Therapeutic antibodies targeting rankl

Abstract

This invention provides monoclonal antibodies against RANKL, particularly high-affinity humanized RANKL monoclonal antibodies. It also provides functional monoclonal antibodies that cross-react with RANKL in humans, cynomolgus monkeys, and mice. Furthermore, this invention provides the amino acid sequence of the antibodies of this invention, cloning or expression vectors, host cells, methods for expressing or isolating the antibodies, and identification of antibody epitopes. It also provides therapeutic compositions comprising the antibodies of this invention. Finally, this invention provides methods for treating cancer and other diseases with anti-RANKL antibodies.

Description

[0001] This application is a divisional application of Chinese application No. 201811299652.4, filed on October 25, 2018, entitled “Therapeutic antibodies targeting RANKL”. TECHNICAL FIELD

[0002] The present application generally relates to antibodies against RankL and compositions thereof, and therapies using anti-RankL antibodies to treat diseases associated with bone loss. BACKGROUND

[0003] The receptor activator of NF kappa-B ligand (RANKL), also known as osteoprotegerin ligand (OPGL) and tumor necrosis factor ligand superfamily member 11 (TNFSF11), is a member of the TNF superfamily (Anderson DM, et.al, Nature 390(6656): 175-179). RANKL can be expressed on the cell surface or in soluble form (Findlay DM, et.al, Osteoporos Int 22(10): 2597-2602). By binding to the receptor RANK on osteoclasts and their precursors, it mediates osteoclast formation, activation and survival (Hsu H, et.al, Proc Natl Acad Sci U S A. 1999 Mar 30; 96(7): 3540-2545). There is also increasing evidence that tumor cells interact within the bone, stimulating the receptor activator of the RANK-RANKL system, leading to cancer-induced bone destruction (Roodman GD, N Engl J Med 2004; 350: 1655-1664).

[0004] Early animal studies demonstrated the potential of RANKL as a therapeutic target for treating osteoclast-mediated bone loss (Lacey DL, et.al, Cell. 1998; 93: 165-176; Ann E. Kearns, et.al, Endocrine Reviews 29(2): 155-192). Animal studies and clinical trials using OPG Fc fusion protein, an endogenous RANKL inhibitor, provided strong evidence that RANKL is critically important in bone remodeling (Ann E. Kearns, et.al, Endocrine Reviews 29(2): 155-192; Bekker PJ, J Bone Miner Res. 2001; 16: 348-360).

[0005] Denosumab is a fully human monoclonal antibody against RANKL approved by FDA, and its clinical studies show significant reduction in vertebral, non-vertebral and hip fractures, and provide evidence consistent with the use of denosumab in postmenopausal women with osteoporosis (David W. Dempster, Clin Ther. 2012; 34:521-536). Three key randomized III phase trials also show that denosumab is superior to zoledronic acid in preventing skeletal-related events (SREs) and has good safety and convenience for patients with bone metastases of advanced cancer (Clin Ther. 2012; 34:521-536; Prasad Narayanan, Journal of Cancer. 2013; 2(4):272-277). SUMMARY

[0006] The present application provides an isolated antibody, particularly a monoclonal antibody or a humanized monoclonal antibody.

[0007] In one aspect, the present application provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to human, monkey and mouse RANKL.

[0008] In one aspect, the present application provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment

[0009] a) binds to human RANKL, K D is 6.98E-10 M or less; and

[0010] b) binds to monkey RANKL, K D is 5.32E-11 M or less.

[0011] The foregoing antibody or antigen-binding fragment has at least one of the following properties:

[0012] a) binds to human RANKL, K D is 6.98E-10 M to 6.33E-11 M, and binds to monkey RANKL, K D is 5.32E-11 M to 9.03E-12 M;

[0013] b) inhibits RANKL-mediated cellular function;

[0014] c) inhibits differentiation of RAW cells into osteoclast-like cells.

[0015] The present disclosure provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% homology to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14,

[0016] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0017] The present disclosure provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14,

[0018] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0019] The present disclosure provides an antibody or antigen-binding fragment thereof comprising:

[0020] a) a heavy chain variable region having an amino acid sequence having at least 70%, 80%, 90%, or 95% homology to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7; and

[0021] b) a light chain variable region having an amino acid sequence having at least 70%, 80%, 90%, or 95% homology to a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14,

[0022] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0023] The present disclosure provides an antibody or antigen-binding fragment thereof comprising:

[0024] a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7; and

[0025] b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14,

[0026] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0027] In various embodiments, the antibody or antigen-binding fragment thereof comprises:

[0028] a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; and

[0029] b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 8,

[0030] wherein the antibody or antigen-binding fragment specifically binds RANKL;

[0031] or the antibody or antigen-binding fragment thereof comprises:

[0032] a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; and

[0033] b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 9,

[0034] wherein the antibody or antigen-binding fragment specifically binds RANKL;

[0035] or the antibody or antigen-binding fragment thereof comprises:

[0036] a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 3; and

[0037] b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 10,

[0038] wherein the antibody or antigen-binding fragment specifically binds RANKL;

[0039] or the antibody or antigen-binding fragment thereof comprises:

[0040] a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 4; and

[0041] b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 11,

[0042] wherein the antibody or antigen-binding fragment specifically binds RANKL;

[0043] or the antibody or antigen-binding fragment thereof comprises:

[0044] a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 5; and

[0045] b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 12

[0046] wherein the antibody or antigen-binding fragment specifically binds RANKL;

[0047] or the antibody or antigen-binding fragment thereof comprises:

[0048] a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 6; and

[0049] b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 13,

[0050] wherein the antibody or antigen-binding fragment specifically binds RANKL;

[0051] or the antibody or antigen-binding fragment thereof comprises:

[0052] a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 7; and

[0053] b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 14,

[0054] wherein the antibody or antigen-binding fragment specifically binds RANKL;

[0055] The sequences of the antibodies are shown in Table 1 and the sequence listing.

[0056] Table 1 Amino acid sequences of antibodies

[0057]

[0058]

[0059] In another aspect, the present application provides an antibody or antigen-binding fragment thereof comprising complementarity determining regions (CDRs) having amino acid sequences selected from the group consisting of SEQ ID NOs: 15-36,

[0060] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0061] In another aspect, the present application provides an antibody or antigen-binding fragment thereof comprising:

[0062] a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; and

[0063] a light chain variable region comprising CDR1, CDR2 and CDR3 sequences,

[0064] wherein the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17 and conservative modifications thereof,

[0065] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0066] Preferably, the heavy chain variable region CDR3 sequence of the aforementioned antibody, or antigen-binding fragment thereof, comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, and conservative modifications thereof.

[0067] Preferably, the heavy chain variable region CDR2 sequence of the aforementioned antibody, or antigen-binding fragment thereof, comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, and conservative modifications thereof.

[0068] Preferably, the light chain variable region CDR2 sequence of the aforementioned antibody, or antigen-binding fragment thereof, comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, and conservative modifications thereof.

[0069] Preferably, the heavy chain variable region CDR1 sequence of the aforementioned antibody, or antigen-binding fragment thereof, comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, and conservative modifications thereof.

[0070] Preferably, the light chain variable region CDR1 sequence of the aforementioned antibody, or antigen-binding fragment thereof, comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, 35, 36, and conservative modifications thereof.

[0071] In more preferred embodiments, the present application provides an antibody, or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to RANKL and comprises: a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:

[0072] a) the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, and 32, the CDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, and 25, and the CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, and 17;

[0073] b) and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, 35, and 36, the CDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, and 29, and the CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, and 21,

[0074] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0075] Another preferred antibody or antigen-binding fragment thereof comprises:

[0076] a) a heavy chain variable region CDR1 comprising SEQ ID NO: 31 ;

[0077] b) a heavy chain variable region CDR2 comprising SEQ ID NO: 23;

[0078] c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16;

[0079] d) a light chain variable region CDR1 comprising SEQ ID NO: 34;

[0080] e) a light chain variable region CDR2 comprising SEQ ID NO: 27;

[0081] f) a light chain variable region CDR3 comprising SEQ ID NO: 19;

[0082] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0083] Another preferred antibody or antigen-binding fragment thereof comprises:

[0084] a) a heavy chain variable region CDR1 comprising SEQ ID NO: 32;

[0085] b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24;

[0086] c) a heavy chain variable region CDR3 comprising SEQ ID NO: 17;

[0087] d) a light chain variable region CDR1 comprising SEQ ID NO: 35;

[0088] e) a light chain variable region CDR2 comprising SEQ ID NO: 28;

[0089] f) a light chain variable region CDR3 comprising SEQ ID NO: 20;

[0090] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0091] Another preferred antibody or antigen-binding fragment thereof comprises:

[0092] a) a heavy chain variable region CDR1 comprising SEQ ID NO: 32;

[0093] b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24;

[0094] c) a heavy chain variable region CDR3 comprising SEQ ID NO: 17;

[0095] d) a light chain variable region CDR1 comprising SEQ ID NO: 35;

[0096] e) a light chain variable region CDR2 comprising SEQ ID NO: 28;

[0097] f) a light chain variable region CDR3 comprising SEQ ID NO: 20;

[0098] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0099] Another preferred antibody or antigen-binding fragment thereof comprises:

[0100] a) a heavy chain variable region CDR1 comprising SEQ ID NO: 30;

[0101] b) a heavy chain variable region CDR2 comprising SEQ ID NO: 25;

[0102] c) a heavy chain variable region CDR3 comprising SEQ ID NO: 15;

[0103] d) a light chain variable region CDR1 comprising SEQ ID NO: 36;

[0104] e) a light chain variable region CDR2 comprising SEQ ID NO: 29;

[0105] f) a light chain variable region CDR3 comprising SEQ ID NO: 21;

[0106] wherein the antibody specifically binds RANKL.

[0107] Another preferred antibody or antigen-binding fragment thereof comprises:

[0108] a) a heavy chain variable region CDR1 comprising SEQ ID NO: 30;

[0109] b) a heavy chain variable region CDR2 comprising SEQ ID NO: 22;

[0110] c) a heavy chain variable region CDR3 comprising SEQ ID NO: 15;

[0111] d) a light chain variable region CDR1 comprising SEQ ID NO: 33;

[0112] e) a light chain variable region CDR2 comprising SEQ ID NO: 26;

[0113] f) a light chain variable region CDR3 comprising SEQ ID NO: 19;

[0114] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0115] Another preferred antibody or antigen-binding fragment thereof comprises:

[0116] a) a heavy chain variable region CDR1 comprising SEQ ID NO: 32;

[0117] b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24;

[0118] c) a heavy chain variable region CDR3 comprising SEQ ID NO: 17;

[0119] d) a light chain variable region CDR1 comprising SEQ ID NO: 35;

[0120] e) a light chain variable region CDR2 comprising SEQ ID NO: 28;

[0121] f) a light chain variable region CDR3 comprising SEQ ID NO: 20;

[0122] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0123] Another preferred antibody or antigen-binding fragment thereof comprises:

[0124] a) a heavy chain variable region CDR1 comprising SEQ ID NO: 32;

[0125] b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24;

[0126] c) a heavy chain variable region CDR3 comprising SEQ ID NO: 17;

[0127] d) a light chain variable region CDR1 comprising SEQ ID NO: 35;

[0128] e) a light chain variable region CDR2 comprising SEQ ID NO: 28;

[0129] f) a light chain variable region CDR3 comprising SEQ ID NO: 20;

[0130] wherein the antibody or antigen-binding fragment specifically binds RANKL.

[0131] The CDR sequences of the antibodies are shown in Table 2 and the Sequence Listing.

[0132] Table 2 CDR sequences of antibodies

[0133]

[0134] The antibody of the present application can be a chimeric antibody.

[0135] The antibody of the present application can be a humanized antibody.

[0136] The antibody of the present application can be a fully human antibody.

[0137] The antibody of the present application can be a rat antibody.

[0138] The antibody or antigen-binding fragment of the present application has at least one of the following properties:

[0139] a) binds to human RANKL, K D is 6.98E-10 M to 6.33E-11 M, and binds to monkey RANKL, K D is 5.32E-11 M to 9.03E-12 M;

[0140] b) inhibits RANKL-mediated cellular function;

[0141] c) inhibits RAW cell differentiation into osteoclast-like cells.

[0142] In another aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

[0143] The present application provides a cloning or expression vector comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

[0144] The present application also provides a host cell comprising one or more cloning or expression vectors.

[0145] In another aspect, the present application provides a method comprising culturing the above-mentioned host cell and isolating the antibody; wherein the antibody is prepared by immunizing a mouse having human RankL protein.

[0146] The present application provides a transgenic animal such as a mouse comprising human immunoglobulin heavy and light chain transgenes, wherein the animal expresses the antibody of the present application.

[0147] The present application provides a hybridoma prepared from the animal of the present application, wherein the hybridoma produces the antibody.

[0148] In yet another aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the present application, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0149] The present application provides a method for preparing an anti-RANKL antibody or antigen-binding fragment thereof, comprising:

[0150] (a) providing:

[0151] (i) an antibody sequence of a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 30-32, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 22-25, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 15-17; and / or

[0152] (ii) an antibody sequence of a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 33-36, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 26-29, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18-21; and

[0153] (b) expressing the altered antibody sequence as a protein.

[0154] The present application also provides a method of preventing or treating a bone loss-related disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the above-described antibody or antigen-binding fragment of the present application.

[0155] The present application also provides a combination method of preventing or treating a bone loss-related disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the above-described antibody or antigen-binding fragment of the present application, and administering to the subject a therapeutically effective amount of an immune checkpoint antibody.

[0156] The immune checkpoint antibody of the present application includes a CTLA-4 antibody, a PD-1 antibody, or a PD-L1 antibody.

[0157] The present application also provides use of the antibody or antigen-binding fragment thereof in the manufacture of a medicament for preventing or treating a disease related to bone loss.

[0158] The disease includes Crohn's disease, male infertility, chronic kidney disease, gout, rheumatoid arthritis, anorexia nervosa, thalassemia major, hyperthyroidism, or cancer.

[0159] The cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer.

[0160] Advantages of the present application

[0161] The antibody disclosed in the present application has high binding affinity and specifically binds to human and monkey RANKL proteins, and is effective in preventing or treating a disease related to bone loss.

[0162] One of the antibodies not only binds to human and monkey RANKL, but also to murine RANKL, which greatly facilitates the preclinical validation of its efficacy in mouse tumor models. BRIEF DESCRIPTION OF DRAWINGS

[0163] Figure 1 Binding of antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, WBP114_7.80, and Prolia to human RANKL is shown.

[0164] Figure 2 Blocking assay of WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, WBP114_7.80, and Prolia is shown.

[0165] Figure 3 Dependent binding of antibody WBP114_5.20 to mouse RANKL is shown.

[0166] Figure 4 NF-κΒ reporter gene assay of selected antibodies is shown.

[0167] Figure 5 RAW264.7 cell differentiation assay of selected antibodies is shown.

[0168] Figure 6 Epitope mapping of selected antibodies against Prolia is shown.

[0169] Figure 7 ELISA binding assay of humanized antibodies is shown.

[0170] Figure 8 ELISA blocking assay of humanized antibodies is shown.

[0171] Figure 9 NF-κΒ reporter gene assay of humanized antibodies is shown.

[0172] Figure 10 RAW264.7 cell differentiation assay of humanized antibodies is shown. DETAILED DESCRIPTION

[0173] The application is further described below by way of specific embodiments and experimental data. Although specific terms are used in the following description for the sake of clarity, these terms are not intended to limit the scope of the application, but rather to describe a particular embodiment thereof.

[0174] The terms "receptor activator of nuclear factor-kappa B ligand," "RANKL," "TNF-related activation-induced cytokine," "TRANCE," "osteoprotegerin ligand," "OPGL," "osteoclast differentiation factor," "ODF" are used interchangeably and include variants, isoforms, species homologs of human RANKL or RANKL of other species, and analogs having at least one common epitope with RANKL.

[0175] As used herein, the term "antibody" includes intact antibodies and any antigen binding fragment (i.e., "antigen binding portion") or single chains thereof. An "antibody" refers to a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), and regions that are more conserved, termed framework regions (FR). 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.

[0176] The term "antibody" as used herein refers to an immunoglobulin or fragment or derivative thereof and includes any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chained, chimeric, synthetic, recombinant, hybrid, mutated, grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, FV, scFv, Fd, dAb and other antibody fragments that retain antigen binding function, i.e., the ability to specifically bind to RANKL. Typically, such fragments will include an antigen binding fragment.

[0177] The terms "antigen binding fragment," "antigen binding domain," and "binding fragment" refer to an antibody molecule that comprises the amino acids responsible for the specific binding between the antibody and the antigen. For example, in the case of a large antigen, the antigen binding fragment can bind only a portion of the antigen. The portion of the antigen molecule responsible for the specific interaction with the antigen binding fragment is referred to as an "epitope" or "antigenic determinant."

[0178] An antigen-binding fragment typically includes both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to include both. For example, a so-called Fd antibody fragment consists of only a VH domain, but still retains some antigen-binding function of the intact antibody.

[0179] The term "epitope" is defined as an antigenic determinant to which the binding fragment specifically binds / recognizes. The binding fragment can specifically bind / interact with a conformational or contiguous epitope unique to the target structure, such as human RANKL and murine RANKL. A conformational or discontinuous epitope is characterized by two or more discrete amino acid residues of a polypeptide antigen that are separated in the primary sequence but come together on the surface of the molecule when the polypeptide is folded into the native protein / antigen. The two or more discrete amino acid residues that make up the epitope exist in separate portions of one or more polypeptide chains. When the polypeptide chains are folded into a three-dimensional structure, the residues come together on the surface of the molecule to make up the epitope. In contrast, a contiguous or linear epitope is composed of two or more discrete amino acid residues that exist in a single linear segment of a polypeptide chain.

[0180] The term "cross-reactivity" as described herein refers to the binding of an antigenic fragment of the same target molecule of human, monkey, and / or murine (mouse or rat) origin. Thus, "cross-reactivity" is to be understood as an interspecies reaction with the same molecule X expressed in different species. The cross-reactivity specificity of a monoclonal antibody recognizing human RANKL, monkey, and / or murine RANKL (mouse or rat) can be determined by FACS analysis.

[0181] The term "subject" as used herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "patient" or "subject" are used interchangeably unless specifically indicated otherwise.

[0182] The terms "treatment" and "treatment methods" refer to both therapeutic treatment and prophylactic / preventive measures. Those in need of treatment include individuals already having a particular medical condition, as well as individuals who can be predisposed to the condition.

[0183] The term "conservative modification" to a nucleotide and amino acid sequence, i.e., a modification that does not significantly affect or alter the binding properties of the antibody encoded by the nucleotide sequence or comprising the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into a sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include those 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).

[0184] The experimental methods used in the following examples are conventional unless otherwise specified.

[0185] Example

[0186] Example 1: Antibody production

[0187] 1. Immunization and generation of antibody hybridomas

[0188] 1.1 Immunization

[0189] Balb / c mice were injected with human RankL protein via the footpad approximately every 3 days. After 6 injections, a first titer test was performed.

[0190] 1.2 Serum titer test

[0191] An ELISA assay was used to measure the titer of antibodies in mouse serum. To measure the titer of antibodies, plates (Nunc) were coated with human RankL at 1 pg / mL overnight at 4°C and then blocked with blocking buffer (1 x PBS / 2% BSA) for 1 hour at room temperature. Rat serum was titrated starting at 1 : 100 dilution in blocking buffer and incubated for 1 hour at room temperature. Plates were then washed and followed by a 1 hour incubation with secondary antibody goat anti-mouse IgG Fc HRP. After washing, TMB substrate was added and the interaction was stopped with 2M HC1. Absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0192] The titer of antigen-specific antibodies in serum was determined by ELISA assay. Mice with serum titers of 312500 or higher were selected for hybridoma fusion.

[0193] 2. Generation of hybridomas

[0194] Lymph nodes and spleen were collected from immunized mice under sterile conditions and lymphocytes were prepared using Ficoll-Paque PLUS gradient centrifugation. Isolated cells were then fused with myeloma cells P3 at a 1 : 1 ratio using an electrofusion device (BTX ECM2001). After fusion, cells were transferred to 1 / 2 HA medium. 5 x 10 5 cells were seeded per 96-well plate.

[0195] 3. Antibody screening

[0196] Hybridoma supernatants were used for primary screening. Antigen-specific hybridomas were then screened by ELISA blocking assay.

[0197] Binding assay by ELISA: Plates (Nunc) were coated overnight at 4°C with 1 pg / mL of human RankL. After blocking and washing, hybridoma supernatants were transferred to the plates and incubated for 1 hour at room temperature. Plates were then washed, followed by a 1-hour incubation with secondary antibody goat anti-mouse IgG Fc HRP. After washing, TMB substrate was added and the interaction was stopped with 2 M HC1. Absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0198] Blocking assay by ELISA: Plates (Nunc) were coated overnight at 4°C with 5 pg / mL of Rank-Fc fusion protein. Hybridoma supernatants were mixed with 250 ng / mL RankL-His and incubated overnight at 4°C. Prolia was used as a positive control. After blocking and washing, the mixture was added to the plates and incubated for 1 hour. Plates were then washed, followed by a 1-hour incubation with anti-His Ab-HRP. After washing, TMB substrate was added and the interaction was stopped with 2 M HC1, and absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0199] Antibodies that can block the binding of human Rank ligand to human Rank were selected for further characterization. Selected antibodies with binding and blocking activity were purified from hybridoma supernatants. At the same time, selected hybridoma lines were subcloned. Hybridoma subclones were verified by binding and blocking ELISA assays and their isotypes were also detected.

[0200] 4. Subcloning

[0201] Hybridoma cells of each selected cell line were seeded in 96-well plates at a density of 0.5, 1, and 5 cells / well. Single clones were picked and tested in a binding ELISA. Three subclones of each hybridoma line were selected and frozen.

[0202] 5. Antibody purification

[0203] After pH adjustment to 7.0, the harvested hybridoma supernatant was loaded onto a protein A column (MabSelect SuRe, GE). The antibody was eluted by glycine and then immediately neutralized with 1 M Tris. The antibody concentration was tested by Nano Drop (Thermal-Fisher). The purity of the protein was assessed by SDS-PAGE (Invitrogen, NuPAGE 4-12% Bis-Tris Gel) and HPLC-SEC (Agilent).

[0204] 6. Antibody isotype

[0205] Antibody isotype was identified by ELISA. Plates (Nunc) were coated with 1 pg / mL goat anti-mouse IgGl / anti-mouse IgG2a / anti-mouse IgG2b / anti-mouse IgG3 / anti-mouse IgM antibodies at 4°C overnight. After blocking and washing, the hybridoma supernatant was transferred to the coated plates and incubated at room temperature for 1 hour. The plates were then incubated with secondary antibody goat anti-mouse kappa HRP or goat anti-mouse lambda HRP (Southern Biotech) for 45 minutes. After washing, TMB substrate was added and the interaction was terminated with 2 M HC1. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0206] The isotype of the murine antibodies is shown in Table 3.

[0207] Table 3. Isotype of murine antibodies

[0208]

[0209]

[0210] 7. Antibody sequence

[0211] RNA was extracted from hyridoma cells using Trizol reagent (Invitrogen-15596018). cDNA was amplified using 5'-RACE kit (Takara-28001488) and then PCR amplification was performed using 3'-degenerate primer and 3'-adaptor primer (ExTaq: Takara-RR001B). The PCR fragments were inserted into pMD18-T vector (Takara-D101C) and sent for sequencing (Shanghai Biosune).

[0212] The variable region amino acid sequences of the murine anti-RankL antibodies are shown in Table 4 and the variable region DNA sequences are shown in Table 5.

[0213] Table 4. Variable region amino acid sequences of murine anti-RankL antibodies

[0214]

[0215]

[0216] Underlined sequences are CDRs 1-3, respectively.

[0217] Table 5. Variable region DNA sequences of murine anti-RankL antibodies

[0218]

[0219]

[0220] Example 2: Characterization of murine antibodies

[0221] 1. ELISA binding

[0222] Antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, and WBP114_7.80 exhibited sub-nanomolar binding activity and their EC 50 values were comparable to Prolia. The binding EC 50 and maximum binding values are summarized in Tables 6 and Figure 1 .

[0223] Table 6. ELISA binding of antibodies

[0224]

[0225] 2. Blocking assay

[0226] Antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, and WBP114_7.80 can block RANKL binding to RANK. WBP114.5.2.1, WBP114_5.14.1, and WBP114_7.80 showed comparable blocking activity to Prolia. The inhibition IC 50 values are summarized in Tables 7 and Figure 2 .

[0227] Table 7. Antibody blocking analysis

[0228]

[0229] 3. Cross-binding activity to mouse RANKL

[0230] ELISA plates (Nunc) were coated with mouse RankL overnight at 4°C at 5 pg / mL. After blocking and washing, 1 pg / mL antibody sample or Prolia was added and incubated for 1 hour. Plates were then washed and followed by incubation with secondary antibody anti-mouse IgG Fc HRP / anti-human IgG Fc HRP (Bethyl) for 1 hour. After washing, TMB substrate was added and the interaction was stopped with 2 M HC1. Absorbance at 450 nm was read using a microplate reader (Molecular Device). Antibody WBP114_5.20 can bind to murine RANKL (EC Figure 3 ), with an EC 50 of 0.067 nM. WBP114.5.2.1, WBP114_5.14.1, WBP114_7.80 and Prolia do not bind to mouse RANKL (data not shown).

[0231] 4. NF-kB reporter assay

[0232] An engineered HK293 cell line containing RANK and NF-kB-luc was used to measure the ability of antibodies to neutralize RANKL-mediated cellular function.

[0233] 293F cells were resuspended in FreeStyle 293 Expression Medium (Invitrogen-12338) at a density of 5 x 10 5 cells / mL. 1 mL / well of 293F cell suspension was transferred to a 24-well plate. The pGl-NF-kB vector was diluted in Opti-MEM I Reduced Serum Medium (Invitrogen-31985). PlasFect was diluted in Opti-MEM I Reduced Serum Medium. After 5 minutes of incubation, the diluted pGl-NF-kB and PlasFect were combined. Mixed gently and incubated at room temperature for 20 minutes. The mixture was added to the cells and incubated at 37°C overnight.

[0234] Transfected 293F-pG1-NF-kB cells were seeded at a density of 5000 cells / well in FreeStyle 293 expression medium in 96-well plates (Corning 3916). Mixtures of RANKL and various concentrations of antibodies were pre-incubated for 30 minutes at 4°C and added to the cells. The final concentration of RANKL was 300 ng / mL. After 24 hours of incubation at 37°C, 50 μL / well Nano-Glo luciferase (Promega - N1120) was added to each well and incubated for 5 minutes at room temperature (protected from light). The fluorescence was read by a MDSpectraMax M5e. The inhibition was calculated as [V(RANKL) - V(sample)] / [V(RANKL) - V(blank)] x 100%. V(RANKL) = RFU of (cells + RANKL), V(sample) = RFU of (cells + RANKL + Ab), V(blank) = RFU of cells.

[0235] Measurement of inhibition of RANKL-induced luciferase activity by antibodies Figure 4 ) IC50values 50 Values are summarized in Table 4. Antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_7.80 showed comparable inhibitory activity to Prolia. Antibody WBP114_5.20 showed slightly lower inhibitory activity than Prolia (Table 8).

[0236] Table 8. NF-kB reporter assay

[0237]

[0238] 5. RAW264.7 cell differentiation assay

[0239] RAW cell differentiation assay was used to examine the inhibition of osteoclast formation by antibodies. RANKL can stimulate RAW cells to differentiate into osteoclast-like cells and the differentiation can be measured by tartrate-resistant acid phosphatase (TRAP) activity.

[0240] RAW264.7 cells were seeded at 2 x 10 4Cells were seeded at a density of 1 cell / well in 96-well plates in DMEM medium containing 10% FBS. The plates were incubated at 37°C overnight. Mixtures of RANKL and various concentrations of antibodies were added to the cells. The final concentration of RANKL was 100 ng / mL. After 4 or 5 days, the medium was removed and 60 μL of lysis buffer (0.1 M citric acid, 0.1 M citric acid trisodium salt, 0.1% Triton X-100) was added to the cells. 40 μL of supernatant was removed and tested for tartaric acid-resistant acid phosphatase using a TRAP assay kit (Beyotime Biotechnology, Cat. No. P0332). The plates were read at 405 nm by MD SpectraMax M5e. The inhibition rate was calculated according to the following formula: [V(RANKL)-V(sample)] / [V(RANKL)-V(blank)]x100% (V(RANKL)=RFU of (cells+RANKL), V(sample)=RFU of (cells+RANKL+Prolia), V(blank)=RFU of cells).

[0241] The inhibition rate was calculated (V(RANKL)-V(sample)) / V(RANKL)x100%, and the IC Figure 5 50 The results are summarized in Table 9.

[0242] Table 9. RAW264.7 cell differentiation assay

[0243]

[0244]

[0245] 6. Affinity of SPR

[0246] The kinetic binding affinity of selected antibodies to human and cynomolgus monkey RANKL was tested by Biacore. Antibody binding affinity to human and cynomolgus monkey RankL was detected by SPR assay using Biacore T200 (GE). Each antibody was captured on a protein A immobilized CM5 sensor chip (GE) or directly immobilized to the chip. Different concentrations of human or cynomolgus monkey RankL were injected onto the sensor chip at a flow rate of 30 μL / min. After each binding cycle, the chip was regenerated by glycine (pH 1.5).

[0247] The sensograms of the blank surface and the buffer channel were subtracted from the test sensor. The experimental data were fitted by a 1:1 model using Langmiur analysis. The molar concentration of the analyte was calculated using a molecular weight of 34 KDa.

[0248] 6.1. Affinity to human RANKL

[0249] ​SPR results showed that the affinities of antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_7.80 to human RANKL were higher than Prolia. The affinity of WBP114_5.20 was slightly lower than Prolia.

[0250] Table 10. Affinity to human RANKL

[0251]

[0252]

[0253] 6.2 Affinity to cynomolgus monkey RANKL

[0254] Table 11. Affinity to cynomolgus monkey RANKL

[0255]

[0256] 7. Epitope mapping

[0257] Antibodies were tested for binding to Prolia by ELISA. ELISA plates (Nunc) were coated with Prolia overnight at 4°C. Antibodies were serially diluted starting from 10 μg / mL and mixed with 20 ng / mL RankL-His protein. After blocking and washing, the mixture was added to the plates and incubated for 1 hour. Plates were then washed and followed by incubation with secondary antibody anti-His HRP. After washing, TMB substrate was added and the interaction was stopped with 2 M HC1. Absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0258] Antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, and WBP114_7.80 were tested in a competitive ELISA assay against Prolia. Figure 6 WBP114.5.2.1, WBP114_5.14.1, and WBP114_7.80 bind to RANKL at the same or very close epitope compared to Prolia. Based on the competitive ELISA results and different cross-reactivity properties, antibody WBP114_5.20 can bind to RANKL at a different or partially overlapping epitope with Prolia.

[0259] Example 3: Generation of humanized antibodies

[0260] 1. Generation of recombinant chimeric antibodies

[0261] V region DNA of each murine antibody was cloned into pcDNA3.3 vectors containing human constant region genes. HEK293 cells were transfected with plasmids encoding the heavy and light chains of the antibodies. Supernatants from the transfected cells were harvested by removing the cells and filtering. Antibodies were purified by a protein A column (MabSelect SuRe, GE) and buffer exchanged into PBS. Antibody concentration was detected by Nanodrop. Purity was assessed by SDS-PAGE (Invitrogen, NuPAGE 4-12% Bis-Tris Gel) and HPLC-SEC (Agilent).

[0262] 2. Humanization

[0263] The "Best Fit" method was used to humanize the antibody light and heavy chains. For the light chain, the amino acid sequence of the corresponding V gene was aligned against an internal database of human germline V genes. The sequence of the humanized VL gene was derived by replacing the human CDR sequences of the top hit with the mouse CDR sequences using the Kabat CDR definition. For the heavy chain, four humanized sequences were derived. For the light chain, the first sequence was obtained. Three additional sequences were created by aligning the mouse framework against a database of human germline V genes. The extended CDR definition was used for the framework, where the Kabat CDR1 was extended 5 amino acids N-terminally. The first three hits were used to obtain the sequence of the humanized VH gene. The humanized genes were reverse translated, codon optimized for mammalian expression, and synthesized by GeneArt Costum Gene Synthesis (Life Technologies). The synthesized genes were re-cloned into IgG expression vectors, expressed and purified. The variable region sequences of the humanized anti-RankL antibodies are shown in Tables 12 and 13.

[0264] Table 12. Variable region amino acid sequences of humanized anti-RankL antibodies

[0265]

[0266]

[0267] Table 13. Variable region DNA sequences of humanized anti-RankL antibodies

[0268]

[0269]

[0270] Example 4: Characterization of humanized antibodies

[0271] 1. ELISA binding

[0272] Humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 exhibited comparable binding activity to Prolia. Binding EC 50 Values are summarized in Table 14.

[0273] Table 14. ELISA binding of humanized antibodies

[0274]

[0275] 2. Blocking experiments

[0276] Humanized antibodies can block the binding of RANKL to RANK. WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed comparable blocking activity to Prolia. Inhibition IC 50 Values are summarized in Table 15.

[0277] Table 15. Blocking experiments of humanized antibodies

[0278]

[0279] 3. SPR affinity

[0280] Humanized antibodies were tested by Biacore for their kinetic binding affinity to human and cynomolgus monkey RANKL.

[0281] 3.1 Affinity to human RankL

[0282] Humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 maintained their original affinity to human RankL. WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 had more than 10-fold higher affinity to human RankL than Prolia (Table 16).

[0283] Table 16. Affinity to human RANKL

[0284]

[0285]

[0286] 3.2 Affinity to cynomolgus monkey RANKL

[0287] Humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed picomolar affinities to cynomolgus monkey RankL.

[0288] Table 19. Affinity for cynomolgus monkey RANKL

[0289]

[0290] 4. NF-kB reporter assay

[0291] Inhibition of RANKL-induced luciferase activity by the antibodies was measured. Figure 9 IC50values are summarized in Table 17. Humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed slightly better inhibitory activity than Prolia. 50

[0292] Table 17. NF-kB reporter assay

[0293]

[0294] 5. RAW264.7 cell differentiation test

[0295] Inhibition of RAW264.7 cell differentiation by the humanized antibodies was measured. Figure 10 IC50values are summarized in Table 18. Humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed comparable inhibitory activity to Prolia. 50

[0296] Table 18. RAW264.7 cell differentiation test

[0297]

[0298] ​​

Claims

1. An antibody or an antigen-binding fragment thereof, comprising: Heavy chain variable regions containing CDR1, CDR2, and CDR3 sequences; and The light chain variable region contains CDR1, CDR2, and CDR3 sequences. The heavy chain variable region CDR1 sequence is shown in SEQ ID NO: 32, the heavy chain variable region CDR2 sequence is shown in SEQ ID NO: 24, and the heavy chain variable region CDR3 sequence is shown in SEQ ID NO:

17. The light chain variable region CDR1 sequence is shown in SEQ ID NO: 35, the light chain variable region CDR2 sequence is shown in SEQ ID NO: 28, and the light chain variable region CDR3 sequence is shown in SEQ ID NO: 20; The antibody or antigen-binding fragment therein specifically binds to RANKL.

2. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the heavy chain variable region is as shown in SEQ ID NO: 3; and the light chain variable region is as shown in SEQ ID NO:

10.

3. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the heavy chain variable region is as shown in SEQ ID NO: 7; and the light chain variable region is as shown in SEQ ID NO:

14.

4. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment binds to human RANKL.

5. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment... a) Combining with human RANKL, K D It is below 6.98E-10.

6. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a chimeric antibody.

7. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a humanized antibody or a rat antibody.

8. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a fully human antibody.

9. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8.

10. A cloning or expression vector comprising the nucleic acid molecule as described in claim 9.

11. A host cell comprising one or more clones or expression vectors as described in claim 10.

12. A method for producing an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 8, comprising culturing a host cell as claimed in claim 11 and isolating the antibody.

13. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

14. A method for preparing an anti-RANKL antibody or an antigen-binding fragment thereof, comprising: (a) Provide: (i) An antibody sequence comprising the CDR1 sequence shown in SEQ ID NO: 32, the CDR2 sequence shown in SEQ ID NO: 24, and the CDR3 sequence shown in SEQ ID NO: 17; and (ii) An antibody sequence comprising the CDR1 sequence shown in SEQ ID NO: 35, the CDR2 sequence shown in SEQ ID NO: 28, and the CDR3 sequence shown in SEQ ID NO: 20; and (b) Express the gene sequence encoding the antibody sequence as a protein.

15. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-8 in the preparation of a medicament for inhibiting RANKL-mediated cell function or inhibiting RAW cell differentiation into osteoclast-like cells in diseases such as Crohn's disease, male infertility, chronic kidney disease, gout, rheumatoid arthritis, anorexia nervosa, severe thalassemia, hyperthyroidism, melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, and rectal cancer.

16. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-8 and the immune checkpoint antibody in the preparation of a combination medicine for inhibiting RANKL-mediated cell function or inhibiting RAW cell differentiation into osteoclast-like cells in diseases such as Crohn's disease, male infertility, chronic kidney disease, gout, rheumatoid arthritis, anorexia nervosa, severe thalassemia, hyperthyroidism, melanoma, renal cell carcinoma, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, and rectal cancer.

17. The use as claimed in claim 16, wherein the immune checkpoint antibody is a CTLA-4 antibody, a PD-1 antibody, or a PD-L1 antibody.

18. The use as claimed in claim 15 or 16, wherein the melanoma is a malignant melanoma of the skin or eye.

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