Therapeutic antibodies targeting rankl

Abstract

The present invention provides monoclonal antibodies to RANKL, in particular high affinity humanized monoclonal antibodies to RANKL. The present invention also provides functional monoclonal antibodies that cross-react with RANKL of human, cynomolgus monkey and mouse. The present invention further provides amino acid sequences of antibodies of the present invention, cloning or expression vectors, host cells and methods for expressing or isolating antibodies, identifying epitopes of antibodies. Therapeutic compositions comprising antibodies of the present invention are also provided. The present invention also provides methods of treating cancer and other diseases with anti-RANKL antibodies.

Description

[0001] This application is a divisional application of Chinese application number 201811299652.4, filed on October 25, 2018, entitled "Therapeutic Antibody Targeting RANKL". Technical Field

[0002] This invention generally relates to antibodies against RankL and compositions thereof, as well as therapies using anti-RankL antibodies to treat diseases associated with bone loss. Background Technology

[0003] RANKL, a receptor activator of NF-κB 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 a soluble form (Findlay DM, et al., Osteoporos Int 22(10):2597-2602). It mediates osteoclast formation, activation, and survival by binding to the RANK receptor on osteoclasts and their precursors (Hsu H, et al., Proc Natl Acad Sci US A. 1999 Mar 30; 96(7):3540-2545). Increasing evidence also suggests that tumor cells interact within bone, stimulating receptor activators 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 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 the OPG Fc fusion protein (an endogenous RNAKL inhibitor) have provided strong evidence for the crucial role of RANKL 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 an FDA-approved fully human monoclonal antibody derived from RANKL. Clinical studies have shown a significant reduction in vertebral, non-vertebral, and hip fractures, providing evidence consistent with the use of denosumab in postmenopausal women with osteoporosis (David W. Dempster, Clin Ther. 2012; 34:521-536). Three pivotal randomized phase III trials have also demonstrated that denosumab is superior to zoledronic acid in preventing skeletal-related events (SREs) and has a good safety and convenience profile in patients with advanced cancer bone metastases (Clin Ther. 2012; 34:521-536; Prasad Narayanan, Journal of Cancer. 2013; 2(4):272-277). Summary of the Invention

[0006] This invention provides isolated antibodies, particularly monoclonal antibodies or humanized monoclonal antibodies.

[0007] On one hand, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to RANKL in humans, monkeys and mice.

[0008] On one hand, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment...

[0009] a) Combined with human RANKL, K D Below 6.98E-10M; and

[0010] b) Combining with monkey RANKL, K D Below 5.32E-11M.

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

[0012] a) Combined with human RANKL, K D The values ​​range from 6.98E-10M to 6.33E-11M, and are associated with monkey RANKL, K. D From 5.32E-11M to 9.03E-12M;

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

[0014] c) Inhibit RAW cells from differentiating into osteoclast-like cells.

[0015] This invention provides an antibody or an 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] The antibody or antigen-binding fragment therein specifically binds to RANKL.

[0017] This invention provides an antibody or an 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] The antibody or antigen-binding fragment therein specifically binds to RANKL.

[0019] This invention provides an antibody or an antigen-binding fragment thereof, comprising:

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

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

[0022] The antibody or antigen-binding fragment therein specifically binds to RANKL.

[0023] This invention provides an antibody or an antigen-binding fragment thereof, comprising:

[0024] a) The heavy chain variable region, having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, and 7; and

[0025] b) The light chain variable region, wherein the amino acid sequence is selected from the sequence in the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14.

[0026] The antibody or antigen-binding fragment specifically binds to RANKL.

[0027] In various implementations, the antibody or its antigen-binding fragment comprises:

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

[0029] b) Having a variable region of a light chain containing an amino acid sequence selected from SEQ ID NO: 8,

[0030] The antibody or antigen-binding fragment specifically binds to RANKL;

[0031] Or antibodies or their antigen-binding fragments include:

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

[0033] b) A light chain variable region having an amino acid sequence selected from SEQ ID NO: 9,

[0034] The antibody or antigen-binding fragment specifically binds to RANKL;

[0035] Or antibodies or their antigen-binding fragments include:

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

[0037] b) A light chain variable region having an amino acid sequence selected from SEQ ID NO: 10,

[0038] The antibody or antigen-binding fragment specifically binds to RANKL;

[0039] Or antibodies or their antigen-binding fragments include:

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

[0041] b) A light chain variable region having an amino acid sequence selected from SEQ ID NO: 11,

[0042] The antibody or antigen-binding fragment specifically binds to RANKL;

[0043] Or antibodies or their antigen-binding fragments include:

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

[0045] b) A light chain variable region having an amino acid sequence selected from SEQ ID NO: 12

[0046] The antibody or antigen-binding fragment specifically binds to RANKL;

[0047] Or antibodies or their antigen-binding fragments include:

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

[0049] b) A light chain variable region having an amino acid sequence selected from SEQ ID NO: 13,

[0050] The antibody or antigen-binding fragment specifically binds to RANKL;

[0051] Or antibodies or their antigen-binding fragments include:

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

[0053] b) A light chain variable region having an amino acid sequence selected from SEQ ID NO: 14,

[0054] The antibody or antigen-binding fragment specifically binds to RANKL;

[0055] The sequence of the antibody is shown in Table 1 and the sequence listing.

[0056] Table 1. Amino acid sequences of the antibodies

[0057]

[0058]

[0059] On the other hand, the present invention provides an antibody or an antigen-binding fragment thereof, comprising a complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-36.

[0060] The antibody or antigen-binding fragment specifically binds to RANKL.

[0061] On the other hand, the present invention provides an antibody or an antigen-binding fragment thereof, comprising:

[0062] Heavy chain variable regions containing CDR1, CDR2, and CDR3 sequences; and

[0063] The light chain variable region contains CDR1, CDR2, and CDR3 sequences.

[0064] The heavy chain variable region CDR3 sequence contains amino acid sequences selected from the group consisting of SEQ ID NO: 15, 16, and 17, along with their conserved modifications.

[0065] The antibody or antigen-binding fragment specifically binds to RANKL.

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

[0067] Preferably, the heavy chain variable region CDR2 sequence of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 23, 24, 25 and its conserved modifications.

[0068] Preferably, the light chain variable region CDR2 sequence of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 29 and its conserved modifications.

[0069] Preferably, the heavy chain variable region CDR1 sequence of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 31, 32 and its conserved modifications.

[0070] Preferably, the antibody of the present invention, wherein the light chain variable region CDR1 sequence of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 33, 34, 35, 36 and its conserved modifications.

[0071] In a more preferred embodiment, the present invention 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 amino acid sequences of SEQ ID NO: 30, 31 and 32, the CDR2 sequence comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 22, 23, 24 and 25, and the CDR3 sequence comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 15, 16 and 17.

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

[0074] The antibody or antigen-binding fragment specifically binds to RANKL.

[0075] Preferred antibodies or their antigen-binding fragments include:

[0076] a) Contains the heavy chain variable region CDR1 of SEQ ID NO: 30;

[0077] b) The heavy chain variable region CDR2 containing SEQ ID NO: 22;

[0078] c) The heavy chain variable region CDR3 containing SEQ ID NO: 15;

[0079] d) Contains the light chain variable region CDR1 of SEQ ID NO: 33;

[0080] e) Contains the light chain variable region CDR2 of SEQ ID NO: 26;

[0081] f) Contains the light chain variable region CDR3 of SEQ ID NO: 18;

[0082] The antibody or antigen-binding fragment specifically binds to RANKL.

[0083] Another preferred antibody or its antigen-binding fragment includes:

[0084] a) The heavy chain variable region CDR1 containing SEQ ID NO: 31;

[0085] b) The heavy chain variable region CDR2 containing SEQ ID NO: 23;

[0086] c) The heavy chain variable region CDR3 containing SEQ ID NO: 16;

[0087] d) Contains the light chain variable region CDR1 of SEQ ID NOs: 34;

[0088] e) Contains the light chain variable region CDR2 of SEQ ID NO: 27;

[0089] f) Contains the light chain variable region CDR3 of SEQ ID NO: 19;

[0090] The antibody or antigen-binding fragment specifically binds to RANKL.

[0091] Another preferred antibody or its antigen-binding fragment includes:

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

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

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

[0095] d) Contains the light chain variable region CDR1 of SEQ ID NO: 35;

[0096] e) Contains the light chain variable region CDR2 of SEQ ID NO: 28;

[0097] f) Contains the light chain variable region CDR3 of SEQ ID NO: 20;

[0098] The antibody or antigen-binding fragment specifically binds to RANKL.

[0099] Another preferred antibody or its antigen-binding fragment includes:

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

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

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

[0103] d) Contains the light chain variable region CDR1 of SEQ ID NO: 36;

[0104] e) Contains the light chain variable region CDR2 of SEQ ID NO: 29;

[0105] f) Contains the light chain variable region CDR3 of SEQ ID NO: 21;

[0106] The antibody specifically binds to RANKL.

[0107] Another preferred antibody or its antigen-binding fragment includes:

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

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

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

[0111] d) Contains the light chain variable region CDR1 of SEQ ID NO: 33;

[0112] e) Contains the light chain variable region CDR2 of SEQ ID NO: 26;

[0113] f) Contains the light chain variable region CDR3 of SEQ ID NO: 18;

[0114] The antibody or antigen-binding fragment specifically binds to RANKL.

[0115] Another preferred antibody or its antigen-binding fragment includes:

[0116] a) The heavy chain variable region CDR1 containing SEQ ID NO: 31;

[0117] b) The heavy chain variable region CDR2 containing SEQ ID NO: 23;

[0118] c) The heavy chain variable region CDR3 containing SEQ ID NO: 16;

[0119] d) Contains the light chain variable region CDR1 of SEQ ID NO: 34;

[0120] e) Contains the light chain variable region CDR2 of SEQ ID NO: 27;

[0121] f) Contains the light chain variable region CDR3 of SEQ ID NO: 19;

[0122] The antibody or antigen-binding fragment specifically binds to RANKL.

[0123] Another preferred antibody or its antigen-binding fragment includes:

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

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

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

[0127] d) Contains the light chain variable region CDR1 of SEQ ID NO: 35;

[0128] e) Contains the light chain variable region CDR2 of SEQ ID NO: 28;

[0129] f) Contains the light chain variable region CDR3 of SEQ ID NO: 20;

[0130] The antibody or antigen-binding fragment specifically binds to RANKL.

[0131] The CDR sequence of the antibody is shown in Table 2 and the sequence listing.

[0132] Table 2. CDR sequences of antibodies

[0133]

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

[0135] The antibody of this invention can be a humanized antibody.

[0136] The antibody of this invention can be a fully human antibody.

[0137] The antibody of this invention can be a rat antibody.

[0138] The antibody or antigen-binding fragment described in this invention has at least one of the following properties:

[0139] a) Combined with human RANKL, K D The values ​​range from 6.98E-10M to 6.33E-11M, and are bound to rankl, K in monkeys. D From 5.32E-11M to 9.03E-12M;

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

[0141] c) Inhibit RAW cells from differentiating into osteoclast-like cells.

[0142] In another aspect, the present invention provides nucleic acid molecules encoding antibodies or antigen-binding fragments thereof.

[0143] The present invention provides cloning or expression vectors containing nucleic acid molecules encoding antibodies or their antigen-binding fragments.

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

[0145] On the other hand, the present invention provides a method comprising culturing the aforementioned host cells and isolating antibodies; wherein the antibodies are prepared by immunizing mice with human RankL protein.

[0146] The present invention provides transgenic animals, such as mice, comprising human immunoglobulin heavy and light chain transgenes, wherein the animals express the antibodies of the present invention.

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

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

[0149] This invention provides a method for preparing anti-RANKL antibody or its antigen-binding fragment, comprising:

[0150] (a) Provide:

[0151] (i) an antibody sequence of a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NO: 30-32, a CDR2 sequence selected from the group consisting of SEQ ID NO: 22-25, and a CDR3 sequence selected from the group consisting of SEQ ID NO: 15-17; and / or

[0152] (ii) An antibody sequence comprising 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) Express the altered antibody sequence as a protein.

[0154] The present invention also provides a method for preventing or treating bone loss-related diseases in subjects, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment of the present invention to the subject in need.

[0155] The present invention also provides a combined method for preventing or treating bone loss-related diseases in subjects, comprising administering a therapeutically effective amount of the above-described antibody or antigen-binding fragment of the present invention to the subject as needed, and administering a therapeutically effective amount of an immune checkpoint antibody to the subject.

[0156] The immune checkpoint antibodies in this invention include CTLA-4 antibodies, PD-1 antibodies, or PD-L1 antibodies.

[0157] The present invention also provides the use of the antibody or its antigen-binding fragment in the preparation of a medicament for the prevention or treatment of diseases associated with bone loss.

[0158] The diseases mentioned include Crohn's disease, male infertility, chronic kidney disease, gout, rheumatoid arthritis, anorexia nervosa, severe thalassemia, hyperthyroidism, or cancer.

[0159] The cancers mentioned are selected from melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, and rectal cancer.

[0160] Beneficial effects of the invention

[0161] The antibodies disclosed in this invention have high binding affinity and specifically bind to human and monkey RANKL proteins; they are effective in preventing or treating diseases related to bone loss.

[0162] One of the antibodies binds not only to human and monkey RANKL, but also to mouse RANKL, which could greatly facilitate preclinical validation of its efficacy in mouse tumor models. Attached Figure Description

[0163] Figure 1 The 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 The blocking assays for WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, WBP114_7.80, and Prolia are shown.

[0165] Figure 3 The antibody WBP114_5.20 is shown to bind in a RANKL-dependent manner to mouse RANKL.

[0166] Figure 4 The NF-κB reporter gene assay for the selected antibody is shown.

[0167] Figure 5 The RAW264.7 cell differentiation assay using the selected antibody is shown.

[0168] Figure 6 This shows the epitope binding of the selected antibody against Prolia.

[0169] Figure 7 The ELISA binding assay of the humanized antibody is shown.

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

[0171] Figure 9 The NF-κB reporter gene assay of the humanized antibody was shown.

[0172] Figure 10 RAW264.7 cell differentiation assay showing humanized antibodies. Detailed Implementation

[0173] The present invention will be further described below through specific embodiments and experimental data. Although technical terms are used below for clarity, these terms are not intended to define or limit the scope of the invention.

[0174] The terms “receptor activator of nuclear factor-κB ligand”, “RANKL”, “TNF-associated activation-induced cytokine”, “TRANCE”, “osteoplastin ligand”, “OPGL”, “osteoclast differentiation factor”, and “ODF” are used interchangeably, including variants, isotypes, species homologs of human RANKL or RANKL of other species, and analogs having at least one common epitope of RANKL.

[0175] As used herein, the term "antibody" includes both intact antibodies and any antigen-binding fragment (i.e., "antigen-binding moiety") or its single chain. An "antibody" is a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or its antigen-binding moiety. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and more conserved regions interspersed with framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0176] The term "antibody," as used herein, refers to an immunoglobulin or fragments thereof or derivatives thereof, and includes any polypeptide containing an antigen-binding site, whether or not it is produced in vitro or in vivo. This term includes, but is not limited to, polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and 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., capable of specifically binding to RANKL. Typically, such fragments will include antigen-binding fragments.

[0177] The terms "antigen-binding fragment," "antigen-binding domain," and "binding fragment" refer to an antibody molecule containing 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 may bind only a portion of the antigen. The part of the antigen molecule responsible for the specific interaction with the antigen-binding fragment is called an "epitaph" or "antigenic determinant."

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

[0179] The term "epitope" is defined as an antigenic determinant that specifically binds to / recognizes a binding fragment. This binding fragment can specifically bind to / interact with a conformational or continuous epitope unique to the target structure, such as human RANKL and mouse RANKL. A conformational or discontinuous epitope is characterized in that the polypeptide antigen is two or more discrete amino acid residues separated in the primary sequence, but aggregates together on the surface of the molecule when the polypeptide folds into its native protein / antigen. The two or more discrete amino acid residues constituting the epitope are present in independent portions of one or more polypeptide chains. When the polypeptide chain folds into a three-dimensional structure, these residues aggregate on the molecular surface to form the epitope. In contrast, a continuous or linear epitope, consisting of two or more discrete amino acid residues, is present in a single linear segment of the polypeptide chain.

[0180] The term “cross-reactivity” as described in this article refers to the binding of antigen fragments to the same target molecule in human, monkey, and / or mouse (mouse or rat) sources. Therefore, “cross-reactivity” should be understood as interspecies reactivity with the same molecule X expressed in different species. The cross-reactivity specificity of monoclonal antibodies recognizing human RANKL, monkey, and / or mouse RANKL (mouse or rat) can be determined by FACS analysis.

[0181] As used herein, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably.

[0182] The terms “treatment” and “method of treatment” refer to therapeutic treatments and preventative / preventive measures. Those who require treatment include individuals who already have a specific medical condition, as well as those who may eventually develop that condition.

[0183] The term "conserved modification" refers to nucleotide and amino acid sequence modifications that do not significantly affect or alter the antibody-binding properties encoded by the nucleotide sequence or comprising the amino acid sequence. Such conserved sequence modifications include nucleotide and amino acid substitutions, additions, and deletions. Modifications can be introduced into the sequence using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitutions include substitutions in which amino acid residues are replaced by amino acid residues having similar side chains. 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0184] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0185] Example

[0186] Example 1: Antibody Production

[0187] 1. The formation of immune and antibody hybridomas

[0188] 1.1 Immunity

[0189] Balb / c mice were injected with human RankL protein via their footpads approximately every 3 days. The first titer test was performed after 6 injections.

[0190] 1.2 Serum titer detection

[0191] An ELISA assay was used to measure antibody titers in mouse serum. To measure antibody titers, plates (Nunc) were coated overnight at 4°C with 1 μg / mL human RankL, and then blocked for 1 hour at room temperature with blocking buffer (1×PBS / 2% BSA). Rat serum, initially titrated 1:3, was diluted 1:100 in blocking buffer and incubated for 1 hour at room temperature. The plates were then washed and incubated for 1 hour with the secondary antibody, goat anti-mouse IgG Fc HRP. After washing, TMB substrate was added, and the interaction was terminated with 2M HCl. The absorbance was read at 450 nm using a molecular device.

[0192] The titer of antigen-specific antibodies in serum was determined by ELISA. Mice with serum titers of 312,500 or higher were selected for hybridoma fusion.

[0193] 2. The formation of hybridomas

[0194] Lymph nodes and spleens were collected from immunized mice under aseptic conditions, and lymphocytes were prepared using Ficoll-Paque PLUS gradient centrifugation. The isolated cells were then fused with myeloma P3 cells at a 1:1 ratio using an electrofusion device (BTX ECM2001). After fusion, the cells were transferred to 1 / 2 HA medium. 5 × 10⁶ cells were seeded per 96-well plate. 5 Each cell.

[0195] 3. Antibody screening

[0196] Hybridoma supernatant was used for initial screening. Then, antigen-specific hybridomas were screened by ELISA blocking assay.

[0197] Binding assay by ELISA: Plates (Nunc) were coated overnight at 4°C with 1 μg / mL human RankL. After blocking and washing, the hybridoma supernatant was transferred to plates and incubated at room temperature for 1 hour. The plates were then washed and incubated with the secondary antibody (goat anti-mouse IgG Fc HRP) for 1 hour. After washing, TMB substrate was added, and the interaction was terminated with 2M HCl. The absorbance was read at 450 nm using a molecular device.

[0198] ELISA blocking assay: Plates (Nunc) were coated overnight at 4°C with 5 μg / mL Rank-Fc fusion protein. Hybridoma supernatant was 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 plate and incubated for 1 hour. The plate was then washed and incubated with anti-His Ab-HRP. After washing, TMB substrate was added, and the interaction was terminated with 2M HCl. The absorbance was read at 450 nm using a molecular device.

[0199] Antibodies capable of blocking the binding of human Rank ligand to human Rank were selected for further characterization. The selected antibodies with both binding and blocking activities were purified from the hybridoma supernatant. Simultaneously, subcloning of selected hybridoma lines was performed. Hybridoma subclones were validated by binding and blocking ELISA assays, and their isotypes were also detected.

[0200] 4. Subcloning

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

[0202] 5. Antibody purification

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

[0204] 6. Antibody isotype

[0205] Antibody isotypes were identified by ELISA. Plates (Nunc) were coated overnight at 4°C with 1 μg / mL of goat anti-mouse IgG1 / anti-mouse IgG2a / anti-mouse IgG2b / anti-mouse IgG3 / anti-mouse IgM antibodies. 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 for 45 minutes with secondary antibodies, either goat anti-mouse κHRP or goat anti-mouse λHRP (Southern Biotech). After washing, TMB substrate was added, and the interaction was terminated with 2M HCl. The absorbance was read at 450 nm using a molecular device.

[0206] The isotypes of mouse antibodies are shown in Table 3.

[0207] Table 3. Homotypes of mouse antibodies

[0208]

[0209]

[0210] 7. Antibody sequence

[0211] RNA was extracted from hyridoma cells using Trizol reagent (Invitrogen-15596018). cDNA was amplified using a 5'-RACE kit (Takara-28001488), followed by PCR amplification using 3'-degenerate primers and 3'-adaptor primers (ExTaq: Takara-RR001B). The PCR fragment was inserted into the pMD18-T vector (Takara-D101C) and sequenced (Shanghai Biosune).

[0212] The amino acid sequence of the variable region of the mouse anti-RankL antibody is shown in Table 4, and the DNA sequence of the variable region is shown in Table 5.

[0213] Table 4. Variable region amino acid sequence of mouse anti-RankL antibody

[0214]

[0215]

[0216] The underlined sequences are CDR1-3.

[0217] Table 5. Variable region DNA sequence of mouse anti-RankL antibody

[0218]

[0219]

[0220] Example 2: Characterization of murine antibodies

[0221] 1. ELISA combined

[0222] Antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, and WBP114_7.80 exhibited sub-nanomolar binding activity, and their EC50 values ​​were [missing information]. 50 The value is comparable to Prolia. Combined with EC 50 The maximum binding values ​​are summarized in Table 6 and Figure 1 middle.

[0223] Table 6. Antibody ELISA binding

[0224]

[0225] 2. Blocking assay

[0226] Antibodies WBP114.5.2.1, WBP114_5.14.1, WBP114_5.20, and WBP114_7.80 can block the binding of RANKL to RANK. WBP114.5.2.1, WBP114_5.14.1, and WBP114_7.80 showed blocking activity comparable to Prolia. Inhibition of IC50... 50 The values ​​are summarized in Table 7 and Figure 2 middle.

[0227] Table 7. Antibody Blocking Analysis

[0228]

[0229] 3. Cross-binding activity against mouse RNAKL

[0230] The ELISA plate (Nunc) was coated overnight at 4°C with 5 μg / mL mouse RANKL. After blocking and washing, 1 μg / mL antibody sample or Prolia was added and incubated for 1 hour. The plate was then washed and incubated for 1 hour with the secondary antibody anti-mouse IgG Fc HRP / anti-human IgG Fc HRP (Bethyl). After washing, TMB substrate was added, and the interaction was terminated with 2M HCl. The absorbance at 450 nm was read using a molecular device. Binding to mouse RANKL was detected by ELISA. Antibody WBP114_5.20 can bind to mouse RANKL (…). Figure 3 EC 50 The value was 0.067 nM. WBP114.5.2.1, WBP114_5.14.1, WBP114_7.80 and Prolia did not bind to mouse RANKL (data not shown).

[0231] 4. NF-κB reporter gene assay

[0232] The ability of antibodies to neutralize RANKL-mediated cellular function was measured using engineered HK293 cell lines containing RANK and NF-κB-luc.

[0233] 293F cells were fed at a rate of 5 × 10⁻⁶. 5 Cells were resuspended at a density of 1 mL / well in FreeStyle 293 expression medium (Invitrogen-12338). 1 mL / well of 293F cell suspension was transferred to a 24-well plate. The pG1-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 incubation for 5 minutes, the diluted pG1-NF-kB and PlasFect were combined. The mixture was gently mixed and incubated at room temperature for 20 minutes. The mixture was then added to the cells and incubated overnight at 37°C.

[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). A mixture of RANKL and various antibody concentrations was pre-incubated at 4°C for 30 minutes and then added to the cells. The final concentration of RANKL was 300 ng / mL. After incubation at 37°C for 24 hours, 50 μL / well of Nano-Glo luciferase (Promega-N1120) was added to each well and incubated at room temperature for 5 minutes (protected from light). Fluorescence was read using MDSpectraMax M5e. The inhibition rate was calculated as [V(RANKL) - V(sample)] / [V(RANKL) - V(blank)] × 100%. V(RANKL) = RFU of (cells + RANKL), V(sample) = RFU of (cells + RANKL + Ab), V(blank) = RFU of cells.

[0235] Measuring the inhibition of RANKL-induced luciferase activity by the antibody ( Figure 4 Suppressing IC 50 The values ​​are summarized in Table 4. Antibodies WBP114.5.2.1, WBP114_5.14.1, and WBP114_7.80 showed inhibitory activity comparable to Prolia. The inhibitory activity of antibody WBP114_5.20 was slightly lower than that of Prolia (Table 8).

[0236] Table 8. NF-κB reporter gene assay

[0237]

[0238] 5. RAW264.7 Cell Differentiation Assay

[0239] The inhibition of osteoclast formation by antibodies was examined using a RAW cell differentiation assay. RANKL can stimulate RAW cells to differentiate into osteoclast-like cells, and this differentiation can be measured by tartrate-resistant acid phosphatase (TRAP) activity.

[0240] RAW264.7 cells were loaded at a rate of 2 × 10⁻⁶. 4Cells were seeded at a density of 100 cells / well in 96-well plates in DMEM medium containing 10% FBS. The plates were incubated overnight at 37°C. A mixture of RANKL and various concentrations of antibody was 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 citrate, 0.1 M trisodium citrate, 0.1% Triton X-100) was added to the cells. 40 μL of supernatant was removed, and tartrate-resistant acid phosphatase was detected using a TRAP assay kit (Beyotime Biotechnology, catalog number P0332). The plate was read at 405 nm using an MD SpectraMax M5e. The inhibition rate is calculated using the following formula: [V(RANKL)-V(sample)] / [V(RANKL)-V(blank)]×100% (V(RANKL) = RFU of (cells + RANKL), V(sample) = RFU of (cells + RANKL + Prolia), V(blank) = RFU of cells).

[0241] Calculate the inhibition rate ( Figure 5 ), and will suppress IC 50 The summary is in Table 9.

[0242] Table 9. RAW264.7 Cell Differentiation Assay

[0243]

[0244]

[0245] 6. SPR's affinity

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

[0247] The sensor map was obtained by subtracting the blank surface and buffer channel from the test sensor. Experimental data were fitted using a 1:1 model with Langmiur analysis. The molar concentration of the analyte was calculated using a molecular weight of 34 kDa.

[0248] 6.1. Affinity for human RANKL

[0249] SPR results showed that antibodies WBP114.5.2.1, WBP114_5.14.1, and WBP114_7.80 had higher affinity for human RANKL than Prolia. WBP114_5.20 had slightly lower affinity than Prolia.

[0250] Table 10. Affinity to human RANKL

[0251]

[0252]

[0253] 6.2 Affinity with RANKL, the cynomolgus monkey

[0254] Table 11. Affinity to RANKL (the cynomolgus monkey)

[0255]

[0256] 7. Epitope Union

[0257] Antibodies were conjugated to Prolia via ELISA. ELISA plates (Nunc) were coated with Prolia overnight at 4°C. Antibodies were serially diluted starting at 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. The plates were then washed and incubated with the secondary antibody anti-His HRP. After washing, TMB substrate was added, and the interaction was terminated with 2M HCl. The absorbance was read at 450 nm using a 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 Compared to Prolia, WBP114.5.2.1, WBP114_5.14.1, and WBP114_7.80 bind to RANKL at the same or very close epitopes. Based on competitive ELISA results and different cross-reactivity properties, antibody WBP114_5.20 can bind to RANKL at different or partially overlapping epitopes with Prolia.

[0259] Example 3: Generation of humanized antibodies

[0260] 1. Generation of recombinant chimeric antibodies

[0261] The V region DNA of each mouse antibody was cloned into a pcDNA3.3 vector containing the human constant region gene. HEK293 cells were transfected with plasmids encoding the antibody heavy and light chains. The supernatant from the transfected cells was harvested by removing the cells and filtering. The antibodies were purified by protein A column (MabSelect SuRe, GE) and the buffer was exchanged for 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 for both the light and heavy chains of the humanized antibody. For the light chain, the amino acid sequence of the corresponding V gene was aligned to an internal human V gene database. The sequence of the humanized VL gene was derived by replacing the top-hit human CDR sequence with a mouse CDR defined using the Kabat CDR. 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 to a mouse frame from the human V gene database. An extended CDR frame was used, where the Kabat CDR1 was extended by 5 amino acids at the N-terminus. The first three hits were used to obtain the sequence of the humanized VH gene. The humanized gene was reverse-translated, codon-optimized for mammalian expression, and synthesized using GeneArt Costum Gene Synthesis (Life Technologies). The synthesized gene was recloned into an IgG expression vector, expressed, and purified. The variable region sequence of the humanized anti-RankL antibody is shown in Tables 12 and 13.

[0264] Table 12. Variable region amino acid sequence of humanized anti-RankL antibody

[0265]

[0266]

[0267] Table 13. Variable region DNA sequence of humanized anti-RankL antibody

[0268]

[0269]

[0270] Example 4: Characterization of humanized antibodies

[0271] 1. ELISA combined

[0272] Humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 exhibited binding activity comparable to Prolia. (The last sentence appears to be incomplete and unrelated to the preceding text.) 50 The values ​​are summarized in Table 14.

[0273] Table 14. ELISA binding of humanized antibodies

[0274]

[0275] 2. Blocking test

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

[0277] Table 15. Blocking assays with humanized antibodies

[0278]

[0279] 3. SPR Affinity

[0280] The kinetic binding affinity of the humanized antibody to human and cynomolgus monkey RANKL was tested using Biacore.

[0281] 3.1 Affinity for human Rank L

[0282] Humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 maintained their original affinity for human RankL. The affinity of WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 for human RankL was more than 10 times higher than that for Prolia (Table 16).

[0283] Table 16. Affinity to human RANKL

[0284]

[0285]

[0286] 3.2 Affinity with RANKL, the cynomolgus monkey

[0287] Humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed picomolar affinity for RankL cynomolgus monkeys.

[0288] Table 19. Affinity to RANKL (the cynomolgus monkey)

[0289]

[0290] 4. NF-κB reporter gene assay

[0291] Measuring the inhibition of RANKL-induced luciferase activity by the antibody ( Figure 9 Suppressing IC 50 The values ​​are summarized in Table 17. The humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed slightly better inhibitory activity than Prolia.

[0292] Table 17. NF-κB reporter gene analysis

[0293]

[0294] 5. RAW264.7 Cell Differentiation Assay

[0295] Measuring the inhibitory effect of humanized antibodies on RAW264.7 cell differentiation ( Figure 10 ), and IC 50 The values ​​are summarized in Table 18. The humanized antibodies WBP1141-5.2.1-z1-IgG4 and WBP1141-7.80-z1-IgG4 showed inhibitory activity comparable to Prolia.

[0296] Table 18. RAW264.7 Cell Differentiation Assay

[0297]

[0298]

Claims

1. An antibody or antigen-binding fragment thereof, comprising: 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 heavy chain variable region CDR1 sequence is set forth in SEQ ID NO:30, the heavy chain variable region CDR2 sequence is set forth in SEQ ID NO:25, and the heavy chain variable region CDR3 sequence is set forth in SEQ ID NO: 15; the light chain variable region CDR1 sequence is set forth in SEQ ID NO:36, the light chain variable region CDR2 sequence is set forth in SEQ ID NO:29, and the light chain variable region CDR3 sequence is set forth in SEQ ID NO:21; wherein the antibody or antigen-binding fragment specifically binds to RANKL.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region is set forth in SEQ ID NO:4; and the light chain variable region is set forth in SEQ ID NO:

11.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment binds to human and monkey RANKL.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment a) binds to human RANKL, K D is below 4.04E-11; and b) binds to monkey RANKL, K D is 7.94E-12 or less.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment is a chimeric antibody.

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

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

8. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 7.

9. A cloning or expression vector comprising the nucleic acid molecule of claim 8.

10. A host cell comprising one or more cloning or expression vectors of claim 9.

11. A method for producing the antibody or antigen-binding fragment thereof of any one of claims 1 to 7, comprising culturing the host cell of claim 10, and isolating the antibody.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 7, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

13. A method for making an anti-RANKL antibody or antigen-binding fragment thereof, comprising: (a) providing: (i) an antibody sequence for a heavy chain variable region comprising a CDR1 sequence set forth in SEQ ID NO:30, a CDR2 sequence set forth in SEQ ID NO:25, and a CDR3 sequence set forth in SEQ ID NO: 15; and (ii) an antibody sequence for a light chain variable region comprising a CDR1 sequence set forth in SEQ ID NO:36, a CDR2 sequence set forth in SEQ ID NO:29, and a CDR3 sequence set forth in SEQ ID NO:21; and (b) expressing the antibody sequences of (a)(i) and (a)(ii) in a host cell. (ii) an antibody sequence of a light chain variable region comprising a CDR1 sequence as set forth in SEQ ID NO: 36, a CDR2 sequence as set forth in SEQ ID NO: 29, and a CDR3 sequence as set forth in SEQ ID NO: 21; and (b) expressing the gene sequence encoding the antibody sequence as a protein.

14. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7 in the manufacture of a medicament for inhibiting RANKL-mediated cellular function or inhibiting differentiation of RAW cells into osteoclast-like cells in a Crohn's disease, male infertility, chronic kidney disease, gout, rheumatoid arthritis, anorexia nervosa, thalassemia major, hyperthyroidism, melanoma, renal 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 disease.

15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7 and an immune checkpoint antibody in the manufacture of a combination medicament for inhibiting RANKL-mediated cellular function or inhibiting differentiation of RAW cells into osteoclast-like cells in a Crohn's disease, male infertility, chronic kidney disease, gout, rheumatoid arthritis, anorexia nervosa, thalassemia major, hyperthyroidism, melanoma, renal 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 disease.

16. The use of claim 15, wherein the immune checkpoint antibody is a CTLA-4 antibody, a PD-1 antibody, or a PD-L1 antibody.

17. The use of claim 14 or 15, wherein the melanoma is cutaneous or intraocular malignant melanoma.

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