Rankl mutant and use thereof

Abstract

A RANKL mutant according to the present invention inhibits the binding to RANK and binds to LGR4 with high affinity to inhibit the activity of RANK, thereby inhibiting the differentiation into osteoclasts. Therefore, the RANKL protein mutant of the present invention can be helpfully utilized in the prevention or treatment of bone metabolic diseases.

Description

RANKL mutants and their uses

[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 pharmaceutical composition for preventing or treating bone metabolic diseases, 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, the term "LGR4 (leucine-rich repeat-containing G-protein-coupled receptor 4)" or "GPR48" refers to another receptor of RANKL that competes with RANK to bind to RANKL and inhibits canonical RANK signaling during osteoclastogenesis. In the present invention, the RANKL protein mutant can bind to LGR4 with high affinity, and LGR4 signaling can inhibit osteoclastogenesis. Specifically, it was shown that the binding of LGR4 and the RANKL mutant resulted in decreased AKT phosphorylation, increased GSK-3β phosphorylation, and suppressed NFATc1 nuclear translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption.

[0016] In the present invention, "mutant" refers to a modified gene or protein derived through a naturally occurring or artificially engineered genetic modification. Such mutants have structural or functional properties 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 can be usefully utilized in the treatment of osteoporosis by modifying the activity of RANKL and inhibiting the binding of RANKL to RANK or RANKL to OPG. In particular, the mutant of the present invention can bind to LGR4 with high binding affinity instead of binding to OPG, and can also act by inhibiting the osteoclast-activating function of RANKL or enhancing the osteoclast-inhibiting function of OPG.

[0017] 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.

[0018] 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.

[0019] Accordingly, the present invention comprises three 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 is provided comprising 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). In the present invention, the RANKL protein mutant sequence may comprise any one of the amino acid sequences of SEQ ID NOs: 14 to 15.

[0020] Accordingly, the present invention comprises three 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 is provided, comprising 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). In the present invention, the RANKL protein mutant sequence may comprise the amino acid sequence of SEQ ID NO: 16.

[0021] 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.

[0022] 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.

[0023] 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:

[0024] (1) 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.

[0025] (2) Substitution of the 190th R with K; substitution of the 223rd H with Y; substitution of the 224th H with Y; and substitution of the 269th F with Y.

[0026] 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:

[0027] (1) 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 269th F with Y.

[0028] (2) Substitution of the 191st R with K; substitution of the 224th H with Y; substitution of the 225th H with Y; and substitution of the 269th F with Y.

[0029] 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 signaling pathway during bone metabolism by having a weak binding affinity for binding to RANK or OPG while maintaining a high binding affinity for LGR4.

[0030] 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. D It binds to OPG.

[0031] 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.

[0032] In one embodiment of the present invention, the RANKL mutant can bind with high binding affinity to LGR4. The mutant has a K equivalent to that of wild-type RANKL. DBinds to LGR4. In the present invention, "equivalent KD" means that two substances have the same or very similar binding affinity to LGR4. For example, the binding affinity of the RANKL mutant of the present invention to LGR4 may be, but is not limited to, 70% or more, 80% or more, 90% or more, or 95% or more of the binding affinity of wild-type RANKL to LGR4. Alternatively, the binding affinity of the RANKL mutant of the present invention to LGR4 may be, but is not limited to, 130% or less, 120% or less, 110% or less, or 105% or less of the binding affinity of wild-type RANKL to LGR4.

[0033] 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: 28 to 29. In addition, the nucleic acid molecule encoding the RANKL mutant may comprise the nucleotide sequence of SEQ ID NO: 30.

[0034] 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.

[0035] For example, the RANKL protein mutant of the present invention may comprise 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 of SEQ ID NOs: 14 to 16 or any one of the nucleotide sequences of SEQ ID NOs: 28 to 30.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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: 14 to 16.

[0043] 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.

[0044] Additionally, the present invention provides a vector comprising a nucleic acid molecule encoding the RANKL mutant.

[0045] 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.

[0046] 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).

[0047] The present invention also provides a pharmaceutical composition for diagnosing or treating a bone metabolic disease, comprising the RANKL protein mutant, a nucleic acid molecule encoding the same, or a vector comprising the same. Furthermore, the present invention provides a method for preventing or treating a bone metabolic disease, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject.

[0048] In the present invention, "bone metabolic disease" refers to diseases that occur due to an imbalance in the processes of bone formation and destruction. This occurs due to 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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).

[0053] The RANKL mutant of the present invention has impaired binding ability to RANK, thereby inhibiting signal transduction of RANK through interaction between RANK and RANKL, and consequently inhibiting differentiation of osteoclasts. Therefore, the RANKL mutant of the present invention or a composition comprising the same can be useful for preventing or treating bone metabolic diseases.

[0054] In addition, the RANKL mutant of the present invention has an impaired ability to bind to OPG, and can suppress the loss of mtRANKL from the body due to binding of OPG and mtRANKL.

[0055] In the present invention, the pharmaceutical 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.

[0056] In the present invention, the pharmaceutical composition may be formulated and provided in an appropriate form. The formulation may be formulated and used in oral formulations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, and aerosols, or in parenteral formulations such as transdermal agents, suppositories, and sterile injectable solutions, according to conventional methods.

[0057] 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).

[0058] As used herein, "administration" refers to physically introducing a therapeutic agent or a 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062]

[0063] 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.

[0064] The RANKL mutant according to the present invention simultaneously inhibits the binding ability of OPG and RANK, thereby reducing the body's loss of the RANKL mutant due to binding to OPG. In addition, the RANKL mutant according to the present invention inhibits binding to RANK and binds to LGR4 with high affinity, thereby inhibiting RANK activity and thereby suppressing differentiation into osteoclasts. Through this, the RANKL mutant of the present invention can be usefully utilized in the prevention or treatment of bone metabolic diseases.

[0065] Figure 1a shows the entire RANKL region encompassing residues 158 to 316 of mouse RANKL.

[0066] Figure 1b shows the pET-30a vector for inserting and classifying cloned genes.

[0067] Figure 2 shows the results of observing TRAP-positive cells according to concentration-dependent treatment of wild-type RANKL (ACMR-0000) and mutant RANKLs ACMR-030, ACMR-031, ACMR-032, ACMR-033, ACMR-034, ACMR-035, ACMR-036, ACMR-047, ACMR-048, ACMR-049, ACMR-050, ACMR-058, and ACMR-059 produced through Example 1. TRAP-positive cells were imaged under an optical microscope (100X magnification).

[0068] Figure 3 shows the results of TRAP-positive cell generation following high-concentration ACMR-059 treatment. TRAP-positive cells were imaged under an optical microscope (100X magnification).

[0069] Figure 4 shows the inhibitory effects of ACMR-050, ACMR-058, and ACMR-059 on TRAP-positive cells treated with various concentrations of wild-type RANKL (ACMR-0000) (50 ng / mL). TRAP-positive cells were imaged under a light microscope (100X magnification).

[0070] Figure 5 shows the inhibitory effects of ACMR-050, ACMR-058, and ACMR-059 on TRAP-positive cells treated with various concentrations of wild-type human-derived RANKL (ACMR-1000) (50 ng / mL). TRAP-positive cells were imaged under a light microscope (100X magnification).

[0071] Figure 6 shows that TRAP-positive cells were not detected at all even after treatment with high concentrations of human-derived mutant RANKL (ACMR-159). TRAP-positive cells were imaged under a light microscope (100X magnification).

[0072] Figure 7 shows the inhibitory effect of human mutant RANKL (ACMR-159) on TRAP-positive cells treated with various concentrations of wild-type human RANKL (ACMR-1000) (50 ng / mL). TRAP-positive cells were imaged under a light microscope (100X magnification).

[0073] Figure 8 shows the TRAP-positive cell area measured by IMAGE J software for analysis of the TRAP-positive cell inhibitory effect of human-derived mutant RANKL (ACMR-159) treated at various concentrations under injection (50 ng / mL) of wild-type human-derived RANKL (ACMR-1000) (p < 0.05).

[0074] Figure 9 shows the trabecular bone structure in micro-CT 3D images of mouse femurs injected with control, wild-type RANKL (WT), wild-type RANKL and mutant RANKL (WT+001), or wild-type RANKL and mutant RANKL (WT+059).

[0075] Figure 10 is a graph showing bone density and bone volume percentage in micro-CT 3D images of the femurs of mice injected with control, wild-type RANKL (WT), wild-type RANKL and mutant RANKL (WT+001), or wild-type RANKL and mutant RANKL (WT+059). The values ​​shown in each graph represent the average values ​​from 10 mice.

[0076] 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).

[0077]

[0078] Experimental Example 1. Production of RANKL, introduction of mutations, and culture

[0079] 1.1. Manufacturing of RANKL

[0080] 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.

[0081] 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.

[0082] 1.2. Introduction of mutations and transformation

[0083] 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. Specifically, MT RANKL (001, SEQ ID NO: 31), in which the RANK binding site in WT RANKL was mutated, and mutant RANKL (030, 031, 032, 033, 034, 035, 036, 047, 048, 049, 050, 058, 059, SEQ ID NOs: 3-15, respectively), in which the RANK binding site in WT RANKL was mutated, were prepared.

[0084] First, mutations in the gene were introduced through a megaprimer. Specifically, 12 mutant amino acid sequences were substituted at amino acid sequence positions 180, 189, 190, 190, 223, 224, 236, 236, and 269, respectively, as shown in Table 3 below, and are represented by the sequence numbers in Table 1. In addition, the nucleotide sequences of the nucleic acids encoding these substituted mutants are shown in SEQ ID NOs: 17 to 29.

[0085] Mutation Set 180K 189D 190R 223H 224H 236Q 269F SEQ ID NO: 3 (ACMR-030) Y SEQ ID NO: 4 (ACMR-031) R Y SEQ ID NO: 5 (ACMR-032) I Y SEQ ID NO: 6 (ACMR-033) K Y SEQ ID NO: 7 (ACMR-034) F Y SEQ ID NO: 8 (ACMR-035) Y Y SEQ ID NO: 9 (ACMR-036) Y Y SEQ ID NO: 10 (ACMR-047) KF Y SEQ ID NO: 11 (ACMR-048) K Y Y SEQ ID NO: 12 (ACMR-049) FY Y SEQ ID NO: 13 (ACMR-050) YY Y SEQ ID NO: 14 (ACMR-058) KFY Y SEQ ID NO: 15 (ACMR-059) KYY Y

[0086] 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 site are identical. Similar to the mRANKL mutant, the substituted hRANKL mutant amino acid sequence is represented by the sequence number listed in Table 2. The nucleotide sequence of the nucleic acid encoding this substituted mutant is shown in SEQ ID NO: 16.

[0087] Mutation Set181K190D191R224H225H237Q270F SEQ ID NO: 16 (ACMR-159) KYY Y

[0088] To replicate the obtained PCR product, it was cloned into the NdeI / XhoI sites of the pET-30a vector (Novagen, Madison, WI, USA) as shown in Figure 1b. After subcloning the RANKL fragment into pET30a, it was confirmed that it had the correct sequence for sequential translation of RANKL and the 6xHis tag. Transformation of the PCR product was performed into E. coli BL21-CodonPlus(DE3)-RIPL (Novagen) by electroporation (5 msec, 12.5 kV / cm). All sequence analyses were performed using the program Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA).

[0089] 1.3. Cultivation

[0090] A single colony containing the recombinant plasmid was inoculated into 20 ml of LB medium supplemented with kanamycin (50 μg / mL) and cultured at 37°C with shaking at 200 rpm for 24 hours. 10 ml of this culture was inoculated into an Erlenmeyer flask containing 1 L of LB medium containing 50 μg / mL kanamycin. 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.

[0091] 1.4. Purification of WT (wild-type) RANKL and MT (mutant) RANKL

[0092] 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. Glycerol (20% v / v, Carlo Erba, France) was then 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 HisTrap HP His tag protein purification columns (Cytiva, 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 columns were 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 using the Bradford assay. An additional washing step was introduced after the initial washing for chromatography to remove endotoxins.This step was performed using W1-TX114 (W1-TX100 with 0.1% Triton X-114) or 1% sodium deoxycholate (buffer W1-DOC) in 80 volumes of the resin bed, at 25 °C with W1-TX100 and W1-DOC, or at 4 °C with W1-TX114. The eluate from each wash step was collected for endotoxin quantification.

[0093] The prepared recombinant protein samples 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 minutes. 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).

[0094]

[0095] Experimental Example 2. TRAP Analysis

[0096] Five-week-old female mice (BL-6; Orient Bio Co., Seoul, South Korea) were sacrificed for use in vitro 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).

[0097] After obtaining bone marrow cells from mice, bone marrow mononuclear cells were seeded in 96-well plates (10 4Cells were seeded at 10 cells / well and cultured with M-CSF (100 ng / mL) for 3 days. Then, they were cultured for 3 days in the absence or presence of various concentrations of RANKL derivatives in the presence of M-CSF (100 ng / mL). After 6 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).

[0098]

[0099] Experimental Example 3. Micro-computed tomography (Micro-CT)

[0100] Five-week-old female mice (BL-6; Orient Bio Co., Seoul, South Korea) were sacrificed for in vivo studies. Forty mice were divided into four groups: 10 mice served as a control group, 10 mice were injected with 1 mg / kg of wild-type RANKL (WT), 10 mice were injected with 1 mg / kg of wild-type RANKL (WT) and 1 mg / kg of mutant RANKL 001, and the last 10 mice were injected with 1 mg / kg of wild-type RANKL (WT) and 1 mg / kg of mutant RANKL 059 prepared in Experimental Example 1. Each group was administered the drugs intraperitoneally, and each mouse was sacrificed on the second day.

[0101] The right femur of each experimental mouse was dissected, and CT images were acquired using a Quantum GX μCT imaging system (PerkinElmer, Hopkinton, MA, USA) located at the Korea Basic Science Institute in Gwangju. The X-ray source was set to a 45-mm field of view, 90 kV, and 88 mA (voxel size, 90 μm; scanning time, 14 min). CT images were acquired using Quantum GX 3D Viewer software. After scanning, image segmentation was performed using Analyze nalyzeDirect (Overland Park, KS, USA). Briefly, the limb was segmented using semi-automatic and manual tools (e.g., object extraction, region growth, and opposition separator) using the volume editing tool. 3D renderings of the limb were then generated, and bone mineral density (BMD) and bone volume percentage (%) were calculated using the region of interest (ROI) tool.

[0102]

[0103] Example 1: Evaluation of TRAP inhibition in vitro

[0104] To evaluate the TRAP inhibition ability of RANKL in vitro, bone marrow-derived macrophages were treated with WT (wild type) RANKL (ACMR-0000) or mutant RANKL (ACMR-030, ACMR-031, ACMR-032, ACMR-033, ACMR-034, ACMR-035, ACMR-036, ACMR-047, ACMR-048, ACMR-049, ACMR-050, ACMR-058, ACMR-059) prepared in Experimental Example 1, and TRAP assay was performed using the method described in Experimental Example 1 above. Injection of wild-type RANKL (ACMR-0000) induced an increase in TRAP-positive cells in macrophages extracted from mice, and among the mutant RANKLs produced in Example 1, ACMR-030, ACMR-031, ACMR-032, ACMR-033, ACMR-034, ACMR-035, ACMR-036, ACMR-047, ACMR-048, ACMR-049, ACMR-050, and ACMR-058 also induced an increase in TRAP-positive cells. However, in the case of ACMR-059, no TRAP-positive cells were observed at all (Fig. 2).

[0105] Figure 3 shows TRAP-positive cells that were not detected at all even with high concentrations of ACMR-059 treatment. It also shows the TRAP-positive cell inhibitory effect of ACMR-050, ACMR-058, and ACMR-059 treated at various concentrations under wild-type RANKL (ACMR-0000) injection (50 ng / mL). Unlike ACMR-050 and ACMR-058, ACMR-059 shows TRAP-positive cell inhibitory effect at high concentrations starting from 25 ng / mL (Figure 4). Figure 5 shows the TRAP-positive cell inhibitory effect of ACMR-050, ACMR-058, and ACMR-059 treated at various concentrations under wild-type human-derived RANKL (ACMR-1000) injection (50 ng / mL). Unlike ACMR-050 and ACMR-058, ACMR-059 shows TRAP-positive cell inhibitory effect at high concentrations starting from 25 ng / mL. Figure 9 shows TRAP-positive cells that were not detected at all even when treated with high concentrations of wild-type human-derived mutant RANKL (ACMR-159). Figure 7 shows the TRAP-positive cell inhibitory effect of human-derived mutant RANKL (ACMR-159) treated at various concentrations under the injection of wild-type human-derived RANKL (ACMR-1000) (50 ng / mL). ACMR-159 shows TRAP-positive cell inhibitory effect as the treatment increases from 75 ng / mL to higher concentrations. Figure 8 shows the values ​​of TRAP-positive cell area measured for the analysis of TRAP-positive cell inhibitory effect of human-derived mutant RANKL (ACMR-159) treated at various concentrations under the injection of wild-type human-derived RANKL (ACMR-1000) (50 ng / mL). ACMR-159 shows TRAP-positive cell inhibitory effect as the treatment increases from 75 ng / mL to higher concentrations.

[0106] As a result, it is thought that the mouse-derived mutant RANKL ACMR-059 can inhibit osteoclastogenesis through suppression of TRAP-positive cells by wild-type RANKL, and the human-derived mutant RANKL ACMR-159 at the corresponding position is also thought to be able to inhibit osteoclastogenesis through suppression of TRAP-positive cells by wild-type RANKL.

[0107]

[0108] Example 2. Effect of MT RANKL on bone loss in mice

[0109] To investigate the effect of mutant RANKL on osteolysis, healthy mice were co-administered with wild-type (WT) RANKL and mutant RANKL, and then the femur bones were examined using micro-CT (Fig. 9). Mice treated with wild-type (WT) RANKL showed significant bone loss compared to the control (untreated) group, whereas those co-treated with WT RANKL and 001 or WT RANKL and 059 showed minimal bone loss. Bone mineral density (BMD) scores and bone volume percent (BV / TV) were assessed using quantitative micro-CT (Fig. 10). As expected, BV / TV and BMD scores were lower in WT RANKL-treated mice than in control mice, and increased in 001 or 059-treated mice. Notably, in 059-treated mice, BV / TV and BMD scores were restored to almost the levels of control mice, demonstrating the bone loss reversibility effect of mutant RANKL in the RANKL-induced bone loss model.

[0110] Taken together, these results demonstrate that mutant RANKL suppresses RANKL-induced osteolysis in a mouse model through the LGR4 signaling pathway, a compensatory mechanism of RANK, and thus may be a useful therapeutic agent for severe osteoporosis.

Claims

1. 1) At the N-terminus of the RANKL protein comprising the amino acid sequence of sequence number 1, Contains three 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 substitution 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 three 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 a substitution 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. In paragraph 1, The above RANKL protein mutant has K equivalent to wild type RANKL D A mutant RANKL protein that binds to LGR4.

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

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

9. A pharmaceutical composition for preventing or treating bone metabolic diseases, comprising the RANKL protein mutant of paragraph 1, the nucleic acid molecule of paragraph 7, or the vector of paragraph 8.

10. In paragraph 9, A pharmaceutical composition wherein the above bone metabolic disease is osteoporosis, bone formation disorder, or fracture.

11. In paragraph 9, The pharmaceutical composition above is a pharmaceutical composition that inhibits osteoclast differentiation by RANK.

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