al., 1994 J. Biol. Chem. 269:9,090); see also K. Keinanen; M. L. Laukkanen, 1994 FEBS Lett. 346: 123; J. J. Killion; I.J. Fidler, 1994 Immunomethods 4: 273.

Uses and methods of the invention

[00240] The antibodies of the present invention have diagnostic and therapeutic utilities in vitro and in vivo. For example, these molecules can be administered to cells in culture, e.g., in vitro or in vivo, or to a subject, e.g., in vivo, to treat, prevent, or diagnose various disorders. The term "subject" as used herein is intended to include both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals, and non-mammals, e.g., non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles.

[00241] The methods are particularly suitable for the treatment, prevention or diagnosis of sclerostin-related disorders and/or aberrant bone mineral density disorders, e.g. osteoporosis.

[00242] The invention also provides methods for increasing the mineral content and/or mineral density of bones. The compositions of the present invention may also be useful for improving the end result in orthopedic procedures, dental procedures, implant surgery, joint replacement, bone grafting, cosmetic bone surgery, and bone repair such as fracture healing, nonunion healing, delayed union healing, and facial reconstruction. One or more compositions may be administered before, during, and/or after the procedure, replacement, grafting, surgery, or repair.

[00243] As used herein, the term "sclerostin-related disorder" includes disorders in which bone mineral density (BMD) is abnormally and/or pathologically low relative to healthy individuals. Disorders characterized by low BMD and/or bone fragility include, without limitation, primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis (osteonecrosis), fractures and implant healing (dental implants and hip implants), bone loss due to other disorders (e.g., associated with HIV infection, cancers, or arthritis). Other "sclerostin-related disorders" include, without limitation, rheumatoid arthritis, osteoarthritis, arthritis, and the formation and/or presence of osteolytic lesions.

[00244] As used herein, the term "sclerostin-related disorder" includes conditions associated with or characterized by aberrant levels of sclerostin. These include cancers and osteoporotic conditions (e.g., osteoporosis or osteopenia), some of which overlap with "sclerostin-related disorders" as defined herein. The term "sclerostin-related cancers" may include myeloma (e.g., multiple myeloma with osteolytic lesions), breast cancer, colon cancer, melanoma, hepatocellular cancer, epithelial cancer, esophageal cancer, brain cancer, lung cancer, prostate cancer, or pancreatic cancer, as well as any metastases thereof.

[00245] The term "sclerostin-related disorder" may also include renal and cardiovascular conditions caused, at least in part, by the expression of sclerostin in the kidney and cardiovascular system. Such disorders include, but are not limited to, renal disorders such as, for example, glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, glomerular lesions associated with systemic diseases such as, for example, systemic lupus erythematosus, Goodpasture syndrome, multiple myeloma, diabetes, polycystic kidney disease, neoplasia, sickle cell disease, and chronic inflammatory diseases), tubular diseases (e.g., acute tubular necrosis and acute renal failure, polycystic kidney disease, medullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug- and toxin-induced tubulointerstitial nephritis, hypercalcemic nephropathy, and hypokalemic nephropathy), acute and rapidly progressive renal failure, chronic renal failure, nephrolithiasis, gout, vascular diseases (e.g., hypertension and nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic renal disease, diffuse cortical necrosis, and renal infarcts) or tumors (e.g., renal cell carcinoma and nephroblastoma).

[00246] Said disorders also include, without limitation, cardiovascular disorders such as, for example, ischemic heart disease (e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, pulmonary ischemic heart disease, valvular ischemic heart disease (e.g., rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (e.g., valvular and vascular obstructive lesions, atrial or ventricular septal defect, and patent ductus arteriosus), or myocardial disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy).

[00247] When antibodies to sclerostin are administered together with another agent, the two may be administered in any order (i.e., sequentially) or simultaneously.

[00248] According to a further embodiment of the invention, the antibodies of the invention can be employed as an adjunct or adjuvant to another therapy, for example a therapy using a bone resorption inhibitor, for example as a therapy for osteoporosis, in particular a therapy using calcium, a calcitonin or an analogue or derivative thereof, for example salmon, eel or human calcitonin, calcilytics, calcimimetics (e.g. cinacalcet), a steroid hormone, for example an estrogen, a partial estrogen agonist or estrogen-progestogen combination, a SERM (Selective Estrogen Receptor Modulator), for example raloxifene, lasofoxifene, bazedoxifene, arzoxifene, FC1271, Tibolone (Livial ®), a SARM (Selective Androgen Receptor Modulator), a RANKL antibody (e.g. denosumab), a cathepsin K inhibitor, vitamin D or an analogue thereof or PTH, a PTH fragment or a PTH derivative, e.g., PTH (1-84) (e.g., Preos™), PTH (1-34) (e.g., Forteo™), PTH (1-36), PTH (1-38), PTH (1-31)NH2 or PTS 893. According to another embodiment, the antibodies of the invention can be used in combination with other current therapeutic approaches for osteoporosis, including bisphosphonates (e.g., Fosamax™ (alendronate), Actonel™ (risedronate sodium), Bonviva™ (ibandronic acid), Zometa™ (zoledronic acid), Aclasta™/Reclast™ (zoledronic acid), olpadronate, neridronate, skelid, bonefos), statins, anabolic steroids, lanthanum salts and strontium, and sodium fluoride.

[00249] In a specific embodiment, the antibodies of the invention may be administered in combination with an LRP4-modulating agent, i.e., an agent that modulates the expression or activity of LRP4, e.g., an LRP4-neutralizing antibody.

[00250] In another specific embodiment, the antibodies of the invention may be administered in combination with a DKK1-modulating agent, i.e., an agent that interferes with or neutralizes Dkk-1-mediated antagonism of Wnt signaling, e.g., a DKK1-neutralizing antibody.

[00251] Accordingly, the invention also provides the use of an antibody or a functional protein of the invention and (i) zoledronic acid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4 antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents (calcilytics), in the manufacture of a medicament for the treatment of a pathological disorder that is mediated by sclerostin or that is associated with an increased level of sclerostin.

[00252] In another embodiment, the invention provides the use of an antibody or a functional protein of the invention in the manufacture of a medicament for the treatment of a pathological disorder that is mediated by sclerostin or that is associated with an increased level of sclerostin, wherein the medicament is used in conjunction with (i) zoledronic acid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4 antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents (calcilytics).

[00253] In another embodiment, the invention provides the use of (i) zoledronic acid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4 antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents (calcilytics), in the manufacture of a medicament for the treatment of a pathological disorder that is mediated by sclerostin or that is associated with an increased level of sclerostin, wherein the medicament is used in conjunction with an antibody or a functional protein of the invention.

[00254] In another embodiment, the invention provides the use of an antibody or a functional protein of the invention in the manufacture of a medicament for the treatment of a pathological disorder that is mediated by sclerostin or that is associated with an increased level of sclerostin, wherein the patient has received a prior administration of (i) zoledronic acid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4 antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents (calcilytics).

[00255] In another embodiment, the invention provides the use of (i) zoledronic acid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4 antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents (calcilytics), in the manufacture of a medicament for the treatment of a pathological disorder that is mediated by sclerostin or that is associated with an increased level of sclerostin, wherein the patient has received a prior administration of an antibody or a functional protein of the invention.

[00256] In one embodiment of the above combinations, hPTH is hPTH(1-34).

[00257] In one embodiment, the antibodies of the invention can be used to detect levels of sclerostin, or levels of cells containing sclerostin. This can be achieved, for example, by contacting a sample (e.g., an in vitro sample) and a control sample with the anti-sclerostin antibody under conditions that allow the formation of a complex between the antibody and sclerostin. Any complexes formed between the antibody and sclerostin are detected and compared in the sample and the control. For example, standard detection methods well known in the art, e.g., ELISA and flow cytometry assays, can be performed using the compositions of the invention.

[00258] Accordingly, in one aspect, the invention further provides methods for detecting the presence of sclerostin (e.g., human sclerostin antigen) in a sample, or for measuring the amount of sclerostin, comprising contacting the sample, and a control sample, with an antibody of the invention, or an antigen binding region thereof, that specifically binds to sclerostin, under conditions that allow the formation of a complex between the antibody or portion thereof and sclerostin. The formation of a complex is then detected, wherein a difference in complex formation between the sample compared to the control sample is indicative of the presence of sclerostin in the sample.

[00259] Also included within the scope of the invention are kits consisting of the compositions (e.g., antibodies, human antibodies, and bispecific molecules) of the invention and instructions for use. The kit may further contain at least one additional reagent, or one or more additional antibodies of the invention (e.g., an antibody that has a complementary activity that binds to an epitope on the target antigen distinct from the first antibody). For example, such kits may comprise an antibody or a functional protein of the invention and one or more of (i) zoledronic acid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4 antibody, (v) hPTH, and (vi) parathyroid hormone releasing agents (calcilytics). Kits typically include a label indicating the intended use of the kit contents. The term "label" includes any printed material, or proprietary material provided with the kit, or otherwise accompanying the kit.

[00260] After the invention has been fully described, it will be further illustrated by the following examples and claims, which are illustrative and are not intended to be limiting. Those skilled in the art will recognize or be able to verify, using at most routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are included within the scope of the present invention and the claims. The contents of all references cited throughout this application, including granted patents and published patent applications, are hereby incorporated by reference.

EXAMPLES FUNCTIONAL TESTS ALKALINE PHOSPHATASE (ALP) ASSAY Cells

[00261] MC3T3 1b cells are a clone of MC3T3-JP cells expressing OSE2-luc. This clone was obtained after stable transfection of MC3T3-JP (mouse osteoblast MC3T3-E1, clone from Japan, Jp; a kind gift from Dr. T. Kokkubo, Novartis, Japan) using 8x-OSE2wt-mOG2luca in pcDNA3.1+.

Culture medium

[00262] MC3T3-1b cells were routinely cultured in minimal essential medium alpha (MEMα; Invitrogen, Catalog No.: 22561021) supplemented with 10% fetal calf serum (FCS; Amimed Catalog No.: 2-01F100-I), 2 mM L-glutamine (Gibco Catalog No.: 25030-024), 50 IU penicillin/50 μg/mL streptomycin (Amimed Catalog No.: 4-01F00-H), and 10 mM Hepes (Gibco Catalog No.: 15630-056) and 0.75 mg/mL G418 (Gibco Catalog No.: 10131-027) (culture maintenance medium).

Stock solutions

[00263] Ten mM ascorbic acid (Wako Pure Chemical Catalog No. 013-12061) in DMEM-LG, 14 nM BMP-2 (R&D Catalog No. 355-BM-010) in 5 mM acetic acid and 0.1% bovine serum albumin (BSA, Sigma Catalog No. A-8806); 1 M β-glycerophosphate (Sigma Catalog No. G9891) in Tyrode's solution; Tyrode's solution: 9.72 g Tyrode's salt (Sigma Catalog No. T2145) and 1 g NaHCO3 in 1 L H2O, ALP substrate buffer: 25 mM glycine and 0.5 mM MgCl2, pH 10.5; ALP substrate solution: 5 mg p-nitrophenyl phosphate (Sigma 104 substrate, Sigma Catalog No. 50942-200TAB) in 3.75 ml ALP substrate buffer, pH 10.5; 1 mM p-nitrophenol (Sigma Catalog No. 104-1) in ALP substrate solution.

Rehearsal

[00264] For ALP assays, MC3T3 1b cells were grown in assay culture medium (which corresponds to the culture maintenance medium in the absence of G418). MC3T3 1b cells were seeded in 200 μL at 3 x 104 cells/mL if induction began 72 hours later, or 2 x 104 cells/mL if induction began 96 hours later. Plates were incubated for 72 hours or 96 hours at 37°C and 5% CO2 before induction was initiated using complete medium (by supplementing the culture medium with 10 mM b-glycerophosphate (bGP) and 50 μM ascorbic acid (AA)). Antibodies to be tested were diluted with complete medium. The antibody, together with BMP-2 (0.7 nM) and sclerostin (50 nM), was added to the wells (in triplicate) in a final volume of 200 μl of complete medium. On each plate, 4 internal controls were included in triplicate: a solvent control (BSA), BMP-2 control (0.7 nM), BMP-2 + sclerostin (BMP-2 (0.7 nM) and sclerostin (50 nM)) and a sclerostin control (50 nM). The plates were incubated for an additional 72 h at 37°C and 5% CO2. At the end of the induction period, the assay was terminated by removing the medium and adding 150 μl of ALP substrate solution (freshly prepared) to each well. The plates were incubated for 3–30 min. One hundred μL of 1 M NaOH was added to stop the reaction and the plates were shaken on a plate shaker. Optical density (OD) was read against a blank at 405 nm and ALP activity was calculated in nmol/min. Fifty nM sclerostin was used to obtain at least 70% inhibition of BMP-2-induced ALP production [0.7 nM BMP-2].

WNT ASSAY

[00265] This assay was established for the testing of antibodies based on the ability of sclerostin to inhibit Wnt1-mediated induction of the STF reporter gene.

Transfection of HEK293 cells

[00266] On day 1, HEK293 cells were seeded at 1.3-1.4 x 105 cells per well (in a volume of 0.5 ml) of a 24-well poly-D-lysine plate (BD-BioCoat Catalog No.: 356414) in DMEM (Gibco, Catalog No.: 61965-026) containing 10% fetal calf serum (FCS), 1% L-glutamine (Gibco, Catalog No.: 25030.024), 1% non-essential amino acids (Gibco, Catalog No.: 11140) without antibiotics. Transfection was performed on day 2 with Lipofectamine 2000 (Invitrogen, Catalog No.: 11668-019). For each well to be transfected, the following amount of plasmids was added to a final volume of 50 μL of OptiMEM®I (Gibco, Catalog No.: 31985-047): for control wells (pcDNA3+, 480 ng; SuperTopFlash (STF) 20 ng; phRL-CMV, 0.5 ng) and for Wnt1 treatment wells (pcDNA-wnt1, 20 ng; pcDNA3+, 460 ng; SuperTopFlash (STF) 20 ng; phRL-CMV, 0.5 ng).

[00267] In a second tube, 1.6 μL of Lipofectamine 2000 was diluted in 50 μL of OptiMEM®I and incubated at room temperature for 5 minutes. The contents of the two tubes were then mixed by adding the contents of the lipid tube to the DNA tube and incubated for 30 minutes at room temperature to allow DNA-lipid complex formation. The DNA-lipid complex (100 μL) was added equally to the wells and incubated at 37°C in 5% CO2 for 5 hours. At the end of the 5-hour incubation, the cells were ready for treatment with test reagents such as SOST, antibodies, etc.

Establishment of dose-dependent inhibition of SOST

[00268] To establish the dose-inhibition curve, a two-fold dilution series of rhSOST was prepared in DMEM containing 10% FCS, 1% L-glutamine, and 1% non-essential amino acids without antibiotics, starting at 160 nM. The stock rhSOST concentration was 260 μg/mL in DMEM with 1% FCS. Routinely, each condition was tested in duplicates. Therefore, 1 mL of medium for each condition was prepared and 450 μL was added per well after removing medium containing the transfection mixture from the wells. The treatment time was 18-20 hours. At the end of the incubation, the luciferase assay was performed as defined below.

Anti-SOST antibody test

[00269] Antibodies were premixed with SOST prior to addition to the cells. For this purpose, a DMEM medium (10% FCS, 1% L-glutamine and 1% non-essential amino acids without antibiotics) with 20 to 30 nM rhSOST was prepared. Then, different dilutions of tested antibodies were added to the medium containing SOST according to the experimental design. These mixtures were prepared 40 minutes before treatment. Each condition was routinely tested in duplicates. To do so, 1 mL of medium for each condition was prepared and 450 μL was added per well after removing medium containing the transfection mixture from the wells. The treatment time was 18-20 hours. At the end of the incubation, the luciferase assay was performed as defined below.

Luciferase assay

[00270] At the end of the incubation, the medium was removed, and 300 μL of 1X Passive Lysis Buffer (Promega, Catalog No.: E194A) was added to lyse the cells. Luciferase activity was then measured using the Dual-Glo Luciferase System (Promega, Catalog No.: E2940) with 30 μL of lysates in duplicates. The assay was performed according to the instruction booklet provided with the kit. Typically, 30 μL of Dual-Glo luciferase (firefly luciferase; for STF) and 30 μL of Dual-Glo Stop and Glo (Renilla luciferase; for transfection efficiency control) substrates were used. Luminescent signals were measured with the Mithras LB940 instrument (Berthold Technologies).

Data calculation

[00271] The ratio of firefly to Renilla luciferase was calculated. Final results are expressed by setting the value of Wnt1 without SOST to 1.

MINERALIZATION TEST Cells

[00272] MC3T3 1b cells are a clone of MC3T3-JP cells expressing OSE2-luc. This clone was obtained after stable transfection of MC3T3-JP (mouse osteoblast MC3T3-E1, clone from Japan, Jp; a kind gift from Dr. T. Kokkubo, Novartis, Japan) using 8x-OSE2wt-mOG2luc in pcDNA3.1+.

Culture medium

[00273] MC3T3-1b cells were routinely cultured in minimal essential medium alpha (MEMα; Invitrogen, Catalog No.: 22561021) supplemented with 10% fetal calf serum (FCS; Amimed Catalog No.: 2-01F100-I), 2 mM L-glutamine (Gibco Catalog No.: 25030-024), 50 IU penicillin/50 μg/mL streptomycin (Amimed Catalog No.: 4-01F00-H), and 10 mM Hepes (Gibco Catalog No.: 15630-056) and 0.75 mg/mL G418 (Gibco Catalog No.: 10131-027) (culture maintenance medium).

Mineralization test

[00274] Matrix-associated calcium deposited in the wells was determined in MC3T3-1b cells using the Calcium kit (Axon Lab, Catalog No.: AXON0012). Cells were seeded at 6 x 103 cells/well or 2 x 103 cells/well to increase cell responsiveness in 96-well plates in 100 μL of assay culture medium (culture maintenance medium without G418) and incubated for 3 days until they reached confluence. The assay culture medium was then changed and the compounds to be tested were added together with 10 mM b-glycerophosphate (bGP; Sigma Catalog No.: G9891) and 50 μM ascorbic acid (AA; Wako Pure Chemical Catalog No.: 013-12061). Prior to addition to the cells, sclerostin and the Fabs to be tested were preincubated in a separate plate for 2 h at room temperature; meanwhile, 96-well assay plates received 2.1 or 2.8 nM BMP-2 (R&D Systems, Catalog No.: 355-BM-010) prior to receiving the sclerostin-Fab mixture. The cells were incubated for 14 days, and the assay medium was replaced every 3–4 days. Briefly, at the end of the incubation, the cells were washed twice with 200 μL PBS/well, 50 μL 0.5 M HCl was added to each well, and the plates were frozen at −20°C for a minimum of 24 h. At the appropriate time, the plates were thawed at room temperature for 2 hours and 10 μL from each well was transferred to a new 96-well plate. Calcium Working Solution (1:5) was then added (200 μL) and 5-30 minutes later the plates were read at 595 nm in a microplate reader.

[00275] Absorbance was translated into μg of calcium according to a standard curve, the value of the control well (ascorbic acid and beta-glycerophosphate) was subtracted from the data of each well and the final results were expressed as % of mineralization induced by BMP-2.

WESTERN SMAD1 PHOSPHORYLATION Materials

[00276] - mouse osteoblast MC3T3-E1, (clone from Japan, courtesy of Dr. T. Kokkubo, Novartis, Japan)

[00277] - C3H10T1/2 cells (mouse embryo mesenchymal; ATCC, Catalog No.: CCL-226)

[00278] - Non-glycosylated SCL, derived from E. coli (Novartis, PSU5257)

[00279] - Non-glycosylated 15N SCL, derived from E. coli (Novartis, PSU11274)

[00280] - hSOST-APP, glycosylated, derived from HEK-EBNA (Novartis, BTP11100)

[00281] - rhSCL, glycosylated, derived from mouse myeloma cells (R&D Systems, Catalog No.: 1406-ST/CF)

[00282] - anti-hSOST antibody (R&D Systems, Catalog No.:AF1406)

[00283] - PhosphoSafe Extraction Reagent (Novagen, Catalog No.: 71296-3)

[00284] - Protease Inhibitor Cocktail Set III (Calbiochem, Catalog No.: 539134)

[00285] - NuPAGE Novex Tris-Acetate 7% Gel 1.5 mm, 15 wells (Invitrogen, Catalog No.: EA03585)

[00286] - SDS Tris-Acetate Running Buffer 20x (Invitrogen, Catalog No.: LA0041)

[00287] - NuPAGE Antioxidant (Invitrogen, Catalog No.:NP0005)

[00288] - Immobilon-P Transfer Membrane 0.45 μm (Millipore, Catalog No.: IPVH00010)

[00289] - Wattman chromatography paper (Merck, Catalog No.:3587600)

[00290] XCell SureLock Mini-Cell and XCell II Blot Module (Invitrogen)

[00291] - VersaMax microplate reader (Bucher)

Cell culture and extraction

[00292] MC3T3-E1 and C3H10T1/2 cells were routinely cultured in minimal essential medium alpha (MEMα; Invitrogen, Catalog No.: 22561-021) or in DMEM high glucose (Invitrogen, Catalog No.: 41965-039), respectively. All culture media were supplemented with 10% fetal calf serum (FCS; BioConcept Catalog No. 2-01F10-I, lot Z04459P), 2 mM L-glutamine (Invitrogen Catalog No. 25030-024), 50 IU penicillin/50 μg/mL streptomycin (Invitrogen Catalog No. 15140-122), and 10 mM Hepes (Invitrogen Catalog No. 15630-056) (culture maintenance medium).

[00293] Cells were seeded in culture maintenance medium in 6-well plates (3 ml/well) and grown to confluence (with 1.4 x 105 MC3T3-1b cells/well, confluence was reached on day 3; with 1.0 x 105 C3H10T1/2 cells/well, confluence was reached on day 3). After overnight serum depletion in culture medium containing 1% FBS, the medium was replaced with fresh medium supplemented with 1% FBS, BMP-6 (R&D Systems, Catalog No.: 507-BP), and the substance(s) to be tested. Prior to addition to the cells, BMP-6 and the test substance(s) were preincubated for 1 hour at room temperature, as described for a phospho-Smad 1/3/5 in C3H10T1/2 (Winkler, EMBO J., 2003, 22(23): 6,267-76). When anti-sclerostin antibodies were tested, they were preincubated with sclerostin overnight at 4°C before being incubated with 0.2 nM BMP-6 for 1 hour at room temperature and finally added to the confluent cells. After the appropriate treatment time, the cells were washed with 2 mL of ice-cold PBS. One hundred μL/well of PhosphoSafe Extraction Reagent and Protease Inhibitor Cocktail diluted 1:200 were then added to the cells, which were then incubated on ice for 5 minutes. The cells were scraped from the wells and transferred to a microcentrifuge tube. The cell extract was kept on ice for 15 minutes, interrupted by a vortexing step every 5 minutes. The cell extract was then centrifuged for 5 minutes at 16,000 g and 4°C. Finally, the supernatant was transferred to a fresh microcentrifuge tube for protein determination.

[00294] Protein concentration in cell lysate was determined using the BCA Protein Assay Kit according to the manufacturer's instructions. BSA was used as a standard. For denaturation, cell lysate was diluted 1:2 with Laemmli buffer (Bio-Rad, Catalog No.: 161-0737, containing freshly added β-Mercaptoethanol (1:20, Merck, Catalog No.: 1.12006)) and boiled for 5 minutes at 95°C. After cooling, samples were stored at -20°C until further use.

Western blot

[00295] The Smad (H-465) antibody (Santa Cruz, Catalog No.: sc-7153) is a rabbit polyclonal IgG raised against amino acids 1-465 representing full-length Smad1 of human origin. It should recognize Smad1, Smad2, Smad3, Smad5, and Smad8 of human, rat, and mouse origin. The phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428) antibody (Cell Signaling, Catalog No.: 9511) is a rabbit polyclonal IgG raised against a synthetic phosphopeptide corresponding to residues surrounding Ser463/465 of human mad5. It should detect endogenous levels of Smad1 only when dually phosphorylated at serine 463 and serine 465, as well as Smad5 and Smad8 only when phosphorylated at the equivalent sites of human, mouse, rat, mink and xenopus origin. The antibody does not cross-react with other Smad-related proteins.

[00296] The XCell SureLock Mini Cell System (Invitrogen) was prepared according to the manufacturer's instructions. Protein samples (2 μg for a Smad Western assay, 5 μg for a Phospho-Smad Western assay) were loaded in equal final volume onto a 7% NuPAGE Novex Tris-Acetate Gel in 1x Running Buffer. SeeBlue Plus2 Pre-stained Standard (10 μL, 1:10; Invitrogen, Catalog No.: LC5925) and MagicMark XP Western Protein Standard (10 μL, 1:100; (Invitrogen, Catalog No.: LC5602) were used as molecular weight markers. The gel was run for 75 minutes at constant voltage (150 V).

[00297] Blotting pads and filter papers were soaked in 700 mL of 1x NuPAGE Transfer Buffer. A PVDF transfer membrane was first soaked for 30 seconds in methanol and then transferred into 1x NuPAGE Transfer Buffer (Invitrogen, Catalog No.: NP0006, Transfer Buffer: Freshly prepared 10:1 Methanol). The gel cassette plates were separated with a gel knife, a pre-soaked filter paper was placed on top of the gel, and any trapped air bubbles were carefully removed. The plate was turned upside down onto Saran wrap, and after removal of the plate, the pre-soaked transfer membrane was placed on the gel and any air bubbles were removed. A second pre-soaked filter paper was placed on top and any air bubbles were removed. Two pre-embedded blotting blocks were placed on the center of the cathode of the Cell II Blot Module. The gel/membrane sandwich was carefully removed and placed on the blotting blocks (with the gel closest to the center of the cathode). Finally, 3 pre-embedded blotting blocks were placed on the membrane assembly and the anode center was added on top. The blot module was slid onto the guide rails in the lower buffer chamber and the wedge was locked into position according to the manufacturer's instructions. The blot module was filled with 1x NuPAGE Transfer Buffer until the gel/membrane assembly was covered. The outer buffer chamber was filled with 650 mL of deionized water and protein transfer to the PVDF membrane was performed at constant voltage (30 V) for 2 h.

[00298] After blotting, the membrane was first washed for 10 minutes in 0.05% Tween 20 in PBS and then blocked under gentle shaking for 1 hour in 25 mL of SuperBlock T20 Blocking Buffer at room temperature. The primary antibody (Phospho-Smad 1/5/8 or Smad (H-465)) was added to the membrane at a 1:1,000 dilution in SuperBlock T20 Blocking Buffer (Pierce, Catalog No.: 37516) and the membrane was incubated overnight at 4°C under shaking. The membrane was then washed 3 times for 10 minutes with 0.05% Tween 20 (Fluka, Catalog No. 93773) in PBS before being incubated with the RP-conjugated secondary antibody (1:1,000 in SuperBlock T20 Blocking Buffer) for 60 minutes at room temperature with shaking. At least 3 wash steps of 10 minutes each were performed before the membrane was incubated for 5 minutes with SuperSignal West Femto Substrate Working Solution (Pierce, Catalog No. 34095). Finally, the membrane was placed in a plastic bag and imaged with a Fluor-S MultiImager (Bio-Rad) and Camera. An optimal exposure time of 1 to 2 minutes was determined by comparing images obtained from 30 seconds to 5 minutes.

Data analysis

[00299] Chemiluminescence activities were measured using Quantity One (Bio-Rad) and EC50 values were calculated using XLfit4 software. Each phospho-Smad signal was normalized by its corresponding total Smad signal.

LRP6 / SCLEROSTIN ELISA

[00300] Ninety untreated 6-well microtiter plates were coated with 100 μL/well of LRP6/Fc (1 μg/mL, R&D Systems, Catalog No.: 1505-LR) diluted in PBS. As a control for nonspecific binding (NSB), some wells were filled with 100 μL/well of PBS. The plates were covered with plastic wrap and incubated overnight at room temperature. After coating, plates were washed 3 times with 200 μL^o of 0.05% Tween 20 (Fluka, Catalog No. 93773) in PBS, and wells were blocked for 1 h at 37°C by adding 300 μL/well of SuperBlock blocking buffer (Pierce, Catalog No. 37535) in TBS. After incubation, the blocking solution was removed and 100 μL/well of sclerostin (derived from E. coli, Novartis; 1–1,000 ng/ml) diluted in 1% BSA in PBS was added. Plates were incubated for 2 h at room temperature before being washed 3 times with 200 μL/well of 0.05% Tween 20 in PBS. Next, 100 μL/well of anti-sclerostin antibody (1 μg/mL) diluted in 1% BSA in PBS was added, and the plates were incubated for 2 h at room temperature before being washed 3 times with 200 μL/well of 0.05% Tween 20 in PBS. Finally, 100 μL/well of ALP-conjugated goat anti-IgG Ab (1:5,000; Sigma Catalog No.: A-7888) diluted in 1% BSA (Sigma Catalog No.: A-7888) in PBS was added for 1 h at room temperature, and the plates were then washed 3 times with 200 μL/well of 0.05% Tween 20 in PBS. To determine ALP, 100 μL/well of ALP substrate solution (Sigma, Catalog No.: S0942) (1 tablet per 5 mL of 1x diethanolamine substrate buffer; Pierce, Catalog No.: 34064) was added to the plates for 90 min and the optical density measured at 405 nm.

OVEREXPRESSION OF LRP4 IN HEK293 CELLS

[00301] HEK293 cells (ATCC Catalog No.: CRL-1573) were routinely cultured in DMEM/F12 (Invitrogen Catalog No.: 21331-020) supplemented with 10% FCS (BioConcept Catalog No.: 2-01F10-I, lot Z04459P), 2 mM L-glutamine (Invitrogen Catalog No.: 25030-024), 100 IU penicillin/100 μg/mL streptomycin (Invitrogen Catalog No.: 15140-122), and 10 mM HEPES (Invitrogen Catalog No.: 15630-056). Hek293 cells were seeded at 5 x 104 cells/well in a 48-well poly-D-Lysine plate format and incubated for 24 h prior to transfection with Lipofectamine 2000 (Invitrogen Catalog No. 11668-019). For each well to be transfected, the following amount of plasmids was added to a final volume of 25 μL of OptiMEM®I (Gibco, Catalog No.: 31985-047) and mixed gently: for control wells (pmaxGFP, 62.5 ng, pcDNA3+, 125 ng; SuperTopFlash (STF) 62.5 ng; SV40-driven Renilla luciferase plasmid, 0.75 ng) and for Wnt1 treatment wells (pmaxGFP, 62.5 ng, pcDNA-wnt1, 62.5 ng; pcDNA3+, 62.5 ng; SuperTopFlash (STF) 62.5 ng; SV40-driven Renilla luciferase plasmid, 0.75 ng) and for LRP4-Wnt1 (pcDNA3+-LRP4, 62.5 ng, pcDNA3+-wnt1, 62.5 ng; pcDNA3+, 62.5 ng; SuperTopFlash (STF) 62.5 ng; SV40-driven Renilla luciferase plasmid, 0.75 ng). In a second tube, 0.8 μL of Lipofectamine 2000 was diluted to a final volume of 25 μL of OptiMEM®I and incubated at room temperature for 5 minutes. The contents of the two tubes were then mixed by adding the contents of the lipid tube to the DNA tube and incubated for 30 minutes at room temperature to allow DNA-lipid complex formation. The DNA-lipid complex (50 μL) was added equally to the wells and incubated at 37°C in 5% CO2 for 5 hours. At the end of the 5-hour incubation, the cells were ready for a 20-hour treatment with test reagents such as SOST, DKK1 (R&D Catalog No. 1096-DK), antibodies, etc. The luciferase assay was performed as described under "Wnt assay" but with the addition of 150 μL of Passive Lysis Buffer instead.

OVEREXPRESSION OF LRP4 IN C28A2 CELLS

[00302] C28a2 cells (from Mary Goldring, Harvard Institutes of Medicine, Boston, MA, USA) were cultured in the same medium as HEK293 cells (see above), except that HEPES was not present and a non-essential amino acid supplement (Gibco Catalog No. 11140) was added. C28a2 cells were seeded at 1 x 105 cells/well in a 24-well plate format in antibiotic-free culture medium, and incubated overnight prior to transfection with Lipofectamine 2000 (Invitrogen Catalog No. 11668-019). For each well to be transfected, the following amount of plasmids (600 ng/well in total) was added to a final volume of 50 μL of OptiMEM®I (Gibco, Catalog No.: 31985047) and mixed gently: for control wells (SuperTopFlash (STF) 100 ng; SV40-driven Renilla luciferase plasmid, 2 ng and pcDNA3+, 500 ng to compensate) and for Wnt1 treatment wells (pcDNA-wnt1 plasmid, 100 ng; LRP5 plasmid, 100 ng; SuperTopFlash (STF) 100 ng; SV40-driven Renilla luciferase plasmid, 2 ng and pcDNA3+, 300 ng) and for LRP4-Wnt1 (pcDNA-wnt1 plasmid, 100 ng; LRP5 plasmid, 100 ng; LRP4 plasmid, 100 ng; SuperTopFlash (STF) 100 ng; SV40-driven Renilla luciferase plasmid, 2 ng; and pcDNA3+, 200 ng). In a second tube, 1.6 μL of Lipofectamine 2000 was diluted to 48.4 μL of OptiMEM®I and incubated at room temperature for 5 minutes. The contents of the two tubes were then mixed by adding the contents of the lipid tube to the DNA tube and incubated for 30 minutes at room temperature to allow DNA-lipid complex formation. The DNA-lipid complex (100 μL) was added equally to the wells and incubated at 37°C in 5% CO2 for 2 hours. At the end of the 2-h incubation, the transfection medium was exchanged for 450 μL/well of antibiotic-free medium, and the cells were incubated for 24 h. The cells were then treated for 20 h with test reagents such as SOST or DKK1 (R&D Catalog No.: 1096-DK). The luciferase assay was performed as described under “Wnt assay”.

KNOCKDOWN FOR LRP4 IN HEK293 CELLS

[00303] Hek293 Wnt1/STF/Renilla cells (a stable clone generated by stable transfection of Hek293 cells (ATCC Catalog No.: CRL-1573)) with Wnt1 expression plasmid, SuperTopFlash reporter plasmid, and an SV40-driven Renilla luciferase plasmid) were routinely cultured in DMEM 4,500 g/L glucose (Invitrogen Catalog No.: 41965-035) supplemented with 10% FCS (Amimed Catalog No.: 2-0F100-I), 2 mM L-glutamine (Invitrogen Catalog No.: 25030-081), 100 IU/mL penicillin/100 μg/mL streptomycin (Gibco catalog no. 15140-163), 6 μg/mL puromycin (Invitrogen catalog no. ant-pr-1), 150 μg/mL zeocin (Invitrogen catalog no. 45-0430), and 150 μg/mL hygromycin (Invitrogen catalog no. 10687-010). Selection antibiotics were left out during knockdown experiments. Cells were seeded at 0.6 x 105 cells/well in a poly-D-Lysine 24-well plate format and allowed to attach overnight prior to transfection of LRP4 siRNAs with HiPerFect (Qiagen catalog no. 301707). The LRP4 siRNA sense and antisense sequences used were as follows: LRP4a: TAAATTATCATAAAGTCCTAA /AGGACTTTATGATAATTTATT; (SEQ ID NOs: 160/161). LRP4b: ATAGTGGTTAAATAACTCCAG /GGAGTTATTTAACCACTATTT; (SEQ ID NOs: 162/163). LRP4c: TAAATTCTCGTGATGTGCCAT /GGCACATCACGAGAATTTATT; (SEQ ID NOs: 164/165). LRP4d: TTTCTTATAGCACAGCTGGTT /CCAGCTGTGCTATAAGAAATT; (SEQ ID NOs: 166/167). LRP4e: TAGACCTTTCCATCCACGCTG /GCGTGGATGGAAAGGTCTATT; (SEQ ID NOs: 168/169).

[00304] For each well to be transfected, two Eppendorf tubes were prepared: the first contained 0.2 μL of a 20 nM stock of one of the LRP4 siRNAs or 0.1 μL of a 20 nM stock of two different LRP4 siRNAs (final total siRNA concentration/well is 6.6 nM) and 50 μL of Optimem, and the second, 3 μL of HiPerFect and 47 μL of Optimem. The contents of the second tube were added to the first, vortexed briefly and left for 10 minutes at room temperature. One hundred μL of this mixture was then added to the respective well and the cells were incubated at 37°C under 5% CO2 for 30 hours. Next, the transfection mixture was removed and replaced with fresh antibiotic-free culture medium (450 μL/well) and allowed to incubate for an additional 24 h. Before treatment with SOST or DKK1, the medium was removed, replaced with fresh antibiotic-free culture medium containing the appropriate dilution of SOST or DKK1 (R&D Catalog No.: 1096-DK), and the cells were incubated for 20 h at 37°C under 5% CO2. Luciferase determination was then performed as described under “Wnt assay”.

Animal models

[00305] Eight-month-old female mice (n = 16/group, Charles River, France) received twice-weekly intravenous administration of anti-sclerostin antibody ANTIBODY A (25 mg/kg, h/mIgG2a) (MOR05813) or control antibody (anti-PC-h/mIgG2a). Control groups received daily subcutaneously 100 micrograms/kg PTH(1-34) or PBS vehicle. Treatment lasted 2.5 weeks for all animals. Half of the animals (n = 8/group) were sacrificed at that time point for histomorphometric analysis. Treatment continued for the remaining animals (n = 8/group) up to 5 weeks.

[00306] Bone mass and tibial geometry of the animals were measured at the beginning of treatment by peripheral quantitative computed tomography (pQCT) and micro-computed tomography (microCT). The animals were equally distributed according to body weight and total tibial bone mineral density assessed by pQCT into groups. Changes in bone mineral density, mass and geometry were assessed after 2.5 and 5 weeks of treatment. Body weight was monitored weekly. Animals that were sacrificed after 2.5 weeks of treatment received administration of two fluorochrome markers for marking bone mineralization 10 and 3 days before necropsy. Blood was collected at necropsy. Dual-energy X-ray absorptiometry (DEXA) measurements were performed at necropsy on the removed tibia, femur and lumbar vertebrae. The bones were fixed, dehydrated and embedded for microtome cutting and histomorphometric analysis of bone formation dynamics.

TREATMENT PROTOCOL

[00307] Control antibody: anti-PC-h/mIgG2a,

[00308] Concentration: 2.5 mg/mL, application volume: 10 mL/kg

[00309] Vehicle: 50 mM Citrate, 140 mM NaCl

[00310] Anti-sclerostin antibody: anti-SOST-MOR05813, h/mIgG2a, 2.45 mg/mL, application volume: 10 mL/kg

[00311] Vehicle: 50 mM Citrate, 140 mM NaCl

[00312] hPTH (1-34) (Bachem, Bubendorf, Switzerland) 100 μg/kg

[00313] Vehicle: PBS + BSA 0.1%TREATMENT GROUPS:1 - Isotype control iv. = anti-PC-mIgG2a2 - Anti-SOST-MOR05813 iv.3 - Vehicle control sc. = PBS + BSA 0.1%4 - hPTH(1-34) sc.

MAINTENANCE CONDITIONS

[00314] Animals were housed in groups of four to five animals at 25°C with a 12:12 h light-dark cycle. They were fed a standardized laboratory diet containing 0.8% phosphorus and 0.75% calcium (NAFAG 3893.0.25, Kliba, Basel, Switzerland). Food and water were provided ad libitum.

DOUBLE ANIMAL WELFARE DECLARATION

[00315] Animal experimentation was carried out in accordance with the regulations in force in the Canton of Basel, Switzerland.

METHODS PERIPHERAL QUANTITATIVE COMPUTED TOMOGRAPHY (PQCT)

[00316] Animals were placed in a lateral position under inhalation narcosis (Isofluorane, 2.5%). The left leg was stretched and fixed in this position.

[00317] Bone mass, density and cross-sectional geometry were monitored in the proximal tibial metaphysis at the level of the midfibular head and 1.8 mm distal to the proximal end of the tibia, as detected on exploratory scanning using a Stratec-Norland XCT-2000 fitted with an Oxford 50 AM X-ray tube and a 0.5 mm diameter collimator. The following setting was chosen for the measurements: voxel size: 0.1 x 0.1 x 0.5 mm; scan speed: exploratory view - 10 mm/s; final scan - 3 mm/s, 1 lock, delineation mode 1, layering mode 2; cortical threshold: 610 mg/cm3, internal threshold: 610 mg/cm3.

MICRO-COMPUTED TOMOGRAPHY (microCT)

[00318] Animals were placed in a lateral position under inhalation narcosis (Isofluorane, 2.5%). The left leg was stretched and fixed in this position.

[00319] Cancellous bone structure was assessed in the proximal metaphysis of the left tibia using a Scanco vivaCT20 (Scanco Medical AG, Switzerland). Non-isometric voxels had a dimension of 10.5 × 10.5 × 10.5 μm. From cross-sectional images, the cancellous bone compartment was delineated from the cortical bone by tracing its contour. In all other slices, boundaries were interpolated based on tracing to define the volume of interest. 143 slices within the secondary cancellous area (starting below the inferior lateral edges of the growth plate) were assessed. A cutoff value of 370 was used for the three-dimensional assessment of structural parameters.

DUAL-ENERGY X-RAY ABSORPTIOMETRY (DEXA)

[00320] Ex vivo DEXA measurements were performed on the left tibia, left femur, and lumbar vertebrae 1–4. Ethanol (70%) was used for soft tissue simulation. Measurements were performed using a standard Hologic QDR-1000 instrument adapted for small animal measurement. A 0.9 cm diameter collimator with ultra-high resolution mode (0.0254 cm line spacing, 0.0127 cm resolution) was used.

FLUOROCHROME LABELING:

[00321] Alizarin (20 mg/kg, subcutaneous, "alizarin complexone", Merck, Dietikon, Switzerland) was applied 10 days before necropsy.

[00322] Calcein (30 mg/kg, subcutaneous, Fluka, Buchs, Switzerland) - 3 days before necropsy.

HISTOLOGY AND HISTOMORPHOMETRY

[00323] After dissection, the right femur and lumbar vertebrae five and six were placed for 24 hours in Karnovsky fixative, dehydrated in ethanol at 4°C, and embedded in resin (methyl methacrylate). Using a Microtome 2050 Supercut (Reichert Jung, Arnsberg, Germany), a set of non-consecutive 5 μm-thick microtome sections were cut in the frontal plane of the midbody for assessment of bone formation based on fluorochrome labeling. The sections were examined using a Leica DM microscope (Leica, Heerbrugg, Switzerland) adapted with a camera (SONY DXC-950P, Tokyo, Japan) and adapted Quantimet 600 software (Leica, Cambridge, UK). One section per animal was collected as a sample. The microscopic images of the samples were digitized and evaluated semi-automatically on the screen. Bone perimeter, single- and double-labeled bone perimeter, and interlabel width were measured (magnification ×200). Mineralized perimeter values (percent), mineral apposition rates (micrometers/day) (corrected for section obliquity in the cancellous bone compartment), and daily bone formation rate values (daily bone formation rate/bone perimeter [micrometers/day]) were calculated. All parameters were assessed in the secondary cancellous bone of the distal femoral metaphysis and in one lumbar vertebra. Another set of sections was stained with tartrate-resistant acid phosphatase (TRAP). Osteoclast surface per bone surface (%) was assessed in the secondary cancellous bone.

STATISTICAL ANALYSIS

[00324] Results are expressed as mean +/- SEM. Statistical analysis was performed using Student's t-test (two-tailed; unpaired). Treatment (anti-sclerostin antibody or hPTH(1-34) was tested for difference from control (control antibody or PBS), *, + p < 0.05, **, ++ p < 0.01.

Affinity determination Determination of the affinity of selected human anti-sclerostin Fabs using surface plasmon resonance (Biacore)

[00325] The kinetic constants kon and koff were determined with serial dilutions of the respective Fab binding to covalently immobilized sclerostin antigen using the BIAcore 3000 instrument (BIAcore, Uppsala, Sweden). For covalent immobilization of the antigen, standardized amine coupling chemistry EDC-NHS was used. Kinetic measurements were made in PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4 pH 7.4) at a flow rate of 20 μL/min using a Fab concentration range of 1.5 - 500 nM. The injection time for each concentration was 1 min, followed by a 3 min dissociation phase. For regeneration, 2 x 5 μL of 10 mM glycine pH 1.5 were used. All sensorgrams were fitted using BIA evaluation software 3.1 (BIAcore).

Electrochemiluminescence-based binding analysis (BioVeris) for measurements of sclerostin-binding Fab affinities in lysates

[00326] For measurement of the affinity of sclerostin binding antibody fragments in E. coli lysates (BEL extracts), binding was analyzed by a BioVeris M-384 SERIES® Workstation (BioVeris Europe, Witney, Oxfordshire, UK).

[00327] The experiment was performed in 96-well polypropylene microtiter plates and PBS supplemented with 0.5% BSA and 0.02% Tween 20 as assay buffer. Biotinylated human sclerostin protein was immobilized on Streptavidin M-280 paramagnetic beads (Dynal) according to the supplier's instructions. A 1:25 dilution of the bead stock solution was added per well. One hundred μl of diluted BEL extract and beads were incubated overnight at room temperature on a shaker. For detection, anti-human (Fab)’2 (Dianova) labeled with BV-tagTM according to the supplier's instructions (BioVeris Europe, Witney, Oxfordshire, UK) was used.

[00328] Randomly chosen clones were analyzed with the method described above. The clones generating the highest values were chosen for further analysis in solution equilibrium titration.

Determination of Fab affinities for sclerostin using solution equilibrium titration (SET)

[00329] For KD determination, monomer fractions (at least 90% of the monomer content, analyzed by analytical SEC; Superdex75, Amersham Pharmacia) of Fab were used. Electrochemiluminescence (ECL)-based affinity determination in solution and data evaluation were performed basically as described by Haenel et al., 2005. A constant amount of Fab (25 pM) was equilibrated with different concentrations (3n serial dilutions) of unlabeled human, mouse or Cynomolgus sclerostin (starting concentration: 500 pM) in solution. Biotinylated human sclerostin (0.5 μg/mL) coupled to Streptavidin M-280 paramagnetic beads, Dynal) and anti-human (Fab)’2 antibody (Dianova) labeled with BV-tagTM (BioVeris Europe, Witney, Oxfordshire, UK) were added and incubated for 30 min. Subsequently, the concentration of unbound Fab was quantified by ECL detection using an M-SERIES® 384 analyzer (BioVeris Europe).

[00330] Fab clones with increased affinity were identified by a BioVeris ECL-based high-throughput affinity screening assay. After hit selection, 4 subclones were consolidated by the same method.

Receptor Binding Inhibition Potency Assay Using BioVerisTM

[00331] For the BioVerisTM-based binding inhibition potency assay, recombinant human BMP-2 was directly coupled (NHS/EDC chemistry) to M270 carboxylic acid magnetic beads (Dynal) according to the supplier's instructions. The assay was performed in a 96-well polypropylene microtiter plate (Nunc). Fifty μL/well of purified Fab in assay buffer (PBS + 1% Tween 20 (stringent) or 0.1% Tween 20 (less stringent) + 1% BSA) was diluted in 1:3 dilution steps (starting concentration: 1,000 nM). Fifty μL/well of biotinylated human sclerostin (4 nM) was added to each Fab dilution. After incubation for 90 minutes shaking at 400 rpm in an Eppendorf Thermomixer at 22°C, 25 μL of BMP-2 coated beads (2.7E07 beads per mL) and streptavidin diluted 1:500 labeled with BV-tagTM according to the supplier's instructions (BioVeris Europe) were added to each well and incubated for 30 minutes (800 rpm, 22°C). Detection was performed by the BioVeris M-384 SERIES® Workstation (BioVeris Europe). For EC50 determination, a 4-parameter logistic fit model (XLfit, IDBS) was used.

Production of immunoglobulins Conversion to human IgG1 format and human/mouse IgG2a format

[00332] In order to express full-length IgG, fragments of the variable domain of heavy (VH) and light (VL) chains were subcloned from Fab expression vectors into appropriate pMorph® Ig vectors: chimeric human/mouse pMorph®_h_Ig1 and pMorph®2_h/m_Ig2a. Restriction enzymes EcoRI, MfeI, BlpI were used for subcloning of the VH domain fragment into pMorph®_h_IgG1 or pMorph®2_h/m_IgG2a and EcoRV, BsiWI, HpaI for subcloning of the VL domain fragment into pMorph®_h_IgK, pMorph®_h_IgA and pMorph®2_h/m_IgA vectors, respectively.

[00333] The restriction enzymes EcoRI, MfeI and BlpI were used for subcloning of the VH domain fragment in pMORPH®_h_IgG1: the vector backbone was generated by EcoRI/BlpI digestion and extraction of the 6,400 bp fragment, while the VH fragment (350 bp) was produced by MfeI and BlpI digestion and subsequent purification. The vector and insert were ligated through compatible overhangs generated by EcoRI and MfeI digests, respectively, and through the BlpI site. In this way, the EcoRI and MfeI restriction sites are destroyed.

[00334] The restriction enzymes MfeI and BlpI were used for subcloning of the VH domain fragment in pMORPH®2_h/m_IgG2a. In this new generation of IgG vectors, with other modifications, the EcoRI site (which only allowed subcloning through compatible projections) was replaced by the MfeI site, thus allowing MfeI/BlpI digestion of both the vector and the insert.

[00335] Subcloning of the VL domain fragment into pMORPH®_h_IgK was performed using the EcoRV and BsiWI sites, while subcloning into pMORPH®_h_IgÀ and pMORPH®2_h/m_IgÀ was performed using EcoRV and HpaI.

Transient expression and purification of human IgG

[00336] HEK293 or HKB11 cells were transfected with an equimolar amount of IgG heavy and light chain expression vectors. On days 4 or 5 post-transfection, cell culture supernatant was collected. After adjusting the pH of the supernatant to pH 8.0 and sterile filtration, the solution was subjected to chromatography on a protein A standardized column (20 A Pores, PE Biosystems).

Example 1: Generation of recombinant human and Cynomolgus sclerostin proteins

[00337] The full-length cDNA encoding human sclerostin precursor (GenBank Accession No. AF326739) displaying a natural signal peptide (aa 1-213, NPL 005002) was cloned into the mammalian expression vector pRS5a by insertion at the Asp718 and Xba1 restriction sites. A protein detection and purification tag (APP = EFRH) was added to the C-terminus of the gene.

[00338] After verification of small-scale expression in transient transfection assays in 6-well plates, generation of stable transfection pools was initiated by transfection of four pools (1.0 x 10E6 cells each) by lipofection. Forty-eight hours after transfection, selection of transfectants was initiated by addition of the antibiotic Zeocin™ at a concentration of 100 μg/mL. After all four pools had regained normal growth, recombinant protein titers were assessed by analytical affinity chromatography on anti-APP HPLC and the largest producing pool - Pool 2 - was selected for adaptation to serum-free medium and subsequent scale-up. Simultaneously, large-scale transient expression experiments at the 10-1 scale were also performed using polyethylenimine as the plasmid DNA carrier during transfection and the Wave™ bioreactor system (C20SPS-F, Part No. 100.001, Wave Biotech) as the culture system. Cell culture supernatants from 12-22 liters were collected 7-10 days post-transfection and concentrated by cross-flow filtration and diafiltration prior to purification.

[00339] Human and Cynomolgus SOST were purified by immunoaffinity chromatography. Briefly, 10-20 liter batches of tissue culture supernatants were concentrated to 1-2 liters by cross-flow filtration (10 kDa cutoff) and loaded at 2 mL/min onto a 50 mL anti-APP Sepharose column prepared by coupling proprietary monoclonal anti-Ab1-40/APP (6E10-A5) to CNBr-activated Sepharose 4B according to the manufacturer's instructions (10 mg antibody per mL resin). After basal wash with PBS, bound material was eluted with 100 mM glycine, pH 2.7, neutralized, and sterile filtered. Protein concentration was determined by A280 using computed absorption factors. Purified proteins were finally characterized by SDS-PAGE, N-terminal sequencing and LC-MS. An aliquot of purified human SOST was biotinylated in PBS for 1 h at 37°C using 1 mM sulfo-NHS-lc-biotin (Uptima; UP54398A). Excess reagent was then removed by intense dialysis against PBS.

Human SOST derived from E. coli'

[00340] His6-tagged SOST aa24-213-PreScission (NPL006071, plasmid pXI504) and SOST aa24-213 (NPL006690, plasmid pXI515) were produced by refolding. E. coli Tuner (DE3) were transformed with one of the plasmids.

[00341] Batch PSU5257 was fermented on a 20 liter scale (V9405) using modified TB medium (lab version 112). Cells were induced to an OD600 of 3.90 with 1 mM IPTG and induced for 3 hours and 30 minutes at 37°C. Harvesting was performed using a continuous flow centrifuge, resulting in a liquid cell pellet weighing 190 g.

[00342] Batch PSU11274 was fermented on a 20-liter scale in 16 L of M9-1 minimal medium supplemented with 15N-labeled ammonium chloride. Cells were induced with 1 mM IPTG once until an OD600 of 1.5 was reached, and collected at an OD600 of 3.3 using a continuous-flow centrifuge, resulting in a liquid cell pellet weighing 50 g.

[00343] Liquid cell pellets from the above fermentation were lysed in 8 volumes of 50 mM Tris pH 8.0 (containing 5 mM each of EDTA, DTT, and Benzamidine-HCl) using an Avestin C-50 emulsifier and centrifuged for 30 minutes at 12,000 rpm. The supernatant was discarded and the resulting pellet resuspended in 10 volumes of lysis buffer and centrifuged again. The process was repeated 3 more times, at which time the lysis buffer was replaced with Milli-Q water containing 5 mM DTT. The pellets were washed twice more under these conditions. Inclusion bodies were dissolved in 8 M Guanidine-HCl (containing 100 mM DTT, 50 mM Tris pH 8, and 5 mM EDTA) for 3–4 h at room temperature, and then centrifuged at 20,000 rpm for 30 min, filtered through a 0.45 μM filter. Refolding was initiated by rapid dilution with 88 volumes (PSU11274) and 92 volumes (PSU5257) of ice-cold refolding buffer (0.5 M Tris containing 0.9 M Arginine-HCl, 5 mM GSH, and 0.5 mM GSSG pH 8). The solution was stored at 4°C for 1 week. At this stage, the two preparations were treated slightly differently because of the need to remove the his6 tag from PSU5257 before refolding was complete.

PSU5257

[00344] The diluted folding solution was diafiltered against 1.5 volumes of 50 mM Tris pH 8, 5 mM GSH, 0.5 mM GSSG by 2-fold concentration, and then diluted back to the original volume. This was done 3 times, at which point PreScission protease was added, and the solution left for 48 hours at 4°C. All concentrations/diafiltrations were done using a Pellicon II ultrafiltration cassette with a 10 KDa cut-off membrane. Finally, the solution was concentrated 10-fold.

PSU11274

[00345] The diluted refolding solution was concentrated 10-fold. LC-MS was used to confirm the formation of all 4 disulfide bonds prior to concentration.

[00346] Both preparations were then dialyzed against 2 x 10 volumes of 50 mM sodium acetate pH 5. After successive filtration through a glass fiber and a 3.0 μm membrane to remove precipitated protein, purification was performed using high-performance cation exchange chromatography on SP-Sepharose™. The column was equilibrated with the dialysis buffer and subsequently eluted with a 0 - 1 M NaCl gradient in the same buffer over 20 column volumes. Sclerostin eluted as a single peak with a slight bump, which was discarded. In the case of PSU5257, the main peak was collected, the concentrate subjected to size exclusion chromatography using a Superdex 75™ column, equilibrated with 50 mM Tris, 150 mM NaCl. With PSU11274, the sample was supplied directly after cation exchange.

[00347] Cynomolgus monkey SOST (cySOST) cDNA was amplified by RT-PCR with primers based on the African green monkey SOST sequence (GenBank Accession No. AF326742). The 5′ primer (ATGCAGCTCCCACTGGCCCTGTGTCTTGT) (SEQ ID NO: 170) corresponds to the N-terminus of the signal peptide sequence and is not in the final secreted protein; the 3′ primer (AATCAGGCCGAGCTGGAGAACGCCTACTAG) (SEQ ID NO: 171) corresponds to a region that is conserved among humans, African green monkey, and mouse. The amplified fragment was subcloned into the BamHI/EcoRI sites of pcDNA3.1(+), and the sequence was confirmed. This plasmid also served as a template for a PCR amplification of cyno-SOST to add an APP tag at the C-terminus and attB recombination sites for final cloning into pDESTRS5a according to Gateway technology [cySOST- pDESTRS5a].

[00348] Expression was done by large-scale transient transfection at the 10-liter scale in the Wave™ bioreactor system, harvested after 9 days post-transfection and purified as described above.

Example 2: Generation of antibodies specific to human sclerostin by the HuCAL GOLD® library

[00349] Therapeutic antibodies against human sclerostin protein were generated by selecting clones that have high binding affinities, using as a source of antibody variant proteins a commercially available phage display library, the MorphoSys HuCAL GOLD® library.

[00350] The HuCAL GOLD® library is a Fab library (Knappik et al., 2000) in which all six CDRs are diversified by appropriate mutation, and which employs CysDisplayTM technology for binding of the Fab to the phage surface (WO 01/05950, Lohning et al., 2001).

Panning selection of sclerostin-specific antibodies from the library

[00351] For the selection of antibodies that recognize human sclerostin, several panning strategies were applied.

[00352] In summary, HuCAL GOLD® antibody-phages were divided into three pools comprising different major VH genes.

[00353] These pools were individually subjected to:

[00354] a solid phase panning, in which antigens (human and mouse sclerostin) were coated directly onto Maxisorp 96-well microtiter plates (Nunc, Wiesbaden, Germany) or

[00355] a capture and semi-solution panning, in which the antigen (biotinylated sclerostin), respectively the phage-antigen complex, was captured on transparent N/A or

[00356] pannings in solution with biotinylated sclerostin, in which the phage-antigen complex was captured by streptavidin magnetic beads (Dynabeads M-280; Dynal) for each panning pool.

- Solid phase panning in sclerostin

[00357] For the first round of sclerostin panning, Maxisorp plate wells were coated overnight with 300 μL (5 μg/mL) human sclerostin (produced in HEK cells) diluted in PBS. After two washing steps with 400 μL PBS, the wells were incubated with blocking buffer containing 5% milk powder diluted in PBS.

[00358] Prior to selections, HuCAL GOLD® phages were pre-adsorbed in blocking buffer (5% milk powder/PBS, 0.5% Tween 20) for 2 hours at room temperature to avoid nonspecific antibody selection.

[00359] After washing (2 x 400 μL PBS) of the coated and blocked Maxisorp plate, 300 μL of the pre-adsorbed phages were added to the coated wells and incubated for 2 hours at room temperature, shaking gently. This incubation was followed by 10 cycles of washing with PBS and PBS/Tween 20 0.05% at room temperature.

[00360] Bound phages were eluted by addition of 300 μL of 20 mM DTT in 10 mM Tris/HCl pH 8.0 per well for 10 minutes at room temperature. The eluate was removed and added to 15 mL of E. coli TG1F+ cells grown to an OD600nm of 0.6 - 0.8. Phage infection of E. coli was allowed for 45 minutes at 37°C without shaking. After centrifugation for 5 minutes at 4,120 xg, each of the bacterial pellets was resuspended in 600 μL of 2xYT medium, plated on LB-CG agar plates, and incubated overnight at 30°C. Colonies were then scraped from the plates, and phages were recovered and amplified.

[00361] The second and third rounds of panning were performed identically to the first round of panning, with the only difference being that the washing conditions after phage binding were more stringent. In the second round of panning, for some panning conditions, mouse sclerostin was used as the antigen in order to enrich for cross-reactive mouse antibodies. For some panning conditions, another batch of recombinant human sclerostin (produced in E. coli) was coated.

- Panning of semi-solution in sclerostin

[00362] Two different methods were performed for this type of panning: a semi-solution capture panning and the standard semi-solution panning procedure.

[00363] In detail, for semi-solution capture panning on biotinylated sclerostin, 100 μL of antigen was coated 2 hours at room temperature onto NeutrAvidin (N/A) clear strips (from Pierce, 100 μL activation level, 15 pmol binding capacity per well). The N/A strips were blocked overnight at 4°C with 200 μL Chemiblock and washed twice with PBS.

[00364] Blocked phage solution (Chemiblock/0.05% Tween 20) was added to the blocked N/A wells for 30 minutes, and this step was repeated for an additional 30 minutes to remove NeutrAvidin binders. The pre-cleared phages were then transferred to biotinylated sclerostin immobilized on N/A clear strips, sealed with foil, and incubated overnight with shaking at room temperature.

[00365] The next day, the phage solution was removed from the antigen-coated wells and the wells were washed.

[00366] For standardized semi-solution panning, pre-cleared blocked phages (Chemiblock/0.05% Tween 20) were added to a new pre-blocked 1.5 mL reaction tube (Chemiblock/0.05% Tween 20) and then biotinylated human sclerostin was added to a final concentration of 100 nM, and incubated overnight with shaking at room temperature.

[00367] The next day, the phage-antigen solution from the 1.5 mL reaction tube was added to new blocked wells of the NeutrAvidin strips, allowed to bind for 30 minutes at room temperature, and then washed.

[00368] For both panning procedures, bound phages were eluted after washing by adding 300 μL of 20 mM DTT in 10 mM Tris/HCl pH 8.0 per well for 10 min at room temperature. The eluate was removed and added to 15 ml of E. coli TG1 cells grown to an OD600nm of 0.6-0.8. Phage infection of E. coli was allowed for 45 min at 37°C without shaking. After centrifugation for 5 min at 4,120 x g, each of the bacterial pellets was resuspended in 600 μL of 2xYT medium, plated on LB-CG agar plates, and incubated overnight at 30°C. Colonies were then scraped from the plates, and phages were recovered and amplified.

[00369] The second and third rounds of selection were performed identically to the first round of selection, with the only difference being that the washing conditions after phage binding were more stringent.

- Panning of solution in sclerostin

[00370] For this type of panning, 200 μL of streptavidin magnetic beads (Dynabeads M-280; Dynal) were washed once with PBS and blocked with Chemiblock for 2 h at room temperature. Five hundred μL of phages were blocked with Chemiblock for 1 h on rotation at room temperature. The blocked phages were preadsorbed twice against 50 μL of blocked streptavidin magnetic beads for 30 min. The phage supernatant was transferred to a new blocked 2 mL reaction tube, and different concentrations of biotinylated human sclerostin were added (see Tables 5, 6, and 7) and incubated for 1 h on rotation at room temperature. One hundred μL of the blocked streptavidin magnetic beads were added to each panning pool, and incubated for 10 min on a shaker. The beads were collected with a particle separator (Dynal MPC-E) for approximately 2.5 min, and the solution was carefully removed.

[00371] After wash cycles (Table 2), bound phages were eluted by addition of 300 μL of 20 mM DTT in 10 mM Tris/HCl pH 8.0 per well for 10 minutes at room temperature. The eluate was removed and added to 15 ml of E. coli TG1F+ cells grown to an OD600nm of 0.6 - 0.8. Phage infection of E. coli was allowed for 45 minutes at 37°C without shaking. After centrifugation for 5 minutes at 4,120 x g, each of the bacterial pellets was resuspended in 600 μL of 2xYT medium, plated on LB-CG agar plates and incubated overnight at 30°C. Colonies were then scraped from the plates, and phages were recovered and amplified.

[00372] The second and third rounds of selection were performed identically to the first round, with the only difference that the washing conditions after phage binding were more stringent. In the second and third rounds of selection, for some panning conditions, the antigen concentration was reduced.

Subcloning and expression of selected Fab fragments Microexpression of selected Fab fragments

[00373] To facilitate rapid expression of soluble Fabs, Fab-encoding inserts from selected HuCAL GOLD® phages were subcloned via XbaI and EcoRI from the respective display vector into the E. coli expression vector pMORPH®X9_MH (Rauchenberger et al., 2003). After transformation of the expression plasmids into E. coli TG1 F- cells, single chloramphenicol-resistant clones were picked into the wells of a sterile 96-well microtiter plate prefilled with 100 μL of 2xYT-CG medium and grown overnight at 37°C. Five μL of each E. coli TG-1 culture was transferred to a fresh, sterile 96-well microtiter plate prefilled with 100 μL of 2xYT medium supplemented with 34 μg/mL chloramphenicol and 0.1% glucose per well. The microtiter plates were incubated at 30°C with shaking at 400 rpm on a microplate shaker until the cultures were slightly turbid (approximately 2–4 h) with an OD600nm of approximately 0.5. To these expression plates, 20 μL of 2xYT medium supplemented with 34 μg/mL chloramphenicol and 3 mM IPTG (isopropyl-β-D-thiogalactopyranoside) were added per well (final concentration: 0.5 mM IPTG), the microtiter plates were sealed with a gas-permeable tape, and incubated overnight at 30°C with shaking at 400 rpm.

[00374] Generation of whole cell lysates (BEL extracts): To each well of the expression plates, 40 μL of BEL buffer was added and incubated for 1 hour at 22°C on a microtiter plate shaker (400 rpm).

Expression and purification of HuCAL®-Fab antibodies in E. coli'

[00375] Expression of Fab fragments encoded by pMORPH®X9_Fab_MH in E. coli TG1 F- cells on a larger scale was performed in shake flask cultures using 750 mL of 2xYT medium supplemented with 34 μg/mL chloramphenicol. Cultures were shaken at 30°C until the OD600nm reached 0.5. Fab expression was induced by addition of 0.75 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and cultivation for an additional 20 h at 30°C. Cells were harvested and disrupted using lysozyme, and Fab fragments were isolated by Ni-NTA chromatography. Apparent molecular weights were determined by size exclusion chromatography (SEC) with calibration standards. Concentrations were determined by UV spectrophotometry (Krebs et al., 2001).

Example 2: Identification of sclerostin-specific HuCAL® antibodies Enzyme-linked immunosorbent assay (ELISA) for detection of sclerostin-binding Fabs on directly coated sclerostin

[00376] Maxisorp 384-well plates (Nunc, Rochester, NY, USA) were coated with 20 μL of 2.5 μg/mL antigen (human sclerostin - produced in HEK cells, human sclerostin - produced in E. coli cells and mouse sclerostin) in PBS, pH 7.4 overnight at 4°C.

[00377] Plates were blocked with PBS/0.05% Tween 20 (PBST) containing 5% milk powder for 1 hour at room temperature. After washing the wells with PBST, BEL extract, purified HuCAL GOLD® Fabs or control Fabs diluted in PBS were added to the wells and incubated for 1 hour at room temperature. To detect primary antibodies, the following secondary antibodies were applied: AffiniPure F(ab')2 fragment conjugated to alkaline phosphatase (AP), goat anti-human IgG, anti-mouse (Jackson ImmunoResearch). For detection of AP conjugates, fluorogenic substrates such as AttoPhos (Roche) were used according to the manufacturer's instructions. Between all incubation steps, the wells of the microtiter plates were washed with PBST three times and five times after the final incubation with the secondary antibody. Fluorescence was measured on a TECAN Spectrafluor plate reader.

Evaluation of capture with biotinylated sclerostin

[00378] This type of ELISA was used to evaluate HuCAL GOLD® Fabs after performing solution pannings.

[00379] Maxisorp 384-well plates (Nunc, Rochester, NY, USA) were coated with 20 μL of 5 μg/mL sheep anti-human IgG, specific for Fd fragment (“The Binding Site”, Birmingham, UK), diluted in PBS, pH 7.4.

[00380] After blocking with 3% BSA in TBS, 0.05% Tween 20 for 2 hours at room temperature, periplasmic extracts or purified HuCAL GOLD® Fabs were added and incubated 1 hour at room temperature. After washing the plates five times with PBST, 20 μL of biotinylated sclerostin for specific binding or biotinylated transferrin for nonspecific binding were added to the wells.

[00381] Subsequently, biotinylated sclerostin antigen was allowed to bind to the captured HuCAL® Fab fragments and, after washing, incubated with streptavidin (Zymex) conjugated to alkaline phosphatase. For detection of AP-streptavidin, fluorogenic substrates such as AttoPhos (Roche) were used according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Fluorescence emission at 535 nm was recorded with excitation at 430 nm.

Results:

[00382] After panning, the enriched phage pools were subcloned by the pMORPH®23 library vector (which allows efficient antibody display on the phage surface) into the pMORPH®X9_Fab_MH expression vector, which mediates the periplasmic expression of soluble Fabs. Single clones were picked out and soluble Fabs were expressed by these single clones. In total, approximately 4,600 clones were analyzed in a primary assay that was performed by binding of Fabs directly from bacterial lysates to human and mouse sclerostin immobilized on Maxisorp microtiter plates or by capturing Fabs by means of anti-Fd antibody to Maxisorp microtiter plates, followed by binding of biotinylated human sclerostin. Detection was performed in ELISA after labeling with an anti-human (Fab)’2 antibody labeled with alkaline phosphatase or with the use of streptavidin-alkaline phosphatase.

[00383] Hits obtained by primary screening on recombinant sclerostin with signals >5-fold relative to background level were further analyzed for binding to human sclerostin from HEK and E. coli and to mouse sclerostin, both coated directly and in solution using biotinylated sclerostin. Hits recognizing all sclerostin derivatives were selected and sequenced.

Characterization of HuCAL GOLD® Fabs Affinity determination using Biacore

[00384] In order to further characterize the anti-sclerostin antibodies, the affinity to human, mouse and Cynomolgus sclerostin was determined. Recombinant sclerostin protein was immobilized on a CM5 Biacore chip and Fabs were applied to the mobile phase at different concentrations. For reliable determination of monovalent affinities, only batches of these Fabs showing >90% monomeric fraction and a qualitative size exclusion chromatography were used for Biacore measurements.

[00385] Affinities for recombinant human, Cynomolgus and mouse sclerostin were determined on Biacore. Affinities range from a subnanomolar range to over 500 nM for human sclerostin, up to nearly 5000 nM for Cynomolgus sclerostin and up to 4,500 nM for mouse sclerostin.

Example 3: Identification of human anti-sclerostin Fab candidates that inhibit sclerostin binding to immobilized BMP-2 using the BioVerisTM device

[00386] All generated and purified Fabs were tested in the BioVeris-based binding inhibition potency assay for their ability to inhibit the binding of sclerostin to recombinant human BMP-2. Two different stringency conditions (0.1% Tween 20 versus 1% in buffer system) were tested. Nine out of twenty-seven Fabs were able to inhibit the binding of sclerostin to immobilized BMP-2 under stringent buffer conditions.

Example 4: Reversal of sclerostin inhibition of BMP-2-induced ALP production in MC3T3 cells. IgG conversions of parental ligands and IgG characterization

[00387] Twenty-one candidates were converted to a human IgG1 format by subcloning into the pMORPH®_h_IgG1 expression vector and the corresponding light chain constructs of the pMORPH®_h_Ig vector series, respectively. Expression was performed by transient transfection of HEK293 or HKB11 cells and full-length immunoglobulins were purified from cell culture supernatant. The functionality of IgG1s after purification was assessed by ELISA binding to immobilized human, mouse and Cynomolgus sclerostin. IgGs in primary bioassay

[00388] All purified hIgGs were titrated and tested in the ALP assay. The assay was reliable with respect to sclerostin inhibition by BMP-2, but selected candidates may not show sclerostin inhibition in all experiments (assay-to-assay activity variation, plate-to-plate variation, high variance within triplicate wells). Therefore, only a ranking of the activity of the IgGs was possible.

Example 5: Affinity maturation of selected anti-sclerostin Fabs by parallel exchange of LCDR3 and HCDR2 cassettes

[00389] The results of the testing of the IgGs in the ALP assay allowed only one ranking. By this ranking, 4 Fabs were selected at a high risk for affinity maturation. All candidates were selected to be optimized as single lead candidates in L-CDR3 and H-CDR2.

[00390] To increase the affinity and biological activity of the four selected antibody fragments, the LCDR3 and HCDR2 regions were optimized in parallel by cassette mutagenesis using site-directed mutagenesis (Virnekas et al., 1994), in which the framework regions were kept constant. Prior to cloning of the maturation libraries, all parental Fab fragments were transferred from the pMORPH®x9_MH expression vector into the CysDisplay™ pMORPH®25 maturation vector via the XbaI/EcoRI restriction sites. This vector provides the phage protein pIII fused at the N-terminus to a cysteine residue as well as a C-terminal cysteine fused to the Fd chain of the antibody and thus allows the disulfide-linked display of the respective Fab fragments on the phage surface.

[00391] For generation of HCDR2 libraries, the HCDR2 region of each parental Fab was removed and replaced with a 590 bp DNA seal. This DNA seal facilitates separation of single-digest vector bands from double-digest vector bands and reduces the background level of high-affinity parental Fabs during maturation pannings. In a subsequent step, the seal was removed from the Fab-encoding plasmids of each parental clone and replaced with the highly diversified HCDR2 maturation cassette.

[00392] In parallel, the LCDR3 region of five of the four parental clones was replaced by a diversified LCDR3 maturation cassette, without intermediate cloning of a seal.

[00393] The sizes of the maturation libraries ranged from 1 x 107 to 4 x 108 clones with a background cloning level below 1%, and quality control by sequencing revealed a good quality of each library. Only the LCDR3 library from the parental clone MOR04520 had a reduced size (< 106) and many incorrect clones and was therefore not included in the maturation pannings.

[00394] For each LCDR3 and HCDR2 maturation library, antibody display phages were prepared, and phage titers determined by spot titration.

Panning strategies for affinity maturation

[00395] Antibody display phages from the following maturation libraries were subjected to separate panning and evaluations:

[00396] Leader 1: MOR04518 (L-CDR3 maturation)

[00397] Leader 1: MOR04518 (H-CDR2 maturation)

[00398] Leader 2: MOR04520 (H-CDR2 maturation)

[00399] Leader 3: MOR04532 (L-CDR3 maturation)

[00400] Leader 3: MOR04532 (H-CDR2 maturation)

[00401] Leader 4: MOR04799 (L-CDR3 maturation)

[00402] Leader 4: MOR04799 (H-CDR2 maturation)

[00403] For each leader library, three different pannings were performed. For each panning strategy, different stringency conditions were applied. To increase the panning stringency and to select for improved off-rates, intensive washing and competition with purified parental Fab or with soluble recombinant sclerostin were performed. After maturation pannings, the phagemid-enriched pools were subcloned into the pMORPH®x9_MH expression vector. Approximately 2,900 unique clones were picked and the Fabs were expressed by IPTG induction.

Affinity Classification and Evaluation by BioVerisTM for Increased Affinities

[00404] For identification of sclerostin-specific Fabs with increased affinity, bacterial lysates from approximately 2,900 unique clones were diluted and checked for Fab binding to biotinylated human sclerostin immobilized on streptavidin-coated beads. Binding was analyzed with a BioVeris Workstation. Those clones generating the highest signals indicate increased affinity and were therefore chosen for further analysis by solution equilibrium titration.

[00405] For this purpose, 176 unique clones were selected and preliminary affinities were determined by 4-point solution equilibrium titration (SET) in BioVeris. From these data, 24 clones showing the best affinities were selected. These Fabs were purified on the mg scale.

[00406] Final affinities were determined using an 8-point SET measurement and human, mouse and Cynomolgus sclerostin.

Fabs optimized in primary bioassay

[00407] The optimized Fabs were tested in the cellular ALP assay. Most of them were inactive; and variable reversal of sclerostin inhibition was observed with some Fabs. Therefore, a selection of clones were converted to human/mouse IgG2a format and tested in the same assay, but these clones in human/mouse IgG2a format could not fully reverse sclerostin inhibition and the desired EC50 criterion (<10 nM) could not be achieved. The best matured candidate, MOR05177 (VL matured from parental MOR04518), showed at 50 nM Fab or at 140 - 467 nM human/mouse IgG2a 75-85% restoration of the ALP signal.

Example 6: Identification of anti-sclerostin antibodies by parental and optimized Fabs and IgGs in a novel primary bioassay

[00408] Based on new publications (Wu et al. JBC 2005, He et al. JBC 2005, Bezooyen et al. ASBMR 2005, Winkler et al. JBC 2005), which show that sclerostin has an impact on Wnt signaling directly or indirectly, a new functional bioassay was developed.

[00409] This assay is based on the ability of sclerostin to inhibit Wnt1-mediated STF activation in HEK293 cells.

[00410] In this assay, all Fabs identified in the initial screenings plus all Fabs identified in affinity maturation were tested. It became apparent that the increased affinity of matured Fabs was reflected in increased potency and efficacy in the wnt-1 assay.

[00411] Testing of all parental Fabs identified two additional antibodies as promising plate models for further affinity maturation. Figure 1 shows the activity of MOR05813_IgG2 lambda, one of the most potent antibodies identified in the wnt-1 assay.

Example 7: Characterization of parental and affinity-matured Fabs in other bioassays Activity of parental and affinity-matured Fabs in the mineralization assay

[00412] The mineralization assay is based on the ability of MC3T3 cells to form a mineralized matrix, which indicates their capacity to undergo osteogenic differentiation. Strong mineralization (> 1 μg calcium deposited per well (96-well format)) was measured after 14 days at all BMP-2 concentrations tested. The addition of increasing concentrations of sclerostin induced a dose-dependent inhibition of BMP-2-induced mineralization (2.1 nM) (IC50: 120 nM) (data not shown).

[00413] Figure 2 shows an example of mineralization in the presence of MOR05813_Fab. The antibody could restore BMP-2-induced mineralization to a maximum of 80% of the initial response, while an anti-lysozyme Fab used as a negative control had no effect.

Activity of parental and affinity-matured Fabs in LRP6/sclerostin ELISA

[00414] The LRP6/sclerostin ELISA is based on the ability of sclerostin to bind to LRP6. To further characterize the Fabs produced, a selection of Fabs and IgGs were tested in this assay. In the presence of an R&D anti-sclerostin antibody (1,000 ng/ml = approximately 7 nM), the binding of sclerostin (0.9 nM) to LRP6 was inhibited by 68% compared to the control (data not shown).

[00415] MOR05813_IgG2 lambda inhibited sclerostin binding to LRP6 by up to 90% at 90 nM, while an anti-lysozyme IgG used as a negative control had no effect on sclerostin binding to LRP6 (Figure 3).

Activity of parental and affinity-matured Fabs in the phospho-Smad1 assay

[00416] The phospho-Smad1 (Western) assay is based on the ability of BMP-6 to induce Smad1 phosphorylation within 15 minutes in MC3T3-E1 cells. MOR05318_IgG2 lambda inhibited the action of sclerostin on BMP-6-induced Smad1 phosphorylation with an EC50 of 115 nM and restored Smad1 phosphorylation to a maximum of 66% of BMP-6-induced phosphorylation. The negative control anti-lysozyme IgG (IgG C) had no effect (Figure 4).

Example 8: Effect of LRP4, a novel SOST interacting partner, on anti-SOST antibody activity

[00417] With the exception of LRP4 siRNA, knockdown of LRP4 mRNA did not affect STF activity in the absence of SOST. However, it reduced the ability of SOST to inhibit STF activity by 10–30%. This was true for all five siRNAs tested alone (Figure 5A) or in different combinations (data not shown). Upon overexpression, LRP4 decreased the IC50 of SOST by 5- and 16-fold in the HEK and c28a2 supertopflash assays, respectively (Figure 5B and C). This effect was specific in that overexpressed LRP4 had no effect on the IC50 of DKK1. LRP4 knockdown (siRNA) decreased the inhibitory action of SOST on Wnt-1-induced STF, while it did not decrease the inhibitory action of DKK1 (Figure 5D). In turn, LRP4 decreased the action of the anti-SOST antibody by increasing the EC50 of MOR05813_IgG2a from 17.2 nM to 30 nM (Figure 5E). These data suggest that LRP4 is a facilitator of SOST action.

Example 9: New maturation

[00418] To increase the affinity and biological activity of two newly selected antibody fragments based on the results obtained by bioassays, the LCDR3 and HCDR2 regions were optimized in parallel by cassette mutagenesis using site-directed mutagenesis (Virnekas et al., 1994), in which the framework regions were kept constant. Prior to cloning of the maturation libraries, all parental Fab fragments were transferred from the pMORPH®X9_MH expression vector into the CysDisplay™ pMORPH®25 maturation vector via the XbaI/EcoRI restriction sites. This vector provides the phage protein pIII fused N-terminally to a cysteine residue, as well as a C-terminal cysteine fused to the Fd chain of the antibody, and thus allows the disulfide display of the respective Fab fragments on the phage surface.

[00419] For generation of HCDR2 libraries, the HCDR2 region of each parental Fab was removed and replaced with a 590 bp DNA seal. This DNA seal facilitates separation of single-digest vector bands from double-digest vector bands and reduces the background level of high-affinity parental Fabs during maturation pannings. In a subsequent step, the seal was removed from the Fab-encoding plasmids of each parental clone and replaced with the highly diversified HCDR2 maturation cassette.

[00420] In parallel, the LCDR3 region of five of the four parental clones was replaced by a diversified LCDR3 maturation cassette, without intermediate cloning of a seal.

[00421] The sizes of the maturation libraries ranged from 2 x 107 to 2 x 108 clones with a background cloning level below 1%, and quality control by sequencing revealed good quality of each library. For each LCDR3 and HCDR2 maturation library, antibody display phages were prepared, and phage titers determined by spot titration. Panning strategies for further affinity maturation

[00422] Antibody display phages from the following maturation libraries were subjected to separate panning and evaluations:

[00423] Leader 1: MOR04525 (L-CDR3 maturation)

[00424] Leader 1: MOR04525 (H-CDR2 maturation)

[00425] Leader 2: MOR04529 (L-CDR3 maturation)

[00426] Leader 2: MOR04529 (H-CDR2 maturation)

[00427] For each leader library or pool, three different pannings were performed, and for each panning strategy different stringency conditions were applied. To increase the stringency of the panning and to select improved off-rates, competition with soluble recombinant sclerostin protein was performed during prolonged incubation and washing periods.

[00428] After panning, the phagemid-enriched pools were subcloned into the pMORPH®X9_MH expression vector. Approximately 1,600 unique clones were picked and Fabs expressed by IPTG induction.

[00429] The affinity criterion of greater than 100 pM for human sclerostin was met for derivatives of both parental Fabs. The required cross-reactivity with Cynomolgus sclerostin and with mouse sclerostin of greater than 500 pM was met for derivatives of both parental Fabs. MOR04525 generated further clones that have higher affinities for all three species.

Example 10: Characterization of anti-sclerostin antibodies in in vivo studies

[00430] Eight-month-old female mice (n = 16/group, Charles River, France) received twice-weekly intravenous administration of anti-sclerostin antibody MOR05813 (24.5 mg/kg, mIgG2a) or isotype control antibody (anti-PC-mIgG2a). Control groups received daily subcutaneously 100 micrograms/kg PTH(1-34) or vehicle (PBS + BSA 0.1%). Treatment lasted 2.5 weeks for all animals. Half of the animals (n = 8/group) were sacrificed at that time point for histomorphometric analysis. These animals received fluorochrome tracers 10 and 3 days prior to necropsy for histomorphometric assessment of bone formation dynamics. Treatment continued for the remaining animals (n = 8/group) up to 5 weeks.

[00431] Animals were monitored for changes in bone mass, density, and geometry. In vivo peripheral quantitative computed tomography [pQCT] demonstrated that MOR05177 is a potent bone anabolic in the proximal tibia of aged mice, increasing bone mineral content (Figure 6) and bone density (Figure 7). The bone anabolic effect occurred in both the cortical (Figure 8) and cancellous (Figure 9) bone compartments. These data suggest that the observed inhibitory effect of MOR05813 on sclerostin action in the Wnt signaling reporter assay in non-osteoblastic cells translates into induction of bone formation responses as a consequence of sclerostin inhibition in vivo. The magnitude of the bone anabolic response in mice was comparable to bone anabolism induced by a high dose of hPTH(1-34).

[00432] MicroCT analysis demonstrated increases in trabecular bone volume (figure 9), mainly related to a thickening of trabecular bone structures, consistent with bone anabolism (figure 10).

[00433] Bone mineral density increased further in animals that were treated for up to 5 weeks (Figure 11). Bone mineral density, as assessed ex vivo by DEXA, was increased in the appendicular (tibia, femur) and axial (lumbar vertebrae) skeleton (Figures 12 - 14). The effect was comparable to that measured in the positive control group treated daily with 100 micrograms/kg hPTH(1-34).

[00434] Fluorochrome histomorphometric marker-based analyses of bone formation dynamics demonstrated that bone mass gain was caused by a substantial increase in bone formation rates in the appendicular (Figure 15) and axial (Figure 18) skeleton. The effects were comparable to those of a high dose of PTH(1-34). Increases in bone formation rates were related to increases in mineral apposition rates (Figure 16) and mineralization surface (Figure 17). Bone resorption was not increased by treatment, as demonstrated by measurement of osteoclast surface (Figure 19).

Example 11: Evaluation of antibodies that cross-block the sclerostin-binding antibodies of the present invention Biacore cross-blocking assay

[00435] The following will generally describe a Biacore assay suitable for determining whether an antibody or other binding agent cross-blocks or is capable of cross-blocking antibodies according to the invention. It will be appreciated that the assay may be used with any of the sclerostin binding agents described herein.

[00436] The Biacore machine (e.g. BIAcore 3000) is operated in accordance with the manufacturer's recommendations.

[00437] Sclerostin can be coupled, for example, to a Biacore CM5 chip, as routinely used in amine coupling chemistry, e.g., EDC-NHS amine coupling, to create a sclerostin coated surface. In order to obtain measurable binding levels, typically 200-800 sclerostin resonance units can be coupled to the chip (this amount generates measurable binding levels and is, at the same time, rapidly saturable by the concentrations of the test reagent used).

[00438] An alternative way of attaching sclerostin to the BIAcore chip is to use a "tagged" version of sclerostin, e.g., sclerostin with a His tag at the N-terminus or C-terminus. In this format, an anti-His antibody would be coupled to the Biacore chip and then the His-tagged sclerostin would pass over the surface of the chip and be captured by the anti-His antibody.

[00439] The two antibodies to be evaluated for their ability to cross-block each other are mixed in a stoichiometric amount, e.g., in a one-to-one molar ratio, of binding sites in a suitable buffer to create the test mixture. The buffer used is typically a buffer that is commonly used in protein chemistry, such as PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4). When calculating concentrations based on binding site, the molecular weight of an antibody is assumed to be the total molecular weight of the antibody divided by the number of target (i.e., sclerostin) binding sites on that antibody.

[00440] The concentration of each antibody in the test mixture must be high enough to ensure saturation of the binding sites for that antibody on the sclerostin molecules that are bound to the BIAcore chip. The antibodies in the mixture are at the same molar concentration (based on binding) and that concentration would typically be between 1.0 mM and 1.5 mM (based on binding site).

[00441] Separate solutions containing the separated antibodies are also prepared. The buffer used for these separate solutions must be the same buffer and at the same concentration as was used for the test mixture.

[00442] The test mixture is passed over the sclerostin-coated BIAcore chip and binding recorded. The bound antibodies are then removed by treating the chip with, for example, an acid, for example, 30 mM HCl for about 1 minute. It is important that the sclerostin molecules that are bound to the chip are not damaged.

[00443] The solution of the first antibody alone is then passed over the sclerostin-coated surface and binding is recorded. The chip is then treated to remove all bound antibody without damaging the sclerostin bound to the chip, for example as acid treatment mentioned above.

[00444] The solution of the second antibody alone is then passed over the sclerostin-coated surface and the amount of binding recorded.

[00445] Maximum theoretical binding can be defined as the sum of the sclerostin binding of each antibody separately. This value is then compared with the actual binding of the measured antibody mixture. If the actual binding is lower than the theoretical binding, the two antibodies are cross-blocking each other.

ELISA-BASED CROSS-BLOCKING ASSAY

[00446] Cross-blocking of an anti-sclerostin antibody or other sclerostin binding agent can also be detected using an ELISA assay.

[00447] The general principle of the ELISA assay involves coating an anti-sclerostin antibody onto wells of an ELISA plate. An excess amount of a second anti-sclerostin antibody, potentially exhibiting cross-blocking, is then added in solution (i.e., not bound to the ELISA plate). A limited amount of sclerostin is then added to the wells.

[00448] The antibody that has been coated on the wells and the antibody in solution will compete for binding of the limited number of sclerostin molecules. The plate is then washed to remove sclerostin that has not bound to the coated antibody and to also remove the second antibody, in solution phase, as well as any complexes formed between the second antibody, in solution phase, and sclerostin. The amount of bound sclerostin is then measured using an appropriate sclerostin detection reagent. An antibody in solution that is capable of cross-blocking the coated antibody will be capable of causing a decrease in the number of sclerostin molecules to which the coated antibody can bind relative to the number of sclerostin molecules to which the coated antibody can bind in the absence of the second antibody, in solution phase.

[00449] This assay is described in more detail below for two antibodies designated Ab-X and Ab-Y. When Ab-X is chosen to be the immobilized antibody, it is coated onto the wells of the ELISA plate, and then the plates are blocked with a suitable blocking solution to minimize nonspecific binding of reagents that are subsequently added. An excess amount of Ab- is then added to the ELISA plate such that the moles of sclerostin binding sites of Ab-Y per well are at least 10-fold greater than the moles of sclerostin binding sites of Ab-X that were used, per well, during coating of the ELISA plate. Sclerostin is then added such that the moles of sclerostin added per well are at least 25-fold lower than the moles of sclerostin binding sites of Ab-X that were used for coating each well. After an appropriate incubation period, the ELISA plate is washed and sclerostin detection reagent is added to measure the amount of sclerostin specifically bound by the coated anti-sclerostin antibody (in this case, Ab-X). The baseline signal for the assay is defined as the signal obtained in the wells with the coated antibody (in this case, Ab-X), the second solution-phase antibody (in this case, Ab-Y), sclerostin buffer alone (i.e., without sclerostin), and sclerostin detection reagents. The positive control signal for the assay is defined as the signal obtained in the wells with the coated antibody (in this case, Ab-X), second solution-phase antibody buffer alone (i.e., without second solution-phase antibody), sclerostin, and sclerostin detection reagents. The ELISA assay must be performed such that the positive control signal is at least 6 times the baseline signal.

[00450] To avoid any artifacts (e.g., significantly different affinities between Ab-X and Ab-Y for sclerostin) resulting from the choice of which antibody to use as the coating antibody and which to use as the second (competitor) antibody, the cross-blocking assay needs to be performed in two formats: 1) format 1, where Ab-X is the antibody that is coated on the ELISA plate and Ab-Y is the competing antibody that is in solution, and 2) format 2, where Ab-Y is the antibody that is coated on the ELISA plate and Ab-X is the competing antibody that is in solution.

Example 12: ELISA to detect the effect of MOR05813_IgG2lambda on SOST binding of LRP6 LRP6 / SCLEROSTIN ELISA

[00451] Ninety untreated 6-well microtiter plates were coated with 100 μL/well of LRP6/Fc (1 μg/mL, R&D Systems, Catalog No.: 1505-LR) diluted in PBS. As a control for nonspecific binding (NSB), some wells were filled with 100 μL/well of PBS. The plates were covered with plastic wrap and incubated overnight at room temperature. After coating, plates were washed 3 times with 200 μL^o of 0.05% Tween 20 (Fluka, Catalog No. 93773) in PBS, and wells were blocked for 1 h at 37°C by adding 300 μL/well of SuperBlock blocking buffer (Pierce, Catalog No. 37535) in TBS. After incubation, the blocking solution was removed and 100 μL/well of sclerostin (derived from E. coli, Novartis; 1–1,000 ng/mL) diluted in 1% BSA in PBS was added. Plates were incubated for 2 h at room temperature before being washed 3 times with 200 μL/well of 0.05% Tween 20 in PBS. Next, 100 μL/well of anti-sclerostin antibody (1 μg/mL) diluted in 1% BSA in PBS was added, and the plates were incubated for 2 h at room temperature before being washed 3 times with 200 μL/well of 0.05% Tween 20 in PBS. Finally, 100 μL/well of ALP-conjugated goat anti-IgG Ab (1:5,000; Sigma Catalog No.: A-7888) diluted in 1% BSA (Sigma Catalog No.: A-7888) in PBS was added for 1 h at room temperature, and the plates were then washed 3 times with 200 μL/well of 0.05% Tween 20 in PBS. To determine ALP, 100 μL/well of ALP substrate solution (Sigma, Catalog No.: S0942) (1 tablet per 5 mL of 1x diethanolamine substrate buffer; Pierce, Catalog No.: 34064) was added to the plates for 90 min and the optical density measured at 405 nm.

Activity of parental and affinity-matured Fabs in LRP6/sclerostin ELISA

[00452] The LRP6/sclerostin ELISA is based on the ability of sclerostin to bind to LRP6. To further characterize the Fabs produced, a selection of Fabs and IgGs were tested in this assay. In the presence of an R&D anti-sclerostin antibody (1,000 ng/ml = approximately 7 nM), the binding of sclerostin (0.9 nM) to LRP6 was inhibited by 68% compared to the control (data not shown). MOR05813_IgG2 lambda inhibited sclerostin binding to LRP6 by up to 90% at 90 nM, while an anti-lysozyme IgG used as a negative control had no effect on sclerostin binding to LRP6 (Figure 20).

Example 13: Co-treatment using MOR05813 MOR05813 + zoledronic acid

[00453] Eight-month-old female mice (n = 10/group, Charles River, France) were ovariectomized to induce bone loss by estrogen deprivation or left intact. Animals received twice-weekly intravenous administration of anti-sclerostin antibody MOR05813 (24 mg/kg, h/mIgG2a) or control antibody (anti-PC-h/mIgG2a, intact and OVX control groups). Additional groups received a single application of zoledronic acid alone (100 μg/kg) or in combination with anti-sclerostin antibody MOR05813. Antibody treatment lasted 3.5 weeks (7 applications).

[00454] Bone mass and tibial geometry of the animals were measured before oophorectomy by peripheral quantitative computed tomography (pQCT). The animals were equally distributed according to body weight and total tibial bone mineral density into groups. Changes in bone mineral density, mass and geometry were assessed at the end of the treatment period.

[00455] Results are expressed as mean +/- SEM. Statistical analysis was performed using RS1 (1999 series for Windows, Domain Manufacturing Corp., USA). Data were subjected to one-way analysis of variance (ANOVA). Equality of variances was tested by Levene's F-test and differences between groups using Bonferroni-adjusted Dunnett's test. Treated groups were tested for significance of differences relative to the control antibody-treated OVX group (p < 0.05*, p < 0.01**).

[00456] Oophorectomy induced bone loss that could be blocked by antibody treatment in aged mice (Figure 21). When the antibody is used in combination with a single intravenous injection of the bisphosphonate zoledronic acid, bone loss is blocked and bone gain is induced, as indicated by increases in total bone mineral content (Figure 21A) and density (Figure 21B), as well as increases in cortical thickness (Figure 21C) and cancellous bone mineral density (Figure 21D).

Pretreatment with MOR05813 + alendronate

[00457] Female OF1/IC mice aged 4.52 months (n = 10/group, Charles River, France) received twice weekly intravenous administration of anti-sclerostin antibody MOR05813 (10 mg/kg, h/mIgG2a) or control antibody (anti-PC-mIgG2a, intact and OVX control groups). Additional groups received pretreatment with alendronate for 7 weeks (4 μg/kg/day; 5 days/week) before receiving control antibody or anti-sclerostin antibody MOR05813. Antibody treatment lasted 3.5 weeks (7 applications).

[00458] Bone mass and tibial geometry of the animals were measured prior to the start of antibody treatment by peripheral quantitative computed tomography (pQCT). The animals were equally distributed according to body weight and total tibial bone mineral density into groups. Changes in bone mineral density, mass and geometry were assessed at the end of the treatment period.

[00459] Results are expressed as mean +/- SEM. Statistical analysis was performed using RS1 (1999 series for Windows, Domain Manufacturing Corp., USA). Data were subjected to one-way analysis of variance (ANOVA). Equality of variances was tested by Levene's F-test and differences between groups using Bonferroni-adjusted Dunnett's test. Groups pretreated with alendronate were tested for significance of differences relative to groups not pretreated (p < 0.05*, p < 0.01**).

[00460] Long-term pretreatment with the bisphosphonate alendronate has no negative impact on the bone anabolic action of the anti-sclerostin MOR05813, which is reflected by increases in total bone mineral content (Figure 22 A) and density (Figure 22 B), as well as increases in cortical thickness (Figure 22 C) and cancellous bone mineral density (Figure 22 D). Due to the persistent anti-resorptive properties of the bisphosphonate beyond the period of administration, an increase in total bone mineral content (Figure 22 A) and cortical thickness (Figure 22 C) is observed.

MOR05813 + DKK1 or hPTH

[00461] Six-week-old female nude mice (n = 8/group) received twice-weekly intravenous administration of vehicle, anti-sclerostin antibody MOR05813 (10, 20, and 40 mg/kg, IgG2), anti-Dkk1 antibody (10 mg/kg, IgG1), hPTH(1-34) (100 micrograms/kg), or combinations thereof. Antibody treatment lasted for 4 weeks (8 applications).

[00462] Bone mass and tibial geometry of the animals were measured before treatment by peripheral quantitative computed tomography (pQCT). The animals were equally distributed according to body weight and total tibial bone mineral density into groups. Changes in bone mineral density, mass and geometry were assessed at the end of the treatment period.

[00463] Results are expressed as mean +/- SEM. Statistical analysis was performed using RS1 (1999 series for Windows, Domain Manufacturing Corp., USA). Data were subjected to one-way analysis of variance (ANOVA). Equality of variances was tested by Levene's F-test and differences between groups using Bonferroni-adjusted Dunnett's test. Groups were tested for significance of differences from the vehicle-treated group (p < 0.05*, p < 0.01**).

[00464] Bone anabolic effects increase with the dose of anti-sclerostin MOR05813 (figure 23). Co-treatment with an anti-Dkk1 antibody results in a marked increase in total bone mineral content (figure 23 A) and density (figure 23 B) and cortical thickness (figure 23 C) and a synergistic increase in cancellous bone mineral density (figure 23 D). Co-treatment with hPTH(1-34) results in a synergistic increase in all measured parameters (figure 23 A - D). REFERENCES Avsian-Kretchmer O., Hsueh A.J. (2004) "Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists". Mol. Endocrinol. 18(1): 1-12.Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., (1998) "Current Protocols in Molecular Biology", Wiley, New York, USA. Dioszegi M., Lacza C., Wuyts W., Van Den Ende J., Willems P., Paes-Alves A.F., Hill S., Bueno M., Ramos F.J., Tacconi P., Dikkers F.G., Stratakis C., Lindpaintner K., Vickery B., Foernzler D., Van Hul W. (2001) "Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum. Mol. Genet. 10(5): 537-43. Lindpaintner K., Vickery B., Foernzler D., Van Hul W. (2002) "Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease". J. Med Gene 39(2): 91-7. Beighton P., Mulligan J. (2001) "Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein". Am. J. Hum. Genet. 68(3): 577-89.Chen, B.P., Hai, T. "Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host." Gene 139, 73-75. 1994.Chen, Y., Wiesmann, C., Fuh, G., Li, B., Christinger, H. W., McKay, P., de Vos, A. M., Lowman, H. B. (1999). "Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen". J. Mol. Biol. 293, 865881.Gardner J.C., van Bezooijen R.L., Mervis B., Hamdy N.A., Lowik C.W., Hamersma H., Beighton P., Papapoulos S.E. (2005) "Bone mineral density in sclerosteosis; affected individuals and gene carriers." J. Clin. Endocrinol. Metab. 90(12): 6.392-5.Haenel C., Satzger M., Della Ducata D., Ostendorp R. and Brocks B. (2005) "Characterization of High Affinity Antibodies by Electrochemiluminescence-Based Equilibrium Titration." Anal. Biochem. 339(1): 182-4. Keller H., Kneissel M. (2005) "SOST is a target gene for PTH in bone". Bone 37(2): 148-58.Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., and Virnekas, B. (2000). "Fully synthetic human combinatorial antibody libraries (HuCAL®) based on modular consensus frameworks and CDRs randomized with trinucleotides". J. Mol. 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Claims (23)

1. An isolated antibody that (i) binds to a sclerostin polypeptide, and (ii) comprises: (a) a heavy chain variable region CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 4; (b) a heavy chain variable region CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 15; (c) a heavy chain variable region CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 26; (d) a light chain variable region CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 37; (e) a light chain variable region CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 48; and (f) a light chain variable region CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 59. 2. The isolated antibody of claim 1, wherein (i) it binds to human sclerostin polypeptide, and (ii) it can increase bone formation, bone mineral density, and bone mineral content in a mammal. 3. The antibody of claim 1 or 2, wherein said antibody binds to said sclerostin polypeptide with a KD of less than 1 nM. 4. The antibody of any one of claims 1 to 3, wherein said antibody can block the inhibitory effect of sclerostin in a Wnt signaling cellular assay. 5. Antibody according to claim 4, characterized in that said antibody has an IC50 of less than 100 nM, measured in a cellular Wnt signaling assay in HEK293 cell lines in the presence of sclerostin. 6. Antibody according to any one of claims 1 to 5, characterized in that said antibody can block the inhibitory effect of sclerostin in a cellular mineralization assay. 7. The antibody of claim 6, wherein said antibody has an IC50 of less than 500 nM, measured in a BMP2-induced mineralization assay in MC3T3 cells in the presence of sclerostin. 8. The antibody of any one of claims 1 to 7, which inhibits the LRP6/sclerostin interaction in a solution inhibition assay. 9. The antibody of claim 8, having an IC50 of less than 10 nM, measured by LRP6/sclerostin ELISA. 10. The antibody of any one of claims 1 to 9, which can block the inhibitory effect of sclerostin on BMP6-induced Smad1 phosphorylation in a functional cellular assay. 11. Antibody according to claim 10, characterized by the fact that it has an IC50 of less than 500 nM, measured in a Smad1 phosphorylation assay by BMP6 in a MC3T3-E1 cell line in the presence of sclerostin. 12. The antibody of any one of claims 1 to 11, comprising a VH polypeptide sequence of SEQ ID NO: 70. 13. The antibody of any one of claims 1 to 12, comprising a VL polypeptide sequence of SEQ ID NO: 81. 14. The antibody of any one of claims 1 to 13, comprising a VL polypeptide sequence having the sequence of SEQ ID NO: 81 and a VH polypeptide sequence having the sequence of SEQ ID NO: 70. 15. Antibody, characterized by the fact that it comprises a heavy chain sequence of SEQ ID NO: 114 and a light chain sequence of SEQ ID NO: 125. 16. Antibody according to any one of claims 1 to 15, characterized in that it is for use as a medicament. 17. Use of an antibody as defined in any one of claims 1 to 15, characterized in that it is for the manufacture of a medicament for the treatment of a bone-related disease selected from the group comprising primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis (osteonecrosis), fractures and implant healing (dental implants and hip implants), bone loss due to other disorders, such as bone loss associated with HIV infection, cancers or arthritis. 18. Pharmaceutical composition, characterized by the fact that it comprises an antibody, as defined in any one of claims 1 to 15. 19. Pharmaceutical composition according to claim 18, characterized in that it is in combination with one or more than one pharmaceutically acceptable excipient, diluent or carrier. 20. Pharmaceutical composition according to claim 18 or 19, characterized in that it also comprises other active ingredients. 21. An antibody according to any one of claims 1 to 15, or a pharmaceutical composition according to any one of claims 18, 19 or 20, characterized in that it is for use in the treatment or prophylaxis of a pathological disorder that is mediated by sclerostin or that is associated with an increased level of sclerostin. 22. An antibody according to any one of claims 1 to 15, or a pharmaceutical composition according to any one of claims 18, 19 or 20, characterized in that it is for use in the treatment of a bone-related disorder in a mammalian subject. 23. Antibody, or pharmaceutical composition, according to claim 22, characterized by the fact that the bone-related disorder is at least one of primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis (osteonecrosis), fractures and scarring of implants (dental implants and hip implants), bone loss due to other disorders such as, for example, bone loss associated with HIV infection, cancers or arthritis.