Anti-RANKL antibodies and uses thereof

Abstract

To provide antibodies that bind human and murine receptor activators of nuclear factor κB ligand (RANKL).SOLUTION: The present invention provides an anti-RANKL antibody comprising a light chain variable region and a heavy chain variable region, each comprising specific CDR sequences, the antibodies serving as agents for treating conditions associated with bone-related disorders or skeletal abnormalities caused by cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention belongs to the field of medicine. More specifically, the present invention relates to an antibody, including an antibody against receptor activator of nuclear factor kappa B ligand (RANKL), and its pharmaceutical composition. The antibody of the present invention is expected to be useful in treating bone-related disorders such as osteoporosis, osteopenia, and arthritis (such as rheumatoid arthritis), as well as skeletal abnormalities caused by cancers such as non-small cell lung cancer and multiple myeloma.

[0002] Bone-related disorders affect millions of individuals, and often cause painful and debilitating symptoms.Osteoporosis is a common metabolic bone-related disorder, characterized by progressive bone loss, which at least in part results from excessive osteoclastic bone resorption over osteoblastic bone formation.Osteoporosis-related bone loss leads to higher risk of fracture.The long-term consequences of osteoporosis-related bone loss can lead to serious physical consequences, including fractures, chronic pain, physical disability, and / or immobility, and can lead to insufficient structural support of the body by the skeleton.

[0003] RANKL is a member of the TNF superfamily of proteins and plays an important role in bone remodeling. RANKL is expressed by osteoblasts and binds to its cognate receptor RANK on the surface of osteoclasts and osteoclast precursors. RANKL binding to RANK induces the formation, activation, and survival of mature osteoclasts, and stimulates intracellular signaling cascades, resulting in increased bone resorption. Neutralizing antibodies against RANKL are known in the art. For example, U.S. Patent No. 6,740,522 discloses anti-RANKL antibodies, including denosumab, which is marketed under the names Prolia® and Xgeva®, the only approved anti-RANKL therapeutic antibody (approved for the treatment of osteoporosis in postmenopausal women and men at high risk of fracture, and for the prevention of skeletal-related events in patients with bone metastases from solid tumors, respectively).

[0004] Osteoporosis-related fractures are a major health concern and economic burden to the healthcare system. According to the National Osteoporosis Foundation, 9.9 million Americans have osteoporosis and an additional 43.1 million suffer from low bone mineral density. Each year, more than 2 million fractures and 400,000 hospitalizations are attributable to osteoporosis. The U.S. Surgeon General estimates that osteoporosis-related fractures result in direct medical costs of $12 to $18 billion each year.

[0005] Current therapies are not suitable for co-administration or co-formulation with other agents such as bone resorption inhibitors or anabolic compounds. In addition, there is a lack of anti-RANKL antibodies that are useful for studying in vivo preclinical models such as mouse models. Therefore, there is still a need for alternative therapies for bone-related disorders or skeletal abnormalities caused by cancer that can provide better outcomes for patients. Such alternative therapies would preferably be able to show efficacy in treating bone-related disorders such as osteoporosis, osteopenia, and arthritis (such as rheumatoid arthritis), as well as skeletal abnormalities caused by cancers such as non-small cell lung cancer and multiple myeloma. The antibody of the present invention provides an alternative therapy that is expected to meet at least one of the above needs.

[0006] The present invention provides an antibody that binds to human RANKL, the antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), the LCVR comprising complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, the HCVR comprising CDRs HCDR1, HCDR2, and HCDR3, the amino acid sequence of the LCDR1 being SEQ ID NO: 12, the amino acid sequence of the LCDR2 being SEQ ID NO: 13, the amino acid sequence of the LCDR3 being SEQ ID NO: 14, the amino acid sequence of the HCDR1 being SEQ ID NO: 9, the amino acid sequence of the HCDR2 being SEQ ID NO: 10, and the amino acid sequence of the HCDR3 being SEQ ID NO: 11. In some specific embodiments, the Xaa at position 6 of the amino acid sequence shown in SEQ ID NO: 11 is Ala, the Xaa at position 14 of the amino acid sequence shown in SEQ ID NO: 11 is Pro, and the Xaa at position 4 of the amino acid sequence shown in SEQ ID NO: 14 is Trp. In another specific embodiment, Xaa at position 6 of the amino acid sequence represented by SEQ ID NO:11 is Arg, Xaa at position 14 of the amino acid sequence represented by SEQ ID NO:11 is Tyr, and Xaa at position 4 of the amino acid sequence represented by SEQ ID NO:14 is Asn.

[0007] In some specific embodiments, the invention provides antibodies, wherein the LCVR has the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8, and the HCVR has the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 7. In some specific embodiments, the LCVR has the amino acid sequence set forth in SEQ ID NO: 4, and the HCVR has the amino acid sequence set forth in SEQ ID NO: 3. In other specific embodiments, the LCVR has the amino acid sequence set forth in SEQ ID NO: 8, and the HCVR has the amino acid sequence set forth in SEQ ID NO: 7.

[0008] In some specific embodiments, the invention provides antibodies wherein the LC has the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6. In further embodiments, the invention provides antibodies wherein the HC has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:5. In some such embodiments, the LC has the amino acid sequence set forth in SEQ ID NO:2 and the HC has the amino acid sequence set forth in SEQ ID NO:1. In some such embodiments, the LC has the amino acid sequence set forth in SEQ ID NO:6 and the HC has the amino acid sequence set forth in SEQ ID NO:5.

[0009] In some embodiments, the antibodies of the invention bind to human RANKL. In other such embodiments, the antibodies of the invention bind to mouse RANKL. In other such embodiments, the antibodies of the invention bind to human RANKL and mouse RANKL.

[0010] The present invention also relates to nucleic acid molecules and expression vectors encoding the antibodies of the present invention. In one embodiment, the present invention provides a DNA molecule comprising a polynucleotide sequence encoding an HC, wherein the amino acid sequence of the HC is SEQ ID NO: 1. According to some such embodiments, the DNA molecule has a polynucleotide sequence as shown in SEQ ID NO: 15.

[0011] In one embodiment, the present invention also provides a DNA molecule comprising a polynucleotide sequence encoding LC, wherein the amino acid sequence of LC is SEQ ID NO: 2. According to some such embodiments, the DNA molecule has the polynucleotide sequence shown in SEQ ID NO: 16.

[0012] In a further embodiment, the present invention provides a DNA molecule comprising a polynucleotide sequence encoding a HC having the amino acid sequence of SEQ ID NO: 1 and comprising a polynucleotide sequence encoding a LC having the amino acid sequence of SEQ ID NO: 2. In a particular embodiment, the polynucleotide sequence encoding the HC having the amino acid sequence of SEQ ID NO: 1 is set forth in SEQ ID NO: 15, and the polynucleotide sequence encoding the LC having the amino acid sequence of SEQ ID NO: 2 is set forth in SEQ ID NO: 16.

[0013] The present invention also provides a mammalian cell transformed with a DNA molecule, the cell being capable of expressing an antibody comprising an HC and an LC of the present invention, the HC having the amino acid sequence shown in SEQ ID NO: 1, and the LC having the amino acid sequence shown in SEQ ID NO: 2. The present invention also provides a method for producing an antibody comprising an HC and an LC, the method comprising culturing a mammalian cell under conditions in which the antibody of the present invention is expressed. The present invention also provides an antibody produced by the method. The present invention also provides a mammalian cell transformed with a DNA molecule, the cell being capable of expressing an antibody comprising two HCs and two LCs of the present invention, each HC having the amino acid sequence shown in SEQ ID NO: 1, and each LC having the amino acid sequence shown in SEQ ID NO: 2. The present invention also provides a method for producing an antibody comprising two HCs and two LCs, the method comprising culturing a mammalian cell under conditions in which the antibody of the present invention is expressed. The present invention also provides an antibody produced by the method.

[0014] The present invention also provides a pharmaceutical composition comprising the antibody of the present invention and one or more pharma- ceutically acceptable carriers, diluents, or excipients. The pharmaceutical composition of the present invention can be used to treat bone-related disorders, and such treatment comprises administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a patient in need thereof. In some such embodiments, the bone-related disorder is one or more of osteoporosis, osteopenia, and arthritis (such as rheumatoid arthritis). In other such embodiments, the pharmaceutical composition of the present invention can be used to treat skeletal abnormalities caused by cancer, and such treatment comprises administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a patient in need thereof. In certain embodiments, the cancer is one or more of non-small cell lung cancer and multiple myeloma.

[0015] The present invention also provides a method for treating bone-related disorders or skeletal abnormalities caused by cancer, comprising administering a therapeutically effective amount of the antibody of the present invention to a patient in need thereof. In some such embodiments, the bone-related disorder is osteoporosis. In some such embodiments, the bone-related disorder is osteopenia. In some such embodiments, the bone-related disorder is arthritis. In more particular embodiments, the bone-related disorder is rheumatoid arthritis. In other such embodiments, the bone-related disorder is one or more of osteoporosis, osteopenia, and arthritis (such as rheumatoid arthritis). The present invention also provides a method for treating one or more of skeletal abnormalities caused by non-small cell lung cancer and skeletal abnormalities caused by multiple myeloma.

[0016] The present invention also provides the antibody of the present invention or its pharmaceutical composition for use in therapy.More specifically, the present invention provides the antibody of the present invention or its pharmaceutical composition for use in one or more of osteoporosis, osteopenia, and arthritis (such as rheumatoid arthritis).The present invention also provides the antibody of the present invention or its pharmaceutical composition for use in one or more of skeletal abnormalities caused by non-small cell lung cancer and skeletal abnormalities caused by multiple myeloma.

[0017] The present invention also provides the use of the antibody of the present invention or its pharmaceutical composition in the manufacture of a medicament for treating bone-related disorders or skeletal abnormalities caused by cancer.According to some specific embodiments, the present invention provides the antibody of the present invention or its pharmaceutical composition in the manufacture of a medicament for treating at least one of osteoporosis, osteopenia, and arthritis (such as rheumatoid arthritis).The present invention also provides the antibody of the present invention or its pharmaceutical composition in the manufacture of a medicament for treating at least one of skeletal abnormalities caused by non-small cell lung cancer and skeletal abnormalities caused by multiple myeloma.

[0018] In one embodiment, an antibody of the invention binds to human RANKL at one or more of residues 89-97 and one or more of residues 125-132 of SEQ ID NO: 17. In another embodiment, an antibody of the invention binds to an epitope on human RANKL, the epitope comprising at least one amino acid of residues 89-97 and at least one amino acid of residues 125-132 of SEQ ID NO: 17. In a further embodiment, the epitope comprises at least two amino acids of residues 89-97 and at least two amino acids of residues 125-132 of SEQ ID NO: 17. In another further embodiment, the epitope comprises at least three amino acids of residues 89-97 and at least three amino acids of residues 125-132 of SEQ ID NO: 17. In another further embodiment, the epitope comprises at least four amino acids of residues 89-97 and at least four amino acids within the range of residues 125-132 of SEQ ID NO: 17. In other further embodiments, the epitope comprises at least 5 amino acids of residues 89-97 and at least 5 amino acids of residues 125-132 of SEQ ID NO:17.

[0019] In one embodiment, the present invention provides an antibody capable of competing for binding to human RANKL with an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of LCDR1 is SEQ ID NO: 12, the amino acid sequence of LCDR2 is SEQ ID NO: 13, the amino acid sequence of LCDR3 is SEQ ID NO: 14, the amino acid sequence of HCDR1 is SEQ ID NO: 9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, and the amino acid sequence of HCDR3 is SEQ ID NO: 11. In a more specific embodiment, Xaa at position 6 of the amino acid sequence shown in SEQ ID NO: 11 is Ala, Xaa at position 14 of the amino acid sequence shown in SEQ ID NO: 11 is Pro, and Xaa at position 4 of the amino acid sequence shown in SEQ ID NO: 14 is Trp. In another more specific embodiment, Xaa at position 6 of the amino acid sequence shown in SEQ ID NO: 11 is Arg, Xaa at position 14 of the amino acid sequence shown in SEQ ID NO: 11 is Tyr, and Xaa at position 4 of the amino acid sequence shown in SEQ ID NO: 14 is Asn.

[0020] As used herein, an "antibody" may be human, humanized, murine, or mouse-rat chimeric. An antibody is an immunoglobulin molecule that contains two HCs and two LCs interconnected by disulfide bonds. The amino-terminal portion of each HC and LC contains a variable region of about 100-120 amino acids that is primarily responsible for antigen recognition via the CDRs contained in the variable region. The CDRs are interspersed with more conserved regions called framework regions ("FRs"). Each LCVR and HCVR 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 three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3" and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3". The CDRs contain most of the residues that form specific interactions with the antigen. The functional ability of an antibody to bind to a specific antigen is largely influenced by the six CDRs. The amino acid assignment to the CDR domains in the LCVR and HCVR regions of the antibody of the present invention is based on the well-known Kabat numbering system (Kabat, et al., Ann. NY Acad. Sci. 190: 382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)), and North numbering system (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406: 228-256 (2011)). The CDRs of the antibody of the present invention are defined according to Table 1. [Table 1]

[0021] The antibody of the present invention is a monoclonal antibody ("mAb"). The mAb of the present invention is a complete mAb containing two HCs and two LCs. As referred to herein, mAb is an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, not the method by which it is produced. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology, such as CDR grafting, or a combination of such or other techniques known in the art. Methods for producing and purifying antibodies are well known in the art and can be found, for example, in Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, chapters 5-8 and 15, ISBN 0-87969-314-2.

[0022] The monoclonal antibodies of the present invention can be prepared and purified using known methods. For example, the cDNA sequences encoding HC (e.g., the amino acid sequence shown in SEQ ID NO:1) and LC (e.g., the amino acid sequence shown in SEQ ID NO:2) can be cloned and engineered into a GS (glutamine synthetase) expression vector. The engineered immunoglobulin expression vector can then be stably transfected into CHO cells. As one skilled in the art will appreciate, mammalian expression of antibodies will typically result in glycosylation at highly conserved N-glycosylation sites in the Fc region. Stable clones can be verified for expression of antibodies that specifically bind to RANKL. Positive clones can be grown in serum-free medium for antibody production in a bioreactor. The medium into which the antibody is secreted can be purified by conventional techniques. For example, the medium can be conveniently applied to a Protein A or G Sepharose FF column equilibrated with a compatible buffer, such as phosphate-buffered saline. The column is washed to remove non-specifically bound components. The bound antibody is eluted, for example, by a pH gradient, and the antibody fractions are detected, for example, by SDS-PAGE, and then pooled. The antibody can be concentrated and / or sterile filtered using common techniques. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, for example, at -70°C, or lyophilized.

[0023] The monoclonal antibody of the present invention can be used for the treatment of patients. More specifically, the antibody of the present invention is expected to treat bone-related disorders such as osteoporosis, osteopenia, and arthritis (such as rheumatoid arthritis), as well as skeletal abnormalities caused by cancers such as non-small cell lung cancer and multiple myeloma. As used interchangeably herein, "treatment" and / or "treating" and / or "treat" are intended to refer to any manner in which the progression of the disorders described herein may be delayed, hindered, inhibited, controlled, stopped, or reversed, but do not necessarily indicate the complete disappearance of all disorder symptoms. Treatment includes the administration of the antibody of the present invention for the treatment of human diseases or conditions that benefit from reduced RANKL activity, and includes (a) inhibiting further progression of the disease, and (b) relieving the disease, i.e., causing regression of the disease or disorder or alleviating its symptoms or complications.

[0024] As used herein, the term "patient" refers to a human. In certain embodiments, the patient is further characterized as having a disease, disorder, or condition (e.g., a bone-related disorder) that would benefit from a reduction in RANKL activity.

[0025] As used herein, the term "binds" (or "binds") to RANKL refers to the interaction of an antibody with an epitope of human RANKL. Preferably, "binds" refers to the interaction of an antibody of the invention with an epitope as determined by hydrogen-deuterium exchange (see, e.g., Example 4). Preferably, the epitope is a conformational epitope of human RANKL. In one embodiment, the term "binds" (or "binds") to RANKL refers to the interaction with a conformational epitope of human RANKL, the epitope being one or more of residues 89-97 and one or more of residues 125-132 of SEQ ID NO:17. In another embodiment, the epitope is at least two amino acids of residues 89-97 and at least two amino acids of residues 125-132 of SEQ ID NO:17. In another embodiment, the epitope is at least three amino acids of residues 89-97 and at least three amino acids of residues 125-132 of SEQ ID NO: 17. In another embodiment, the epitope is at least four amino acids of residues 89-97 and at least four amino acids within the range of 125-132 of SEQ ID NO: 17. In another embodiment, the conformational epitope is at least five amino acids of residues 89-97 and at least five amino acids of residues 125-132 of SEQ ID NO: 17.

[0026] The term "epitope" as used herein refers to the discrete three-dimensional site of an antigen that is recognized by the monoclonal antibody of the present invention. Epitope can be determined by methods currently known in the art, such as hydrogen-deuterium exchange, alanine scanning, or X-ray crystallography. As used herein, "competing" or "competition" refers to an antibody that can inhibit or prevent any one of the antibodies of the present invention from binding to human RANKL. For example, if there is a reduction or complete loss of binding to human RANKL by any one of the antibodies of the present invention in the presence of another antibody, the antibody can inhibit or prevent binding.

[0027] The monoclonal antibodies of the invention can be prepared by methods well known in the art and can be incorporated into pharmaceutical compositions comprising the monoclonal antibodies of the invention and one or more pharma- ceutically acceptable carriers and / or diluents.

[0028] A pharmaceutical composition comprising the monoclonal antibody of the present invention can be administered by parenteral routes (e.g., subcutaneously, intravenously, intraperitoneally, intramuscularly, or transdermally) to a patient at risk for or exhibiting a disease or disorder described herein. The pharmaceutical composition of the present invention comprises an "effective" or "therapeutically effective" amount of the monoclonal antibody of the present invention, which are used interchangeably herein. An effective amount refers to the amount necessary (at the dosage and duration of administration, and the means of administration) to achieve the desired therapeutic result. The effective amount of a monoclonal antibody may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or adverse effects of the monoclonal antibody of the present invention are outweighed by the therapeutically beneficial effects. EXAMPLES

[0029] Example 1: Antibody Expression and Purification Exemplary antibodies of the invention are expressed and purified essentially as follows: A glutamine synthetase (GS) expression vector containing the polynucleotide sequences shown in SEQ ID NO:15 (encoding an exemplary heavy chain of SEQ ID NO:1) and SEQ ID NO:16 (encoding an exemplary light chain of SEQ ID NO:2) is used to transfect Chinese hamster cell line (CHO, GS knockout) by electroporation. The expression vector encodes the SV Early (Simian Virus 40E) promoter and the GS gene. Expression of GS allows the biochemical synthesis of glutamine, an amino acid required by CHO cells. After transfection, cells are subjected to bulk selection with 50 μM L-methionine sulfoximine (MSX). Inhibition of GS by MSX is used to increase the stringency of selection. Cells in which the expression vector cDNA has integrated into a transcriptionally active region of the host cell genome can be selected against CHO wild-type cells. The transfected pool is plated at low density to allow close-to-clonal outgrowth of stable expressing cells. Master wells are screened for antibody expression and then scaled up in serum-free suspension medium and used for production.

[0030] The clarified medium into which the exemplary antibodies are secreted is applied to a Protein A affinity column equilibrated with a compatible buffer, such as phosphate-buffered saline (pH 7.4). The column is washed to remove non-specifically bound components. The bound antibodies are eluted, for example, by a pH gradient and neutralized, for example, with Tris buffer at pH 8. The antibody fractions are detected, for example, by SDS-PAGE or analytical size exclusion, and then pooled. Soluble aggregates and multimers can be efficiently removed by common techniques, including size exclusion, hydrophobic interaction, Capto multimodal chromatography, ion exchange, or hydroxyapatite chromatography. The antibodies are concentrated and / or sterile filtered using common techniques. The purity of the exemplary antibodies after these chromatographic steps is greater than 98% (monomer). The antibodies may be immediately frozen at -70°C or stored at 4°C for several months.

[0031] Example 2: Binding Affinity Binding affinity is measured using a BIAcore® 2000 instrument. All measurements are performed at 25° C. 2 μg / mL of the exemplary antibody of Example 1 is dissolved in HBS-EP buffer (150 mM sodium chloride, 3 mM EDTA, 0.005% (w / v) surfactant P-20, and 10 mM HEPES, pH 7.4). Protein A is immobilized on flow cells 1-4 of a CM4 sensor chip at a level of 500 response units (Rus) using an amine coupling kit.

[0032] Binding is assessed using multiple analytical cycles. Each cycle is performed at a flow rate of 20 μL / min. Approximately 10 μL of the exemplary antibody of Example 1 is injected at a concentration of 2 μg / mL, aiming at capture of approximately 100-200 Rus. 250 μL of human RANKL (starting at 5 nM and using 2-fold serial dilutions for each cycle) is injected, 10 minutes is allowed for dissociation, and 10 μL of 10 mM glycine hydrochloride, pH 1.5, is used for regeneration. Association and dissociation rates for each cycle are assessed using the "1:1 with mass transfer" binding model in the BIAevaluation software.

[0033] During engineering of the exemplary antibodies of Example 1, the presence of a leucine at the third penultimate position in HCDR3 (e.g., residue 12 of HCDR3 as set forth in SEQ ID NO:11) was identified as essential for conferring the engineered improved affinity of the antibodies of the invention for human RANKL. For example, in directed mutation analysis, when leucine was not present at the third penultimate position in HCDR3 (e.g., isoleucine was substituted for leucine), further amino acid changes to the antibodies of the invention failed to increase affinity for RANKL.

[0034] Following essentially the procedure above, the antibody was synthesized with a K D Binds to human RANKL at 34.43 pM K D It binds to rat RANKL with a K of 6.64 pM. D These data demonstrate that the exemplary antibodies of Example 1 bind to mouse RANKL with a K D have demonstrated high affinity binding to human and mouse RANKL.

[0035] Example 3: Neutralization of RANKL-induced NF-kB-driven luciferase activity in vitro HEK293 cells that stably co-express human RANK and NF-kB-driven luciferase reporter are used to evaluate the ability of the exemplary antibody of Example 1 to neutralize RANKL activity.In this HEK293 cell model, RANK induces NF-kB signal transduction when bound by human RANKL, which leads to luciferase luminescence.The neutralization of RANKL binding to RANK by the exemplary antibody of Example 1 is measured by the reduction of luciferase luminescence.

[0036] HEK293 cells are routinely cultured under selection pressure of 700 μg / mL Geneticin. 25,000 cells / well are added to wells of a 96-well tissue culture plate in assay medium (50 μL of DMEM / F12 (1:3) medium containing 0.5% FBS, 20 nM Hepes, 1× GlutaMax, and 1× penicillin / streptomycin). Cells are incubated overnight at 37° C. (5% CO2 and 95% humidity).

[0037] A dose range of 10 nM to 0.005 nM (1:3 serial dilutions) is prepared for the exemplary antibody of Example 1 using assay medium containing human RANKL at concentrations of 1 nM and 10 nM, and the doses are incubated at room temperature for 15 minutes. Assay medium is used for the "medium only" control. 50 μl of antibody is then added to 50 μl of medium containing cultured cells and incubated overnight at 37°C.

[0038] After overnight incubation, remove the existing growth medium and suspend the cells in 50 µL of BugLite (2.296 g DTT (Sigma), 1.152 g Coenzyme A, and 0.248 g ATP) in 1 L of 1% Trition X-100 lysis buffer (30 mL Triton X-100, 3 mL MgCl, 108.15 mL 1 M Trizma HCL, 41.85 mL 1 M Trizma Base, and 817 mL HO). The cells are then lysed on a plate shaker for 5-10 min with gentle agitation. After cell lysis, luminescence is measured on a plate reader (Envision plate reader). IC for all treatment groups 50 Values ​​are calculated using a three-parameter logistic regression model with GraphPad Prism 6.

[0039] Essentially following the procedure described above, IC of antibodies that bind to human RANKL 50was 0.069 nM. This result demonstrates that the exemplary antibody of Example 1 neutralizes human RANKL-induced NF-kB-driven luciferase luminescence. The medium control did not neutralize human RANKL-induced NF-kB-driven luciferase luminescence in the HEK293 cell model at any of the concentrations tested.

[0040] Example 4: Epitope mapping by hydrogen-deuterium exchange To determine where the exemplary antibody of Example 1 binds to the RANKL protein, hydrogen deuterium exchange with mass spectrometry (HDXMS) is performed. Using this method, the epitopes of several antibodies have been successfully mapped (Obungu et. al. 2009 Biochemistry,48:7251-60 Lu et. al. 2005 Biochemistry44:11106-14).

[0041] RANKL / antibody complexes were made by mixing 10 μg of human RANKL solution with 10 μg of the exemplary antibody of Example 1 and then diluting with 1×PBS. Complexes for protein surface labeling studies are labeled with hydroxysuccinimide acetate ester, and HDXMS analysis is performed by mixing 4 μL of RANKL / antibody complex with 16 μL of 100% D2O (80% D during exchange) and holding the mixture at ambient temperature for 60 seconds. The exchange is quenched with 50 μL of 0.1N HCl at 0° C., immediately treated with 2 μL of 2 mg / mL pepsin solution at 0° C. for 3.5 minutes, and then manually injected into a RP-HPLC column. The digest solution is then subjected to LC / MS / MS analysis by a Waters SYNAPT mass spectrometer coupled with a Waters Acquity UPLC.

[0042] Following essentially the procedure described above, two human RANKL peptides 239-272 and 281-290 showed significant negative delta. This analysis demonstrated that the RANKL epitope of the exemplary antibody of Example 1 is conformational and is amino acids 89-97 and 125-132 of SEQ ID NO: 17. The first region (89-97) is identical in human and rodent RANKL. The other region (125-132) is identical in both human and rodent RANKL, except for a single amino acid difference at position 129, where a serine is replaced by an alanine in rodent RANKL.

[0043] Example 5: An intact female mouse model is used to evaluate the effects on bone mass density in vivo. Intact C57 / B6 female mice (Charles River), 20-22 weeks of age, are maintained on a 12-h light / dark cycle at 22 °C with ad libitum access to food (TD 2014 with 0.72% Ca and 0.61% P, vitamin D 0.99 IU / g, Teklad, Madison, WI) and water.

[0044] Mice are divided into treated animals that receive weekly subcutaneous injections of 10 mg / kg of the exemplary antibody of Example 1 (n=6 animals) or PBS vehicle control. Mice are sacrificed at 4 weeks. Bone mass density (BMD) of the distal and mid femur is monitored by quantitative computed tomography (qCT) using an Aloka LaTheta LTC-100 model CT scanner.

[0045] Following essentially the procedures described above, animals treated with the antibody showed a 23% increase in distal femur BMD and a 4% increase in mid femur BMD compared to control animals. These results demonstrate that animals administered the exemplary antibody of Example 1 weekly showed increased BMD in both the distal and mid femur.

[0046] Example 6: In vivo efficacy analysis in an ovariectomized mouse model An ovariectomized mouse model is used to evaluate the effect on bone mass density in vivo. 20-week-old female C57 / B6 mice (Harlan, Indianapolis, IN) are ovariectomized (or sham-operated controls) and maintained on a 12-h light / dark cycle at 22°C with free access to food (TD 2014 with 0.72% Ca and 0.61% P, Vitamin D 0.99 IU / g, Teklad, Madison, WI) and water. Osteopenia is established in mice by allowing ovariectomized mice to lose bone mass for 6 weeks.

[0047] After a 6-week osteopenia establishment period, mice are divided into treatment groups (n=6 animals per group) or vehicle PBS control group. Each treatment group of mice receives weekly subcutaneous injections of 3mg / kg or 10mg / kg of the exemplary antibody of Example 1 or PBS. Mice are sacrificed at week 4. After sacrifice, skeletal bone mass density (BMD) of the fifth vertebra is assessed by quantitative computed tomography (qCT) using an Aloka LaTheta LTC-100 model CT scanner.

[0048] Following essentially the procedures described above, animals treated with 3 mg / kg antibody had a 2% increase in BMD and animals treated with 10 mg / kg antibody had an 11% increase in BMD compared to controls. These results demonstrate that the exemplary antibody of Example 1 produces a dose-dependent increase in vertebral BMD in ovariectomized mice when administered weekly.

[0049] Example 7: In vivo efficacy analysis in an orchiectomized mouse model An orchiectomized mouse model is used to evaluate in vivo effects on bone mass density and bone mineral content. 16-week-old male C57 / B6 mice (Harlan, Indianapolis, IN) are orchiectomized (or vehicle control group, n=6) and maintained on a 12-hour light / dark cycle at 22°C with free access to diet (TD 2014 with 0.72% Ca and 0.61% P, Vit.D 0.99 IU / g, Teklad, Madison, WI) and water. Osteopenia is established in orchiectomized mice by allowing the mice to lose bone mass for 6 weeks.

[0050] After a 6-week osteopenia establishment period, 9 mice are subcutaneously injected twice a week with the exemplary antibody of Example 1 at a dose of 2 mg / kg. Animals injected with PBS serve as controls. Mice are sacrificed at 2 weeks. Bone mass density (BMD) of the distal femur and bone mineral content (BMC) of the lumbar spine are assessed by quantitative computed tomography (qCT) using an Aloka LaTheta LTC-100 model CT scanner.

[0051] Mice treated with the antibody essentially following the procedure described above showed a 19% increase in BMD at the distal femur and a 13% increase in BMD at the lumbar spine compared to control animals. These results demonstrate that treatment with the exemplary antibody of Example 1 results in increased BMD at the distal femur and lumbar spine in orchiectomized mice. array Exemplary HC (SEQ ID NO:1) QVQLVQSGAEVKKPGSSVKVSCKASGYAFTNYYIEWVRQAPGQGLEWMGVINPGWGDTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARRDTAHGYYALDPW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Exemplary LC (SEQ ID NO:2) DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWDYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Exemplary HCVR (SEQ ID NO:3) QVQLVQSGAEVKKPGSSVKVSCKASGYAFTNYYIEWVRQAPGQGLEWMGVINPGWGDTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARRDTAHGYYALDPWGQGTTVTVSS Exemplary LCVR (SEQ ID NO:4) DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWDYPLTFGGGTKVEIK Exemplary HC (SEQ ID NO:5) QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYYIEWLKQRPGQGLEWIGVINPGWGDTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVFFCARRDTRHGYYALDYW GQGTSVTVSSAKTTPPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRNC GGDCKPCICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEVHTAQTRPPEEQFNSTFRSVSELPILHQDWLNGRTFRCKVTSAAFPSPIEK TISKPEGRTQVPHVYTMSPTKEEMTQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPPTMDTDGSYFLYSKLNVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPG Exemplary LC (SEQ ID NO:6) DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNDYPLTFGAGTRLEIK RADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDSTYSMSSTLSLSKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC Exemplary HCVR (SEQ ID NO:7) QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYYIEWLKQRPGQGLEWIGVINPGWGDTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVFFCARRDTRHGYYALDYWGQGTSVTVSS Exemplary LCVR (SEQ ID NO:8) DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNDYPLTFGAGTRLEIK Exemplary HCDR1 (SEQ ID NO:9) KASGYAFTNYYIE Exemplary HCDR2 (SEQ ID NO: 10) VINPGWGDTNYNEKFKG Exemplary HCDR3 (SEQ ID NO:11) ARRDTXHGYYALDX X at position 6 is Ala or Arg, and X at position 14 is Pro or Tyr. Exemplary LCDR1 (SEQ ID NO: 12) KASQNVGTNVA Exemplary LCDR2 (SEQ ID NO: 13) YSASYRYS Exemplary LCDR3 (SEQ ID NO: 14) QQYXDYPLT X at position 4 is Trp or Asn. Exemplary DNA encoding the HC protein of SEQ ID NO:1 (SEQ ID NO:15) Exemplary DNA encoding the LC of SEQ ID NO:2 (SEQ ID NO:16) GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAAGGCCAGCCAGAATGTGGGCACCAACGTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGCGCCAGCTACA GATACAGCGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAGTACTGGGACTACCCCCTGACCTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC RANKL extracellular domain (SEQ ID NO: 17) MSKLEAQPFAHLTINATDIPSGSHKVSLSSWYHDRGWAKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGDLATEYLQLMVYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNPSLLDPDQDATYFGAFKVRDID

Claims

1. An antibody that binds to human RANKL, comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, the amino acid sequence of the LCDR1 is SEQ ID NO: 12, the amino acid sequence of the LCDR2 is SEQ ID NO: 13, the amino acid sequence of the LCDR3 is SEQ ID NO: 14, the amino acid sequence of the HCDR1 is SEQ ID NO: 9, the amino acid sequence of the HCDR2 is SEQ ID NO: 10, and the amino acid sequence of the HCDR3 is SEQ ID NO: 11, and the Xaa at position 6 of the amino acid sequence shown in SEQ ID NO: 11 is Arg, the Xaa at position 14 of the amino acid sequence shown in SEQ ID NO: 11 is Tyr, and the Xaa at position 4 of the amino acid sequence shown in SEQ ID NO: 14 is Asn.

2. The antibody of claim 1 , wherein the amino acid sequence of the LCVR is SEQ ID NO: 8 and the amino acid sequence of the HCVR is SEQ ID NO:

7.

3. The antibody of claim 1 or 2, comprising a light chain (LC) and a heavy chain (HC), wherein the amino acid sequence of the LC is SEQ ID NO: 6 and the amino acid sequence of the HC is SEQ ID NO:

5.

4. A pharmaceutical composition comprising the antibody of any one of claims 1 to 3 and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

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