Rankl mutant and vaccine composition comprising same

Abstract

An RANKL protein mutant according to the present invention can inhibit RANK activity through inhibition of the ability of RANKL to bind to both OPG and RANK, thereby suppressing differentiation into osteoclasts. Therefore, the RANKL protein mutant of the present invention can be effectively used for the prevention or treatment of bone metabolic diseases.

Description

Mutants of RANKL and vaccine compositions containing the same

[0001] The present invention relates to mutants of RANKL and uses thereof.

[0002] Osteoporosis is a condition in which bone mineral density (BMD) is lower than 2.5 or the T-score (standard deviation from the average bone mass of a typical adult) is lower than -2.5, resulting in weakened bones that are more prone to fractures. If bone density decreases excessively, fractures can easily occur even with small impacts. Osteoporosis is not a symptom itself, but rather various fractures caused by weakened bones, especially femur fractures or spinal fractures, which limit long-term activities and make it difficult to lead a healthy life, and is known to be the cause of 15% of deaths in the elderly.

[0003] Human bones are composed of osteoblasts, osteocytes, and osteoclasts. Among them, osteoblasts play a role in forming bone tissue through a proliferation phase, bone matrix formation phase, and calcification phase. Osteoclasts also play a role in bone resorption. In adult bones, after growth is complete, old bone is removed by osteoclasts and replaced with new bone by osteoblasts. This process of bone remodeling continuously repeats, with osteoclasts resorbing and forming new bone. For example, osteoblasts maintain the homeostasis of bone metabolism in the body by regulating the differentiation of osteoclasts, which are responsible for bone resorption, through the secretion of substances such as receptor activator of nuclear factor-kappa B ligand (RANKL) of NF-κB and its disruptive receptor, osteoprotegerin (OPG). If this homeostasis of bone metabolism is disrupted due to certain causes, bone metabolic diseases such as osteoporosis, osteogenesis disorders, or fractures can occur.

[0004] Current osteoporosis treatment drugs primarily work by inhibiting osteoclast activity to suppress bone density loss or by promoting bone formation to increase bone density. Among these, bisphosphonates, which inhibit osteoclast differentiation, are the most commonly prescribed osteoporosis treatment worldwide. While bisphosphonates offer excellent osteoporosis treatment effects, long-term use can cause potentially fatal side effects, such as osteonecrosis of the jaw and an increased risk of femoral fractures. In addition to bisphosphonates, hormones such as estrogen, calcitonin, and denosumab, as well as monoclonal antibodies, are also used. Other bone formation stimulants, such as parathyroid hormone analogs, also increase bone density. However, these agents are not free from side effects. Anti-cytokine antibody therapies, such as denosumab, face the challenges of high manufacturing costs and immunogenicity due to multiple antibody administration. In other words, all osteoporosis drugs currently in use or being prepared for release cannot restore already reduced bone density, but only prevent further bone density decline. In addition, there is a limitation that prevents long-term treatment with a single drug due to concerns about side effects.

[0005] Accordingly, the inventors of the present invention have made great efforts to solve the above-mentioned problem, and as a result, have developed a mutant of RANKL in which binding to RANKL (Receptor activator of Nuclear factor-kappa B ligand), its receptor activator RANK (Receptor activator of NF-kB), and its disrupting receptor OPG (osteoprotegerin) is inhibited, thereby completing the present invention.

[0006] Accordingly, it is an object of the present invention to provide a mutant of RANKL protein.

[0007] In addition, another object of the present invention is to provide a nucleic acid molecule encoding a RANKL protein mutant.

[0008] In addition, another object of the present invention is to provide a vector comprising a nucleic acid molecule encoding the RANKL protein mutant.

[0009] In addition, another object of the present invention is to provide a host cell comprising the vector.

[0010] Another object of the present invention is to provide a vaccine composition comprising a RANKL protein mutant, a nucleic acid molecule encoding the same, or a vector comprising the same.

[0011] The present invention relates to a mutant of RANKL protein.

[0012] In the present invention, the term "RANKL protein" or "RANKL" refers to receptor activator of Nuclear factor-kappa B ligand (RANKL). RANKL is known as a type II membrane protein and is a member of the tumor necrosis factor (TNF) superfamily. RANKL is a necrosis regulatory gene and can regulate cell proliferation by modulating the protein levels of Id4, Id2, and cyclin D1. RANKL can be expressed in various tissues and organs, including skeletal muscle, thymus, liver, colon, small intestine, adrenal gland, osteoblasts, mammary epithelial cells, prostate, and pancreas.

[0013] NF-κB is a group of proteins involved in regulating inflammatory responses, immune system regulation, apoptosis, cell proliferation, and epithelial cell differentiation. NF-κB regulates the expression of various genes and can form a central axis of the intracellular signaling system. When RANKL binds to the receptor activator of nuclear factor kappa-B (RANK), RANK is activated, which can sequentially activate NF-κB, mitogen-activated protein kinase (MAPK), activating protein 1 (AP-1), and nuclear factor of activated T cells (NFATc1). Ultimately, the expression of osteoclast-inducing factors is promoted by various transcription factors activated by RANKL.

[0014] In the present invention, the term "OPG protein" or "OPG" refers to "osteoprotegerin," a soluble decoy receptor that regulates bone metabolism. OPG, a protein belonging to the TNF receptor superfamily, acts as a natural inhibitor of RANKL, thereby inhibiting the formation and activation of osteoclasts. This process prevents bone loss and reduces the risk of osteoporosis by inhibiting bone resorption and promoting bone formation. OPG is primarily expressed in osteoblasts and bone-derived cells and may function as an important regulatory factor in various disease states associated with bone loss.

[0015] In the present invention, "mutant" refers to a modified gene or protein derived through naturally occurring or artificially engineered genetic modification. Such mutants have structural or functional characteristics different from the original form through one or more amino acid substitutions, insertions, deletions, or any other form of genetic modification. The mutant of the present invention has the characteristics of not binding to RANK on its own, so that it does not promote osteoclast activity, and not binding to OPG, so that it can exist stably in vivo. In addition, it functions as an antigen manufactured to be structurally similar to RANKL, so that it can generate antibodies against RANKL, and can be usefully utilized in the prevention or treatment of osteoporosis.

[0016] Mutations can be categorized by how they work and their magnitude. Mutations can occur at the nucleotide level, including point mutations, where a single nucleotide is changed; insertion mutations, where a portion of a nucleotide is inserted between the original base sequence; deletion mutations, where a portion of an original nucleotide is lost; and chromosomal mutations, such as gene duplications, deletions, chromosomal inversions, interstitial deletions, chromosomal translocations, and loss of heterozygosity.

[0017] For example, the RANKL protein of the present invention may be a mutant in which one or more amino acids in the amino acid sequence are substituted.

[0018] Accordingly, the present invention comprises at least four substitutions selected from the group consisting of a substitution of 180th lysine (Lys) with arginine (Arg), a substitution of 189th aspartic acid (Asp) with isoleucine (Ile), a substitution of 190th arginine (Arg) with lysine (Lys), a substitution of 223rd histidine (His) with phenylalanine (Phe) or tyrosine (Try), and a substitution of 224th histidine (His) with phenylalanine (Phe) or tyrosine (Try) in the N-terminus of a RANKL protein having an amino acid sequence of SEQ ID NO: 1; A RANKL protein mutant comprising at least one substitution selected from the group consisting of a substitution of glutamine (Gln) at position 236 with aspartic acid (Asp), and a substitution of phenylalanine (Phe) at position 269 with lysine (Lys), tyrosine (Try), or histidine (His) is provided. In the present invention, the RANKL protein mutant sequence may comprise any one of the amino acid sequences of SEQ ID NOs: 3 to 6.

[0019] Accordingly, the present invention comprises at least four substitutions selected from the group consisting of a substitution of 181st lysine (Lys) with arginine (Arg), a substitution of 190th aspartic acid (Asp) with isoleucine (Ile), a substitution of 191st arginine (Arg) with lysine (Lys), a substitution of 224th histidine (His) with phenylalanine (Phe) or tyrosine (Try), and a substitution of 225th histidine (His) with phenylalanine (Phe) or tyrosine (Try) in the N-terminus of a RANKL protein having an amino acid sequence of SEQ ID NO: 2; A RANKL protein mutant comprising at least one substitution selected from the group consisting of a substitution of glutamine (Gln) at position 237 with aspartic acid (Asp), and a substitution of phenylalanine (Phe) at position 270 with lysine (Lys), tyrosine (Try), or histidine (His) is provided. In the present invention, the RANKL protein mutant sequence may comprise any one of the amino acid sequences of SEQ ID NOs: 7 to 10.

[0020] Sequence number 1 is the amino acid sequence of the RANKL protein (mRANKL) of mouse (Mus musculus). The amino acid substitution site of the RANKL protein may be related to the site that binds to RANK or OPG.

[0021] Sequence number 2 is the amino acid sequence of the human (Homo sapiens) RANKL protein (hRANKL). The human RANKL protein has a full-length amino acid sequence of 317, which includes one additional amino acid compared to the mouse amino acid sequence of 316, and the substituted RANKL site may be located one position later than the mouse amino acid sequence. The amino acids at the substituted sites may be identical.

[0022] In one embodiment of the present invention, the RANKL protein mutant may be a RANKL protein mutant in which an amino acid at the following sequence position in the amino acid sequence of SEQ ID NO: 1 is substituted:

[0023] (1) Substitution of the 180th K with R; substitution of the 189th D with I; substitution of the 190th R with K; substitution of the 223rd H with F; substitution of the 224th H with Y and substitution of the 236th Q with D.

[0024] (2) Substitution of the 180th K with R; substitution of the 189th D with I; substitution of the 190th R with K; substitution of the 223rd H with F; substitution of the 224th H with Y; and substitution of the 269th F with L.

[0025] (3) Substitution of the 180th K with R; substitution of the 189th D with I; substitution of the 190th R with K; substitution of the 223rd H with F; substitution of the 224th H with Y; and substitution of the 269th F with Y.

[0026] (4) Substitution of the 180th K with R; substitution of the 189th D with I; substitution of the 190th R with K; substitution of the 223rd H with F; substitution of the 224th H with Y; and substitution of the 269th F with H.

[0027] In one embodiment of the present invention, the RANKL protein mutant may be a RANKL protein mutant in which an amino acid at the following sequence position in the amino acid sequence of SEQ ID NO: 2 is substituted:

[0028] (1) Substitution of the 181st K with R; substitution of the 190th D with I; substitution of the 191st R with K; substitution of the 224th H with F; substitution of the 225th H with Y and substitution of the 237th Q with D.

[0029] (2) Substitution of the 181st K with R; substitution of the 190th D with I; substitution of the 191st R with K; substitution of the 224th H with F; substitution of the 225th H with Y; and substitution of the 270th F with L.

[0030] (3) Substitution of the 181st K with R; substitution of the 190th D with I; substitution of the 191st R with K; substitution of the 224th H with F; substitution of the 225th H with Y; and substitution of the 270th F with Y.

[0031] (4) Substitution of the 181st K with R; substitution of the 190th D with I; substitution of the 191st R with K; substitution of the 224th H with F; substitution of the 225th H with Y; and substitution of the 270th F with H.

[0032] In the present invention, "binding affinity" is a measure of the binding strength between a specific ligand and a receptor, and means the relative tendency for the stability and persistence of the binding. "KD" or "dissociation constant" indicates the ligand concentration when the bound ligand-receptor complex dissociates into the ligand and receptor, and is a value that quantitatively expresses the stability of the binding. A high KD value indicates a weaker binding affinity, meaning that the ligand and receptor bind relatively weakly. The RANKL mutant of the present invention has a higher KD value for binding to RANK or OPG compared to the native RANKL protein, meaning that the binding affinity is low. For example, the RANKL mutant of the present invention may exhibit a therapeutic effect by regulating a specific signal pathway during bone metabolism by having a weak binding affinity for binding to RANK or OPG.

[0033] In one embodiment of the present invention, the RANKL mutant may have impaired binding to OPG. The mutant may have a K greater than that of wild-type RANKL. DIt binds to OPG.

[0034] In one embodiment of the present invention, the RANKL mutant may have impaired binding to RANK. The mutant may have a K greater than that of wild-type RANKL. D Combines with RANK.

[0035] The present invention also provides a nucleic acid molecule encoding a mutant of the RANKL protein. In the present invention, the nucleic acid molecule encoding the RANKL mutant may comprise any one of the nucleotide sequences of SEQ ID NOs: 11 to 14. In addition, the nucleic acid molecule encoding the RANKL mutant may comprise any one of the nucleotide sequences of SEQ ID NOs: 15 to 18.

[0036] Even if the present invention describes a 'RANKL protein mutant comprising a specific sequence number' (or a 'nucleic acid molecule encoding a RANKL protein mutant'), if it has the same or corresponding activity as a RANKL protein mutant composed of the amino acid sequence (or nucleotide sequence) of the sequence number, it does not exclude meaningless sequence additions before and after the amino acid sequence (or nucleotide sequence) of the sequence number, mutations that may occur naturally, or silent mutations thereof, and it is clear that even if it has such sequence additions or mutations, it falls within the scope of the present invention.

[0037] For example, the RANKL protein mutant of the present invention may include an amino acid sequence or nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with any one of the amino acid sequences or nucleotide sequences of SEQ ID NOs: 3 to 18, but is not limited thereto.

[0038] In the present invention, "homology" or "identity" refers to the degree to which two given amino acid sequences or nucleotide sequences are related to each other, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0039] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0040] Whether any two base sequences or peptide sequences are homologous, similar, or identical can be determined using a known computer algorithm, such as the "FASTA" program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be used. (including the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, BLAST from the National Center for Biotechnology Information database, or ClustalW can be used to determine homology, similarity, or identity.

[0041] In one embodiment of the present invention, the mutant of RANKL may be one in which a point mutation has occurred in the nucleic acid encoding the amino acid of the RANKL protein. A 'point mutation' is a mutation that occurs at the nucleotide level, in which a single nucleotide is changed to prevent or modify the production of a specific protein at the DNA transcription stage. A point mutation may have effects such as a 'silent mutation' in which the mutated codon instructs the formation of the same amino acid as the existing codon, a 'missense mutation' in which the mutated codon instructs the formation of a different amino acid, and a 'nonsense mutation' in which the formation of an amino acid is interrupted or omitted due to the mutated codon.

[0042] In one embodiment of the present invention, point mutations in the nucleic acid encoding the RANKL protein can be induced by a megaprimer. The megaprimer method is a type of PCR method that is used when the position where a mutation is to be induced is in the middle of the protein and cannot be completed in one step, and a two-step PCR is performed to complete the mutation. Specifically, when PCR amplification is induced using a forward primer containing the nucleotide to be induced and a reverse primer containing the nucleotide at the C-terminus of the protein, DNA without information about the N-terminus located ahead of the forward primer can be obtained. In the next step, when PCR is performed using the megaprimer as the reverse primer and an oligo DNA containing the N-terminus of the protein as the forward primer, DNA encoding a full-length protein with a mutation induced at the desired position can be obtained.

[0043] The above mutant may be in the form of a recombinant protein. The term "recombinant protein" refers to a protein obtained by artificially expressing "recombinant DNA," a new DNA created by inserting a specific gene into a vector using genetic recombination. The "genetic recombination" method refers to a technique that combines a DNA fragment from any organism with another DNA molecule. Genetically, genetic recombination can usually be achieved through transformation, transduction, conjugation (crossing), cell fusion, etc.

[0044] In addition, the "RANKL mutant" may additionally include, in addition to the amino acid sequence forming the active protein, an amino acid sequence manufactured for a specific purpose of increasing a cell-penetrating protein, a targeting sequence, a tag, a labeled residue, half-life, or protein stability. For example, the present invention may be a fusion protein in which a cell-penetrating protein, a targeting sequence, a tag, a labeled residue, an amino acid sequence for increasing half-life, or protein stability is fused to one end of a protein comprising any one of the amino acid sequences of SEQ ID NOs: 3 to 6.

[0045] Examples of the cell-penetrating proteins include TAT (HIV-1 Trans-Activator of Transcription), R9, etc. Examples of the tags include His tag (histidine tag), Strep(II)-tag, GST tag (glutathione-S-transferase tag), MBP tag (maltose binding protein tag), GFP tag (green fluorescent protein tag), SUMO tag (small ubiquitin-related(like) modifier tag), FLAG tag, HA tag, myc tag, etc. Biotin, reporter enzymes, fluorophores, or radioactive isotopes can be used for the labels.

[0046] Additionally, the present invention provides a vector comprising a nucleic acid molecule encoding the RANKL mutant.

[0047] In the present invention, a "vector" is a DNA molecule used as an artificial carrier of a nucleic acid sequence. It can replicate within cells and cause gene expression. In genetic engineering, a vector can be cut at a specific site with a restriction enzyme, the desired nucleic acid sequence can be inserted, and the vector can be cultured by reintroducing it into a host cell. A vector can serve as a vehicle for inserting a nucleic acid sequence. The nucleic acid sequence can be exogenous or heterologous. Types of vectors include plasmids, cosmids, and viruses such as bacteriophages.

[0048] The present invention provides a host cell comprising the vector. The host cell includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. The host cell can be transfected or transformed by the vector, which refers to the process by which an exogenous nucleic acid molecule is transferred or introduced into the host cell. A representative example of a host cell is Escherichia coli (E. coli).

[0049] In one embodiment of the present invention, the mutant may be administered to a subject to produce antibodies. Accordingly, the present invention additionally provides antibodies produced by mutants of the RANKL protein.

[0050] In the present invention, an antibody (immunoglobulin) may be a substance that specifically binds to an antigen and causes an antigen-antibody reaction. The antibody may be a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a chimeric antibody, an antibody conjugate, a human antibody, a humanized antibody, or a fragment thereof.

[0051] In the present invention, the mutant may induce the formation of anti-RANKL antibodies within a subject after administration to the subject. In other words, the mutant may be an antigen, a substance that acts as an immunogen for active immunity. The generated antibody can form an antigen-antibody reaction with RANKL within the subject. When the concentration of RANKL within the subject is reduced by the antibody, the binding between RANKL and RANK is reduced, thereby inhibiting the differentiation of osteoclasts.

[0052]

[0053] The present invention provides a vaccine composition comprising the RANKL protein mutant, a nucleic acid molecule encoding the same, or a vector comprising the same. The present invention also provides a method for treating or preventing a disease or disorder, comprising administering a therapeutically effective amount of the vaccine composition to a subject in need thereof.

[0054] In the present invention, the term "vaccine composition" or "vaccine" refers to a composition administered to generate or artificially increase immunity against a specific antigen. The vaccine composition provides a subject with an enhanced systemic or local immune response induced by a cellular immune response, such as CTL (Cytotoxic T Lymphocyte) or a humoral immune response, such as antibodies.

[0055] The cellular immune response may be CD8+ T lymphocyte-mediated (i.e., a cytotoxic response) or CD4+ T lymphocyte-mediated (a helper response). Cytotoxic and helper cellular immune responses may also be combined. The helper response may involve Th1, Th2, or Th17 lymphocytes (which, as is known in the art, can induce different cytokine responses). The composition may allow for better presentation of antigens present therein via the MHC1 or MHC2 pathway.

[0056] In the present invention, the vaccine composition may be a prophylactic (i.e., intended to protect the recipient against the occurrence of a disease) or a therapeutic (i.e., intended to help the recipient fight an existing disease) vaccine.

[0057] Additionally, in the present invention, the vaccine composition may further comprise an adjuvant. As used herein, "adjuvant" refers to a substance capable of modifying or enhancing an immune response to an antigen. In other words, the immune response to an antigen may be higher or different in the presence of the adjuvant than in the absence of the adjuvant (including when the response is modified, e.g., when a subset of T cells activated in the presence of the adjuvant differs from a subset activated in the absence of the adjuvant). Adjuvants are well known in the art and have been widely used in the field of vaccines. Examples of such adjuvants include alum, emulsions (oil-in-water or water-in-oil, such as Freund's incomplete adjuvant (IFA) and MF59®), pattern recognition receptor (PRR) ligands, toll-like receptor 3 (TLR3) and recombinant ligands (RIG-I-like receptor) such as double-stranded RNA (dsRNA), or synthetic analogs of dsRNA, such as poly(I:C), TLR4 ligands such as bacterial lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA), TLR5 ligands, TLR7 / 8 ligands such as imidazoquinolines, TLR9 ligands such as oligodeoxynucleotides containing specific CpG motifs (CpG ODNs) or nucleotide-binding oligomerization domain-containing protein 2 (NOD2) ligands.

[0058] In the present invention, the vaccine composition comprises at least one antigen or immunogen (e.g., a RANKL protein mutant) in a pharmaceutically acceptable vehicle useful for inducing an immune response in a host.

[0059] In the present invention, the vaccine composition may be administered alone, co-administered with another treatment or preventive therapy, or administered subsequently. In the present invention, the vaccine composition may be administered by a technique well known to those skilled in the medical or veterinary field and in a dosage, taking into account various factors, such as the age, sex, weight, species, and condition of the subject (preferably a mammal) to be administered, and the route of administration. In the present invention, the vaccine composition may be administered 1 to 5 times. For example, the vaccine composition may be administered 2 to 4 times. For example, the vaccine composition may be administered 1, 2, 3, 4, or 5 times.

[0060] The RANKL protein mutant of the present invention can be administered to a subject in the form of a vaccine composition. When the RANKL protein mutant is administered to a subject, the RANKL protein mutant itself can act as an antigen and form anti-RANKL antibodies. The anti-RANKL antibodies bind to wild-type RANKL and reduce the amount of wild-type RANKL. Accordingly, the amount of RANKL binding to RANKL is reduced, and RANK activity is inhibited, thereby inhibiting osteoclast differentiation.

[0061] In addition, the vaccine composition of the present invention can be used for the prevention or treatment of bone metabolic diseases. In the present invention, "bone metabolic diseases" refer to diseases that occur due to an imbalance in the processes of bone formation and destruction, which are caused by an imbalance in the activity of osteoblasts (bone-forming cells) and osteoclasts (bone-destroying cells), which are cells involved in bone metabolic processes. Bone metabolic diseases may include, for example, osteoporosis, osteogenesis disorders, and fractures, and range from simple bone damage to chronic bone diseases. These diseases can compromise the structural stability of bones, increase the risk of fractures, and cause severe pain and disability.

[0062] In the present invention, the bone metabolic disease may be at least one selected from the group consisting of osteoporosis, osteodystrophy, and fracture. The osteoporosis may be caused by RANKL binding to RANK, thereby inducing differentiation of osteoclasts.

[0063] Osteoclasts are cells that destroy bone. When the body needs to extract calcium from bone, osteoclasts can destroy bone. Osteoclasts can destroy bone when the body needs to replenish calcium from bone due to a calcium deficiency in the blood, when bone has microscopic cracks or blemishes, or when old bone needs to be replaced with new bone. This imbalance between osteoclasts and bone-forming osteoblasts can lead to bone metabolic diseases such as osteoporosis.

[0064] In the present invention, “differentiation” means a phenomenon in which structures or functions become specialized while cells divide and proliferate and grow, that is, cells, tissues, etc. of a living organism change in form or function to perform their respective assigned tasks.

[0065] The differentiation and activation of the above osteoclasts can be regulated by RANKL. Osteoclasts are formed into multinucleated bone-resorbing osteoclasts by RANKL-induced RANK activation in osteoclast precursor cells, which in turn stimulates TNF receptor-associated factors and sequentially activates NF-κB, mitogen-activated protein kinase (MAPK), activating protein 1 (AP-1), and nuclear factor of activated T cells (NFATc1).

[0066] The RANKL mutant of the present invention forms anti-RANKL antibodies, which bind to wild-type RANKL and reduce the amount of wild-type RANKL. Accordingly, RANKL signaling through the interaction between RANKL and RANKL can be inhibited, resulting in inhibition of osteoclast differentiation. Therefore, the RANKL mutant of the present invention or a vaccine composition comprising the same can be useful for the prevention or treatment of bone metabolic diseases.

[0067] In addition, the RANKL mutant of the present invention has an impaired ability to bind to OPG, and can suppress the loss of MT RANKL from the body due to binding of OPG and MT RANKL.

[0068] In the present invention, the vaccine composition may further comprise a pharmaceutically acceptable carrier. In the present invention, the "pharmaceutically acceptable carrier" includes any substance that, when combined with the active ingredient of the composition, allows the ingredients to retain biological activity without causing adverse physiological responses, such as unintended immune responses. Pharmaceutically acceptable carriers include water, phosphate buffered saline, emulsions such as oil / water emulsions, and wetting agents. Compositions comprising such carriers are described in Remington's Pharmaceutical Sciences, current Ed., Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; They are formulated by well-known conventional methods, such as those described in Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., 3rd ed. Amer. Pharmaceutical Assoc.

[0069] In the present invention, the vaccine composition may be formulated and provided in an appropriate form. The formulation may be formulated and used, respectively, in the form of oral formulations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, and aerosols, or in the form of parenteral formulations such as transdermal agents, suppositories, and sterile injectable solutions, according to conventional methods.

[0070] In the present invention, “treatment” means any type of intervention or process performed on a subject or administering an active agent to a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, or delaying or preventing the progression, development, severity or recurrence of a disease-related syndrome, complication, symptom or biochemical sign. Treatment can be performed on a subject with a disease or a subject without a disease (e.g., for prophylaxis).

[0071] In the present invention, "administration" refers to physically introducing a vaccine, therapeutic agent, or composition comprising the same into a subject using any of various methods and delivery systems known to those skilled in the art. Preferred routes of administration for the compositions of the present invention include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, intravitreal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, "parenteral administration" generally refers to modes of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intravitreal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation.

[0072] In the present invention, a "therapeutically effective amount" refers to an amount of a drug, alone or in combination with another therapeutic agent, that is effective in "treating" a disease or disorder in a subject or reducing the risk, potential, likelihood, or occurrence of a disease or disorder (e.g., a bone metabolic disorder). A "therapeutically effective amount" includes an amount of a drug or therapeutic agent that provides some improvement or benefit to a subject who has or is at risk of having a disease or disorder (e.g., osteoporosis as disclosed herein). Accordingly, a "therapeutically effective amount" is an amount that reduces the risk, potential, likelihood, or occurrence of a disease or disorder, or provides some alleviation, relief, or reduction in at least one indicator (e.g., a bone metabolic disorder), and / or reduces at least one clinical symptom of the disease or disorder.

[0073] In the present invention, the dosage of the composition may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and may be administered once or several times a day at regular intervals at the discretion of a doctor or pharmacist. For example, the daily dosage based on the active ingredient content is 0.001 to 10,000 mg / kg, 0.01 to 10,000 mg / kg, 0.1 to 10,000 mg / kg, 0.5 to 10,000 mg / kg, 0.001 to 1000 mg / kg, 0.01 to 1000 mg / kg, 0.1 to 1000 mg / kg, 0.5 to 1000 mg / kg, 0.001 to 500 mg / kg, 0.01 to 500 mg / kg, 0.1 to 500 mg / kg, 0.5 to 500 mg / kg, 0.001 to 300 mg / kg, 0.01 to 300 mg / kg, 0.1 to 300 ㎎ / kg, or 0.5 to 300 ㎎ / kg. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0074] In the present invention, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0075]

[0076] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims.

[0077] The RANKL mutant according to the present invention simultaneously inhibits the binding ability of OPG and RANK, thereby reducing the loss of the RANKL mutant from the body due to binding to OPG. In addition, by inhibiting the binding to RANK, the activity of RANK can be inhibited, thereby suppressing differentiation into osteoclasts. Furthermore, the RANKL mutant of the present invention can act as an immunogen that generates anti-RANKL antibodies. Through this, the RANKL mutant of the present invention can be usefully utilized in the prevention or treatment of bone metabolic diseases.

[0078] Figure 1a shows the entire RANKL region encompassing residues 158 to 316 of mouse RANKL.

[0079] Figure 1b shows wild-type WT RANKL, mutant RANKL (001) with a mutation in the RANK binding site, and mutant RANKL (011, 012, 013, 014) with a mutation in the RANK binding site and OPG binding site, indicating the mutation site.

[0080] Figure 1c shows the pGEX-4T-1 vector for inserting and classifying cloned genes.

[0081] Figure 1d shows the SDS-PAGE results of wild-type WT RANKL (WT) produced through Example 1, mutant RANKL (001) in which the RANK binding site is mutated, and mutant RANKL (013) in which the RANK binding site and OPG binding site are mutated.

[0082] Figure 2 shows the binding affinity (Kd value) results measured through MST analysis between wild-type WT RANKL and mutant RANKL (001) in which the RANK binding site is mutated, and mutant RANKL (011, 012, 013, 014) in which the RANK binding site and the OPG binding site are mutated, to OPG (Figure 2a) or RANK (Figure 2a).

[0083] Figure 3 shows the results of Western blot analysis of the serum of mice immunized with 001 (left) and 013 (right) in the commercially purchased wild-type WT RANKL and the wild-type GST-RANKL produced in Experimental Example 1.

[0084] Figure 4a shows that injection of commercially purchased wild-type WT RANKL induced an increase in TRAP-positive cells in macrophages extracted from mice, while sera extracted from mice immunized with 001 or 013 produced in Example 1 induced a decrease in TRAP-positive cells. TRAP-positive cells were imaged under a light microscope (100X magnification).

[0085] Figure 4b shows the area of ​​TRAP-positive cells measured by IMAGE J software (p < 0.05).

[0086] Hereinafter, the present invention will be described in detail through examples. The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0087]

[0088] [Example]

[0089] All chemical reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA), and cell culture media were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0090]

[0091] Experimental Example 1. Production of RANKL, introduction of mutations, and culture

[0092] 1.1. Manufacturing of RANKL

[0093] RNA for cloning RANKL cDNA was extracted from MC3T3-E1 cells (Korea Cell Line Bank, Seoul) expressing RANKL. The extracted RNA was verified by agarose gel electrophoresis. cDNA was prepared using the AccuPower RT PreMix Kit (Bioneer, Daejeon, Korea) according to the manufacturer's instructions. The reaction mixture contained Taq polymerase buffer, 10 mM dNTPs, 25 mM MgCl2, 10 μM primers (RANKL-K158: 5'-CAT ATG AAG CCT GAG GCC CAG CCA TT-3', RANKL-D316: 5'-CTC GAG GTC TAT GTC CTG AAC TTT GAA AGC C-3'), 2.5 U of KOD DNA polymerase (EMD Millipore, Billerica, MA, USA), and 2 μL of RANKL gene construct template, and amplification and cloning of RANKL fragments were performed in the reaction mixture.

[0094] The thermal cycle consisted of a) initial denaturation at 95 °C for 5 min, b) denaturation at 95 °C for 30 s, c) primer annealing at 55 °C for 30 s, and d) denaturation at 70 °C for 30 s, for a total of 40 cycles. The RANKL sequence encoded a full-length 158 amino acid target region encompassing residues 158 to 316, as shown in Figure 1a.

[0095] 1.2. Introduction of mutations and transformation

[0096] Amino acid sequence positions 180, 189-190, 223-224, and 236 of mouse RANKL (mRANKL) were mutated, and at least one of amino acid sequence positions 236 and 269 was mutated (Fig. 1b). Specifically, MT RANKL (001, SEQ ID NO: 19), in which the RANK binding site of WT RANKL was mutated, and mutant RANKL (011, 012, 013, 014, SEQ ID NOs: 3-6, respectively), in which the RANK and OPG binding sites of WT RANKL were mutated, were prepared. Gene mutations were introduced via megaprimers.

[0097] First, the amino acid sequences of four mutants in which lysine (Lys) at position 180, aspartic acid (Asp) at position 189, arginine (Arg) at position 190, histidine (His) at position 223, and histidine (His) at position 224, glutamine (Gln) at position 236, and phenylalanine (Phe) at position 269 were substituted, as shown in Table 3 below, are indicated by the sequence numbers in Table 1. In addition, the nucleotide sequences of nucleic acids encoding these substituted mutants are shown in SEQ ID NOs: 11 to 14.

[0098] Mutation Set 180K189D190R223H224H236Q269F SEQ ID NO: 3 (011)RIKFYD SEQ ID NO: 4 (012)RIKFYL SEQ ID NO: 5 (013)RIKFYY SEQ ID NO: 6 (014)RIKFYH

[0099] In the case of human RANKL protein (hRANKL), the full length is 317, which contains one amino acid additionally compared to the mouse amino acid sequence with a full length of 316, and the substituted RANKL site is located one position after the mouse amino acid sequence. The amino acids of the substituted sites are identical. As with the mRANKL mutants, the amino acid sequences of the substituted hRANKL mutants are each represented by the sequence numbers listed in Table 2. The nucleotide sequences of the nucleic acids encoding these substituted mutants are shown in SEQ ID NOs: 15 to 18.

[0100] Mutation Set 181K190D191R224H225H237Q270F SEQ ID NO: 7 (111)RIKFYD SEQ ID NO: 8 (112)RIKFYL SEQ ID NO: 9 (113)RIKFYY SEQ ID NO: 10 (114)RIKFYH

[0101] To replicate the obtained PCR product, it was cloned into the BamHI / XhoI sites of the pGEX-4T-1 vector (Promega, Madison, WI, USA) (Fig. 1c). After subcloning the RANKL fragment into pGEX-4T-1, it was confirmed that it had the correct sequence for sequential translation of RANKL and GST tags. Transformation of the PCR product was performed by electroporation (5 msec, 12.5 kV / cm) into E. coli BL21-CodonPlus(DE3)-RIPL (Novagen). All sequence analyses were performed using the program Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA).

[0102] 1.3. Cultivation

[0103] A single colony containing the recombinant plasmid was inoculated into 20 ml of LB medium supplemented with ampicillin (50 ng / mL) and cultured at 37°C with shaking at 200 rpm for 24 h. 10 ml of this culture was inoculated into an Erlenmeyer flask containing 1 L of LB medium containing 50 μg / mL of ampicillin. OD 600　 The cells were cultured at 37°C with vigorous shaking at 180 rpm until the value reached approximately 1.0. Protein expression was then induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) at concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM, and cultured for 6 h. After induction, the cultures were centrifuged at 5600 ×g at 4°C for 20 min, and the cell pellets were stored at -20°C.

[0104] 1.4. Purification of WT (wild type) or MT (mutant) RANKL (001, 011, 012, 013, 014)

[0105] After centrifugation of the culture, the pelleted cells were resuspended in 10 mL of lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4). The cell suspension was supplemented with 0.1 mg / mL lysozyme and 0.1 mM phenylmethylsulfonyl fluoride (Calbiochem, La Jolla, CA, USA) and incubated on ice for 1 h. Subsequently, glycerol (20% v / v, Carlo Erba, France) was added to the cell suspension. The cells were sonicated and centrifuged at 15,000 × g at 4 °C for 10 min. The supernatant was passed through a 0.2 μm filter paper and immobilized on a Glutathione Sepharose 4 M resin column (GE Healthcare, Uppsala, Sweden) equilibrated with binding buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4, 10 mM imidazole, 5 mM DTT, pH 7.4). The column was then washed with binding buffer supplemented with 20 mM imidazole. After washing, the protein was eluted with elution buffer (Qiagen). The eluted protein was dialyzed against dialysis buffer (20% v / v glycerol in phosphate-buffered saline [PBS]) in a 10,000 MW Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). The purified protein was concentrated in vacuo (Savant Instruments, Holbrook, NY, USA). Finally, proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentrations were calculated and determined by the Bradford assay. An additional washing step was introduced after the initial washing for chromatography to remove endotoxins. This step was performed by adding W1-TX114 (0.The washing steps were performed with W1-TX100 with 1% Triton X-114) or 1% sodium deoxycholate (buffer W1-DOC) at 25 °C with W1-TX100 and W1-DOC, or with W1-TX114 at 4 °C. The eluates from each washing step were collected for endotoxin quantification.

[0106] The prepared recombinant protein samples (WT RANKL, 001, 011, 012, 013, 014) were added to 2x lysis buffer (0.5 M Tris-HCl, pH 6.8, 0.5% (v / v) bromophenol blue, 10% (v / v) glycerol, 2% (v / v) SDS, and 10% (v / v) β-mercaptoethanol) at a 1:1 (v / v) ratio and boiled for 5 min. The proteins were then analyzed by electrophoresis. After separation, the gel was stained with Coomassie brilliant blue G-250. To determine the purity and recovery of the recombinant proteins, the stained gels loaded with fixed amounts of proteins were imaged at 300 dpi using a digital scanner (EPSON, USA).

[0107]

[0108] Experimental Example 2. Binding Affinity Measurement

[0109] Protein binding affinity was measured using MicroScale Thermophoresis (MST). MST experiments were performed using Monolith NT.115 cells (NanoTemper Technologies, München, Germany) and a red filter. All dilutions were prepared so that no other gradients (salt, glycerol, DMSO, etc.) were generated during buffer mixing. To minimize sample adsorption to the substrate, 0.05% Tween 20 was added to PBS and used to dilute all receptors and ligands. To measure protein-protein binding, WT RANKL or MT RANKL, the ligands for the receptor proteins RANK and OPG, respectively, were mixed in equal proportions. The volume of the fluorescent ligand spiperone-Cy5 was adjusted to obtain final ligand concentrations of 0.125, 5, 7.5, and 12 nM. After incubation for 1 h, samples were loaded into the capillaries and the LED was set to 20% for the 0.125 nm samples and 1% for the 5, 7.5, and 12 nm samples using the intermediate MST power. For receptor titration assays, various concentrations of protein (~10 mg / mL to ~5 μg / mL total protein) were mixed with a specific concentration of the fluorescent ligand, spiperon-Cy5, and added to each protein dilution point to a final concentration of 5, 7.5, or 12 nM. Samples were incubated for 1 h before capillary loading. The LED power was set to 1% and the MST power was set to intermediate. The intersection of the binding curves was determined using the manufacturer's protocol, and the Kd values ​​were obtained from the average of three replicates.

[0110]

[0111] Experimental Example 3. Mouse Immunization and Antibody Detection

[0112] Seventeen-week-old male BALB / c mice were immunized with MT RANKL (001 or 013) for 52 days. MT RANKL was administered mixed with aluminum hydroxide adjuvant. The immunization process consisted of three doses: the first dose 52 days prior, the second 39 days prior, and the third 14 days prior. The dose of MT RANKL was 0.2 mg per mouse. After completion of immunization, serum from each mouse was collected and tested for RANKL antibodies.

[0113] For detection of RANKL antibodies by immunization, tagging-free wild-type RANKL in its native form and the wild-type GST-RANKL protein obtained in Experimental Example 1 were separated by SDS-PAGE and electroporated onto a nitrocellulose membrane (Bio-Rad). After blocking the membrane with 5% (wt / vol) non-fat dry milk in TBST [10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% (vol / vol) Tween 20], the nitrocellulose membrane was reacted with a solution of the primary antibody, diluted 1:5000 in TBST using the serum of the immunized mouse, for 24 hours. The membrane was washed three times with Tris-buffered saline and then reacted with a horse peroxidase:HRP-conjugated secondary antibody in a solution of 1% (wt / vol) non-fat dry milk diluted 1:5000 in TBST for 3 hours. After the reaction, the membrane was washed three times with Tris-buffered saline, and antibodies were detected by immunization using the ECL_system (Amersham Pharmacia Biotech).

[0114]

[0115] Experimental Example 4. TRAP Analysis

[0116] Five-week-old female mice (BL-6; Orient Bio Co., Seoul, South Korea) were sacrificed for use in vitro and in vivo studies. All experimental procedures involving animals were performed in compliance with institutional and governmental requirements and were approved by the Institutional Animal Care and Use Committee of Chosun University, Gwangju, South Korea (CIACUC2018-S0012-1).

[0117] After obtaining bone marrow cells from mice, bone marrow mononuclear cells were seeded in 96-well plates (1 Υ 10 4 Cells were seeded at 10 cells / well and cultured with M-CSF (100 ng / mL). On the third day of culture, WT RANKL (50 ng / mL) and each serum collected in Experimental Example 3 were treated and cultured in M-CSF (100 ng / mL)-treated medium. After 3 days, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and washed with PBS. Then, TRAP activity (Sigma-Aldrich, St. Louis, MO, USA) was stained. After staining, TRAP-positive area was measured by IMAGE J software (v.1.54). Two-tailed paired Student's t test was used for statistical analysis. P<0.05 was considered statistically significant. Data are expressed as mean ± standard deviation (SD) unless otherwise stated. Data were analyzed using the GraphPad Prism version 6.00 software program for Windows (GraphPad, La Jolla, CA, USA).

[0118]

[0119] Example 1: Purification of mutant RANKL 001, 013

[0120] Mutant RANKL (001, 013) identified by SDS-PAGE showed a band of approximately 43 kDa in the total cell protein extract of the sample (Fig. 1d).

[0121]

[0122] Example 2. Confirmation of binding affinity of RANKL mutants to RANK and OPG, respectively.

[0123] In the above experimental examples, MST analysis was performed to determine the binding affinity of wild-type WT RANKL or RANKL mutants (001, 011, 012, 013, 014) to RANK or OPG, respectively (Fig. 2). The MST measurement results showed that the Kd for the binding of WT RANKL to OPG was 315 nM, and the Kd for the binding of 001 to OPG was 5.9 μM, which means that the affinity of the mutant RANKL (001) to OPG was ~18.7 times lower. In addition, 011 and 013 showed an even lower affinity below the measured value (ND).

[0124] In addition, the Kd for binding of WT RANKL to RANK was 1.05 μM, the Kd for binding of 001 to RANK was 5.61 μM, and 011 and 013 showed lower affinity at 17.1 μM. These results showed that 011 or 013 binds little to OPG or RANK than WT RANKL or 001. This suggests that the novel mutant RANKL 011 or 013 may act as an exogenous antigen that does not bind to OPG and gives little RANK signaling.

[0125]

[0126] Example 3. Confirmation of the antibody-forming ability of MT RANKL in vivo.

[0127] The results of Western blot analysis of the sera of mice immunized with wild-type wt RANKL and GST-RANKL produced in Example 1 as 001 (left) and 013 (right) are shown (Fig. 3). To determine whether immunization with MT RANKL (001, 013) induced antibody production, Western blot was performed three times on sera collected from three mice each. As shown in Fig. 3, both GST-RANKL and WT RANKL antibodies were detected in the sera of mice immunized with all 001 or 013 MT RANKL. In particular, stronger WT RANKL antibodies than 001 were detected in all 013-immunized mice. Accordingly, it can be seen that immunization with 013 RANKL resulted in the formation of stronger anti-RANKL antibodies than 001 in the mice.

[0128]

[0129] Example 4. Results of TRAP activity inhibition according to immunization

[0130] To evaluate the effect of RANKL mutant (MT RANKL) on osteoclast formation, TRAP assay was performed in RANKL-treated primary bone marrow-derived macrophages (BMMs) using the method described in the experimental example above.

[0131] As shown in Fig. 4a, BMM differentiated into mature TRAP-positive multinucleated osteoclasts in cells treated with wild-type RANKL (WT RANKL) and control serum, while differentiation of TRAP-positive multinucleated osteoclasts was inhibited in cells treated with serum from mice immunized with 001 or 013 (Fig. 4a). In particular, it was confirmed that the group treated with serum from mice immunized with 013 had a superior effect on inducing osteoclast differentiation compared to 001 (Fig. 4b).

Claims

1. 1) At the N-terminus of the RANKL protein comprising the amino acid sequence of sequence number 1, Contains at least four substitutions selected from the group consisting of: substitution of the 180th K with R; substitution of the 189th D with I; substitution of the 190th R with K; substitution of the 223rd H with F or Y; and substitution of the 224th H with F or Y; A mutant comprising one or more substitutions selected from the group consisting of a substitution of Q at position 236 with D; and a substitution of F at position 269 with L, Y, or H; or 2) At the N-terminus of the RANKL protein comprising the amino acid sequence of sequence number 2, Contains at least four substitutions selected from the group consisting of: substitution of the 181st K with R; substitution of the 190th D with I; substitution of the 191st R with K; substitution of the 224th H with F or Y; and substitution of the 225th H with F or Y; A mutant comprising one or more substitutions selected from the group consisting of a substitution of Q at position 237 with D; and a substitution of F at position 270 with L, Y, or H; RANKL protein mutant.

2. In paragraph 1, The above RANKL protein mutant is a RANKL protein mutant whose binding to OPG is inhibited.

3. In paragraph 2, The above RANKL protein mutant has a higher K than wild-type RANKL. D A mutant RANKL protein that binds to OPG.

4. In paragraph 1, The above RANKL protein mutant is a RANKL protein mutant whose binding to RANK is inhibited.

5. In paragraph 4, The above RANKL protein mutant has a higher K than wild-type RANKL. D A mutant RANKL protein that binds to RANK.

6. A nucleic acid molecule encoding the RANKL protein mutant of paragraph 1.

7. A vector comprising the nucleic acid molecule of paragraph 6.

8. A vaccine composition comprising the RANKL protein mutant of claim 1, the nucleic acid molecule of claim 6, or the vector of claim 7.

9. In paragraph 8, A vaccine composition wherein the mutant is administered to a subject to produce anti-RANKL antibodies.

10. In paragraph 9, A vaccine composition wherein the anti-RANKL antibody binds to RANKL and reduces the concentration of RANKL binding to RANK.

11. In paragraph 8, The above vaccine composition is a vaccine composition that does not promote osteoclast differentiation by RANK.

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