Novel use of antibodies specifically binding to DKK-1

Abstract

In the present invention, DKK1 protein is specifically present on the surface of immune cells, particularly regulatory T cells, and thus can be used as a new target for therapeutic agents for various metabolic diseases, cranial nervous system diseases, and bone diseases. That is, DKK-1 antibodies, or antibodies or aptamers that specifically bind to a ligand of DKK-1 and consequently inhibit DKK-1, can prevent or treat metabolic diseases, cranial nervous system diseases, and bone diseases by regulating Wnt signaling.

Description

New uses of antibodies that specifically bind to DKK-1

[0001] The present invention relates to novel uses of antibodies or aptamers that specifically bind to the DKK-1 (Dickkopf-1) protein, specifically for the treatment of metabolic diseases, neurological diseases, and bone diseases.

[0002] Among immune cells, T cells are produced in the bone marrow and mature in the thymus; they function to regulate antibody production by B cells or to coordinate the functions of innate immune cells. These T cells, also known as T lymphocytes, not only act as helpers for other immune cells but also directly destroy invading substances.

[0003] One of the most important functions of immune cells is that while the immune response to antigenic substances constituting the self surrounding the immune cell is suppressed, the immune cell recognizes non-self antigenic substances and induces an immune response. During development, immune cells induce a non-response to self-antigens through the death of cells that recognize the self, the induction of mutations in receptors specific to self-antigens, or the inactivation of immune cells that recognize self-antigens; this is referred to as immunological unresponsiveness or tolerance. If this self-tolerance fails, an immune response to self-antigens is induced, which can lead to disease; this is known as an autoimmune disease.

[0004] In the early 1970s, the concept of regulatory T cells capable of controlling the effector function of conventional T cells was introduced to induce or maintain the aforementioned self-tolerance (RK Gershon and K. Kondo, Immunology, 1970, 18: 723-37), and research has been steadily conducted to elucidate the immunological characteristics and functions of suppressor T cells.

[0005] In normal individuals, the aforementioned regulatory T cells control the function of palliative T cells to induce excessive immune responses or self-tolerance; however, it has been reported that in autoimmune and chronic inflammatory diseases, the function and number of regulatory T cells are significantly reduced, preventing them from performing their functions properly. Therefore, restoring regulatory T cells to normal levels of function and number in patients with autoimmune and chronic inflammatory diseases can be one of the treatments for these conditions.

[0006] Cell surface proteins capable of targeting regulatory T cells, such as CD25, CTLA4, CD38, CD62L, GITR, and CD45RB, have been suggested through research and animal and clinical trials have been conducted, but no cell surface protein capable of targeting regulatory T cells alone has been identified to date.

[0007]

[0008] One objective of the present invention is to provide novel uses for antibodies that specifically bind to the DKK-1 protein, specifically for the prevention and treatment of obesity, neurological diseases, and bone diseases.

[0009] Another objective of the present invention is to provide a pharmaceutical composition or a method for the prevention or treatment of obesity, neurological diseases, and bone diseases, comprising antibodies that specifically bind to the DKK-1 protein as active ingredients.

[0010]

[0011] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0012] Various embodiments of the present invention are described with reference to the drawings. In the following description, for a complete understanding of the present invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to make the present invention unnecessary or obscure. Reference throughout this specification to one embodiment implies that a particular feature, form, composition, or characteristic described in association with the embodiment is included in one or more embodiments of the present invention. Accordingly, the circumstances of an embodiment expressed at various locations throughout this specification do not necessarily represent the same embodiment of the present invention. Additionally, a particular feature, form, composition, or characteristic may be combined in any suitable way in one or more embodiments.

[0013]

[0014] DKK-1, known as a representative negative regulator of the Wnt signaling pathway, ultimately induces the degradation of β-catenin and the inactivation of TCF-1 by competitively binding to LRP (Low-density lipoprotein receptor-related protein) 5 / 6 or through receptor endocytosis. DKK-1 is the most extensively studied protein among the four proteins in the DKK Family, consisting of 266 amino acids and measuring approximately 26 kDa. Structurally, it interacts with various receptor domains, including LRP6, through two cysteine-rich domains (CRDs). Currently known receptors for DKK-1 include LRP5 / 6, Kremen 1 / 2, and CKAP4, through which it induces various cellular signals. When examining the signaling involved in DDK-1, it inhibits signaling by binding to Co-Receptor LRP5 / 6 of Canonical Wnt Signaling, or conversely, activates signaling, thereby inducing PI3K / Akt Signaling or JNK signaling by LRP5 / 6 through binding with CKAP4 and LRP5 / 6.

[0015] Since DDK-1 is closely associated with cell proliferation through Wnt signaling and CKAP4-PI3K-AKT signaling, it is known that high expression of DDK-1 in cancer indicates a negative prognosis, although the degree varies depending on the type of cancer. Based on this, numerous studies on cancer immunotherapy targeting DDK-1 are being conducted. Furthermore, according to recent papers, the role of DDK-1 in various immune responses has been elucidated, and research results regarding the impact of DDK-1 on autoimmune diseases can be confirmed.

[0016] Furthermore, in vivo experiments confirmed that inflammatory responses caused by allergies and non-healing parasitic infections induce platelet activation, thereby increasing DDK-1 secretion from platelets and providing an environment in which DDK-1 can induce a Type 2 cell-mediated immune response through MAPK and mTOR signaling. Increased DDK-1 levels under physiological conditions generate Th2 cells through the induction of C-Maf and Gata3, and induce the expression of Th2 cell-associated cytokines through p38 MAPK and SGK-1. In other words, DDK-1 preferentially participates in Th2-related functions compared to other effector T cells. In vitro experiments showed that DDK-1 inhibits IFN-γ expression under Th1 polarization conditions while simultaneously increasing the expression of GATA3 and cytokines such as IL-4, IL-5, IL-10, and IL-13. In addition, it was shown that DDK-1 increases IL-10 expression in iTreg while simultaneously downregulating Foxp3 expression. In particular, platelets are known to be a major source of TGF-B1 production. It is hypothesized that in an inflammatory response environment generated by such infections, DDK-1, along with CXCL4, a ligand produced by platelets, lowers Foxp3 expression, thereby inhibiting the production of TGF-B-mediated Foxp3+ Treg cells and allowing Th2 cells to function properly.

[0017] nTreg cells exhibit high levels of DDK-1 mRNA expression compared to naive T cells or effector CD4 T cells. While DDK-1 is generally known to function by binding to receptors as a ligand in a soluble form, DDK-1 in nTreg cells is expressed on the surface; approximately 6% of DDK-1 is expressed in the resting state, and this expression rate increases to 14.1% upon activation. Functionally, DDK-1 is presumed to play a crucial role in the suppressive function of Tregs. During an in vitro suppression assay, treatment with a monoclonal antibody against DDK-1 failed to inhibit the proliferation of effector CD4 T cells. In addition, when colitis was induced in Rag2-deficient mice to examine the inhibitory effect on Tregs, it was found that Tregs in mice with DDK-1 expression reduced by more than 90% did not inhibit the proliferation of effector CD4 T cells as effectively as Wild-Type Tregs, ultimately leading to the development of severe colitis. Finally, an examination of the DDK-1 expression pathway within Tregs revealed that it is expressed independently of the Wnt pathway, mTOR, and Mevalonate pathway, and that the MAPK pathway is required for activated Tregs to express DDK-1.

[0018] Wnt signaling acts as an essential factor in cell proliferation and the formation of new bone in osteoblasts. Therefore, DKK1, which acts as a Wnt antagonist, indicates the possibility of erosion due to increased bone resorption when its concentration in the blood is measured at high levels, whereas, conversely, low concentrations are considered to indicate the formation of new bone. Based on this, an examination of the relationship between DKK1 and the prognosis of rheumatoid arthritis (RA) revealed that blood DKK1 levels increased along with increased bone resorption as the duration of the disease lengthened. Similarly, in Systemic Lupus Erythematosus (SLE), another autoimmune disease, it was confirmed that DKK1 has the potential to serve as a positive marker for diagnosing Lupus Nephritis through its concentration in the serum. As such, clinical results involving patients have confirmed that DDK-1 can act as a marker to determine the presence or absence of various autoimmune diseases, but further research is needed on the specific evidence regarding the mechanism by which this conclusion is reached.

[0019] Meanwhile, DKK-1 (Dickkopf-1) is known as a representative negative regulator of the Wnt / β-catenin signaling pathway, inhibiting cell proliferation, differentiation, survival, and tissue regeneration by binding to LRP5 / 6 receptors and blocking interactions with Wnt ligands. Therefore, anti-DKK-1 antibodies that neutralize the function of DKK-1 can restore excessively suppressed Wnt signaling, thereby exerting therapeutic effects in various degenerative and chronic diseases.

[0020] First, in the case of bone diseases, DKK-1 contributes to a decrease in bone density by inhibiting the differentiation and activity of osteoblasts and inducing osteoclast-dominant bone remodeling. Anti-DKK-1 antibodies block the binding of DKK-1 to LRP5 / 6, thereby restoring Wnt / β-catenin signaling and promoting osteoblast differentiation and bone matrix formation, resulting in increased bone mass and improved bone strength.

[0021] In relation to hair loss, the activation of hair follicle stem cells and the maintenance of the hair growth phase (anagen) are dependent on Wnt signaling, and the overexpression of DKK-1 induces hair follicle catagen and inhibits hair follicle stem cell function. Anti-DKK-1 antibodies can neutralize these inhibitory signals to promote the proliferation and differentiation of hair follicle stem cells and exert an effect on improving hair loss by extending the hair growth phase.

[0022] In pigment disorders, the survival, migration, and melanin synthesis of melanocytes are regulated by Wnt signaling. Increased expression of DKK-1 can induce melanocyte dysfunction and localized pigment loss or imbalance, and anti-DKK-1 antibodies contribute to normalizing melanocyte activity and restoring the balance of pigmentation by restoring Wnt signaling.

[0023] During the wound healing process, DKK-1 can cause delayed healing by inhibiting the proliferation and migration of keratinocytes and fibroblasts. Anti-DKK-1 antibodies improve the speed of wound healing and the quality of tissue regeneration by promoting epithelial cell proliferation, angiogenesis, and collagen synthesis through Wnt signaling activation.

[0024] In neurodegenerative diseases, DKK-1 has been reported to be associated with synaptic loss, neuronal death, and reduced neuroplasticity. Anti-DKK-1 antibodies can delay the progression of neurodegeneration or induce functional recovery by restoring Wnt / β-catenin signaling, thereby promoting neuronal survival and protecting synaptic function.

[0025] Finally, in the case of COPD (Chronic Obstructive Pulmonary Disease), DKK-1 is known to be involved in inhibiting lung epithelial cell regeneration and exacerbating the inflammatory microenvironment. Anti-DKK-1 antibodies can promote the regeneration of damaged lung epithelium and activate Wnt signaling-mediated tissue repair processes, thereby preserving lung structure and mitigating functional decline.

[0026] In summary, anti-DKK-1 antibodies selectively restore Wnt / β-catenin signaling excessively suppressed by DKK-1, thereby promoting regenerative and protective mechanisms in bone, skin, hair follicles, nerves, and lung tissues; thus, they possess therapeutic utility in various diseases such as bone disorders, hair loss, pigmentation abnormalities, delayed wound healing, neurodegenerative diseases, and COPD.

[0027] DKK-1 is a secreted glycoprotein known to bind to LRP6, CKAP4, and TLR-4, respectively, inducing different pathological signaling pathways, and these receptor-mediated signals are involved in the development and progression of various degenerative, inflammatory, and proliferative diseases. Therefore, anti-DKK-1 antibodies that inhibit the binding between DKK-1 and the aforementioned receptors (LRP6, CKAP4, TLR-4) can exert disease-specific therapeutic effects by selectively inhibiting pathological ligand-receptor interactions without directly blocking the receptors themselves.

[0028] First, LRP6 serves as a key co-receptor in Wnt / β-catenin signaling, playing an essential role in osteogenic formation, tissue regeneration, stem cell maintenance, and neuronal survival. However, when DKK-1 binds to LRP6, access to Wnt ligands is blocked, leading to inhibition of osteoblast differentiation, hair follicle degeneration, reduced epithelial regeneration, and decreased neuroplasticity. These mechanisms are commonly observed in conditions such as bone diseases, delayed fracture healing, hair loss, skin aging and impaired wound healing, osteoarthritis, and neurodegenerative diseases. Anti-DKK-1 antibodies can fundamentally improve the pathophysiology of these diseases by inhibiting the binding of DKK-1 to LRP6, thereby restoring the suppressed Wnt / β-catenin signaling. Consequently, this leads to increased osteoblast activity, promotion of tissue regeneration, activation of hair follicle stem cells, and enhanced neuronal survival signals.

[0029] Meanwhile, CKAP4 is reported to act as a functional receptor for DKK-1 to activate the PI3K / AKT signaling pathway, and the DKK-1–CKAP4 axis promotes cell proliferation, survival, migration, and invasion, particularly in tumor cells and fibrosis-related cells. These signals are involved in the growth and metastasis of various solid tumors, such as lung cancer, pancreatic cancer, and liver cancer, as well as the progression of chronic fibrotic diseases such as pulmonary fibrosis and hepatic fibrosis. Anti-DKK-1 antibodies can alleviate the progression of tumors and fibrotic diseases by blocking DKK-1 from binding to CKAP4, thereby suppressing PI3K / AKT-mediated tumor-promoting signals, reducing the proliferation and viability of cancer cells, and simultaneously inhibiting the excessive activation of fibroblasts and the accumulation of pathological stroma.

[0030] Furthermore, TLR-4 serves as a major receptor for innate immune responses, mediating the secretion of inflammatory cytokines and the activation of NF-κB signaling. When DKK-1 binds to TLR-4, the inflammatory response is amplified, leading to chronic tissue damage and disease exacerbation. This DKK-1–TLR-4 axis is known to play a pathological role in chronic inflammatory diseases such as rheumatoid arthritis, COPD and chronic lung inflammation, sepsis, and neuroinflammatory diseases. Anti-DKK-1 antibodies inhibit the binding of DKK-1 to TLR-4, thereby reducing excessive innate immune activation and inflammatory cytokine secretion. By blocking the vicious cycle of tissue damage and chronic inflammation, they preserve the function of joint, lung, and nervous tissues and provide effects that inhibit disease progression.

[0031] Consequently, anti-DKK-1 antibodies can simultaneously block or normalize regeneration-inhibiting signals, tumor-fibrosis-promoting signals, and inflammation-amplifying signals induced by DKK-1 interacting with LRP6, CKAP4, and TLR-4, respectively, and thereby function as a comprehensive and fundamental therapeutic strategy acting at a higher level of pathophysiology across bone, skin, hair follicle, nerve, lung, and tumor-related diseases.

[0032] As described in this specification, the overexpression of DKK-1 (Dickkopf-1) mediates the inhibition of Wnt / β-catenin signaling, thereby inducing pathological mechanisms such as inhibition of bone formation, inhibition of hair follicle stem cell function, delay in skin and mucosal regeneration, reduced neuronal survival, inflammatory response, and promotion of fibrosis; thus, DKK-1 acts as an important pathogenic factor in various degenerative and chronic diseases. The composition and use of the present invention relate to a combination therapy that maximizes complementary therapeutic effects by providing a DKK-1 antibody in a form with appropriately adjusted dosage and administration route, and simultaneously administering it in combination with standard therapeutic agents currently used in clinical practice for individual diseases.

[0033] In the case of fibrosis, tissue fibrosis, such as pulmonary fibrosis and hepatic fibrosis, is characterized by the excessive activation of fibroblasts by TGF-β, PDGF, and other fibrosis-mediating factors, and the accumulation of the Extracellular Matrix (ECM). Current fibrosis treatments, pirfenidone and nintedanib, suppress inflammatory and fibrosis signals, but have limited effects on the pathological activity of fibroblasts and signals inhibiting regeneration. DKK-1 antibodies inhibit the progression of fibrosis by blocking the DKK-1–CKAP4 / TLR-4 axis in fibroblasts, thereby suppressing the excessive activation of the PI3K / AKT and NF-κB pathways. Therefore, the present invention provides that by co-administering a DKK-1 antibody with pirfenidone or nintedanib, the progression of fibrosis can be more effectively inhibited by suppressing DKK-1-mediated signals that are not targeted by the standard treatments and by simultaneously controlling multiple pathological mechanisms of inflammation and fibrosis.

[0034] In the treatment of bone diseases, bisphosphonates, RANKL inhibitors (e.g., denosumab), SERMs (selective estrogen receptor modulators), and PTH analogs (e.g., teriparatide) are currently used to delay or restore bone loss by inhibiting bone resorption or promoting bone formation. However, existing therapies have limitations in the effective regeneration of osteoblasts and the maintenance of long-term bone formation. DKK-1 antibodies directly promote osteoblast differentiation by restoring LRP6-mediated Wnt / β-catenin signaling. Therefore, the present invention enhances osteoblast activity and promotes bone formation by combining DKK-1 antibodies with bisphosphonates or denosumab, and improves the effect of increasing bone density by restoring the balance of bone remodeling.

[0035] In the treatment of hair loss, minoxidil or finasteride promotes hair growth by increasing blood flow around hair follicles or inhibiting DHT. However, there are limitations in fundamentally restoring the activity of hair follicle stem cells. The DKK-1 antibody induces the maintenance of the anagen phase and the promotion of hair growth by releasing the inhibition of Wnt signaling in hair follicle stem cells. Therefore, the present invention provides that by using the DKK-1 antibody in combination with minoxidil or finasteride, the restoration of hair follicle activity and hair growth can be improved more fundamentally and sustainably.

[0036] Regarding pigment disorders, existing treatments for vitiligo or other pigment deficiency diseases (e.g., corticosteroids, calcineurin inhibitors) alleviate melanocyte damage through local inflammation suppression and immune modulation. However, there are limitations in restoring melanocyte function and promoting melanin synthesis. The DKK-1 antibody improves melanocyte survival and melanin-producing ability by restoring Wnt / β-catenin signaling. The present invention provides that the DKK-1 antibody can promote the restoration of pigment cell function and the re-establishment of the balance of pigmentation by combining it with a corticosteroid or calcineurin inhibitor.

[0037] In the case of wound healing, growth factor preparations or regenerative dressings are currently used, focusing primarily on suppressing inflammation and promoting epithelial regeneration. However, inhibition of DKK-1-related Wnt signaling hinders the migration and proliferation of epithelial cells and fibroblasts, leading to delayed healing. DKK-1 antibodies promote epithelial regeneration and angiogenesis by restoring Wnt signaling. Therefore, the present invention provides that the rate of epithelial regeneration and tissue structure restoration can be more effectively enhanced by using DKK-1 antibodies in combination with existing wound healing promoters.

[0038] In neurodegenerative diseases, cholinesterase inhibitors (e.g., donepezil) and NMDA receptor antagonists (e.g., memantine) are primarily used for symptom relief but have limited effectiveness in restoring neuronal survival signals. DKK-1 antibodies activate Wnt / β-catenin signaling in neurons to promote synapse maintenance and cell survival. The present invention provides that by using DKK-1 antibodies in combination with a cholinesterase inhibitor or an NMDA receptor antagonist, it is possible to fundamentally regulate neurodegenerative pathways in addition to symptom relief.

[0039] In COPD (Chronic Obstructive Pulmonary Disease), corticosteroids, β2 agonists, and PDE4 inhibitors are used to suppress inflammation and dilate airways. However, they are limited in their ability to regenerate lung tissue and suppress chronic exacerbations of inflammation. DKK-1 antibodies contribute to the preservation of lung structure and improvement of function by restoring Wnt signaling in lung epithelial cells and reducing DKK-1–TLR-4 mediated inflammatory responses. The present invention provides that inflammation suppression and tissue regeneration can be simultaneously achieved by using DKK-1 antibodies in combination with corticosteroids or PDE4 inhibitors.

[0040] Antibodies that specifically bind to DKK-1 can be usefully employed as therapeutic agents for the treatment and / or prevention of obesity. Dickkopf-1 (DKK-1) is a representative antagonist of the Wnt / β-catenin signaling pathway and plays a crucial role in regulating adipogenesis, energy metabolism, and adipose tissue homeostasis. It is known that increased expression of DKK-1 promotes the differentiation of preadipocytes into mature adipocytes, leading to increased lipid accumulation. Therefore, by inhibiting or neutralizing the activity of DKK-1 through antibodies that specifically bind to it, it is possible to suppress adipogenesis, reduce lipid accumulation, and regulate metabolic processes.

[0041] In some embodiments, by administering the antibody to an individual in need, improvements in metabolic indicators may occur, such as suppression of body weight gain, reduction of body fat mass, and improvement of insulin sensitivity and glucose tolerance. Explained without being bound by a specific theory, the antibody can be understood to restore the Wnt / β-catenin signaling pathway by blocking the binding between DKK-1 and its receptor (e.g., LRP5 / 6), thereby suppressing adipocyte differentiation and promoting energy consumption.

[0042] In addition, in some embodiments, the DKK-1 antibody may be administered alone or in combination with other treatments for obesity or metabolic diseases. The antibody may be prepared in formulations suitable for various systemic administration routes, including intravenous injection, subcutaneous injection, or intramuscular injection. Thus, an antibody that specifically binds to DKK-1 provides a novel and effective therapeutic means for the treatment of obesity and related metabolic diseases.

[0043] The composition and combination therapy according to this specification describe that by combining a standard therapeutic agent used for each disease with a DKK-1 antibody in an appropriate dose ratio, they exhibit significantly enhanced tissue regeneration, inflammation suppression, and pathophysiological modulation effects compared to monotherapy. The present invention provides a novel therapeutic approach that improves the therapeutic effect for each disease and enhances long-term prognosis through the said combination therapy.

[0044] It is known that the DKK-1 protein is highly expressed in human regulatory T cells, while its expression is reduced in iTregs.

[0045] Furthermore, DKK-1 is an anchored protein located outside the human Treg cell membrane, which can then be released into the blood and exist in a soluble state.

[0046] In the present invention, DKK-1 is one of the DKK family genes DKK1, DKK2, DKK3, and DKK4, and encodes a secreted protein. The DKK protein generally consists of 255 to 350 amino acids, and in the case of DKK-1, it has a size of 24 KDa to 29 KDa, and the N-terminus exists in a glycosylated form. As an example of the present invention, DKK-1 may be represented by the amino acid sequence of SEQ ID NO. 53 and may be coded by a nucleotide represented by SEQ ID NO. 54 (Table 1).

[0047] Classification Sequence Information dickkopf-1 (DKK-1) Amino Acid Sequence MMALAGAAGAT RVFVAMVAAA LGGHPLLGVS ATLNSVLNSN AIKNLPPPLG GAAGHPGSAV SAAPGILYPG GNKYQTIDNY QPYPCAEDEE CGTDEYCASP TRGGDAGVQI CLACRKRRKR CMRHAMCCPG NYCKNGICVS SDQNHFRGEI EETITESFGN DHSTLDGYSR RTTLSSKMYH TKGQEGSVCL RSSDCASGLC CARHFWSKIC KPVLKEGQVC TKHRRKGSHG LEIFQRCYCG EGLSCRIQKD HHQASNSSRL HTCQRH (Sequence No. 53) dickkopf-1 (DKK-1) mRNA, complete cds (GenBank: AF177394.2). ctctgcagtc agcgccgcgc cgggaatcct gtacccgggc gggaataagt accagaccat tgacaactac cagccgtacc cgtgcgcaga ggacgaggag tgcggcactg atgagtactg cgctagtccc acccgcggag gggacgcagg cgtgcgct ggtcagcgc ccgaaaacgc tgcatgcgtc acgctatgtg ctgccccggg aattactgca aaaatggaat atgtgtgtct tctgatcaaa atcatttccg aggagaaatt gaggaaacca tcactgaaag ctttggtaat gatcatagca ccttggatgg gtattccaga agaaccacct tcactcac aagaaggttc tgtttgtctc cggtcatcag actgtgcctc aggattgtgt tgtgctagac acttctggtc caagatctgt aaacctgtcc tgaaagaagg tcaagtgtgt accaagcata ggagaaaagg ctctcatgga ctagaaatat tccagcgttg ttagctctc caccatcaag ccagtaattc ttctaggctt cacacttgtc agagacacta a(서열번호 54).

[0048] In the present invention, antibodies that specifically bind to DKK-1 bind more strongly to receptors than ligands such as Wnt3a in the Wnt signaling pathway involved in cell proliferation and wound healing, thereby competitively inhibiting Wnt signaling and playing an important role in the development of the heart, head, hands, etc., during embryonic development. However, in the present invention, DKK-1 is not secreted outside the cell as in general cases, but may exist on the surface of immune cells, particularly regulatory T cells. Through this, immune cells can assist the function of regulatory T cells for self-tolerance.

[0049] According to one embodiment of the present invention, the regulatory T cell extracellular surface protein of DKK-1 is denoted by SEQ ID NO. 55 (Table 2).

[0050] Category Sequence InformationDKK1 extracellular domainTLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYCKNGICVSSDQNHFRG EIEETITESFGNDHSTLDGYSRRTTLSSKMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH (SEQ ID NO: 55)

[0051]

[0052] The antibodies provided in the present invention that specifically bind to DKK-1 can interact with ligands present in effector T cells to reduce the activity of regulatory T cells.

[0053] In the present invention, the term “binding molecule” means an intact immunoglobulin comprising a monoclonal antibody, such as a chimeric, humanized, or human monoclonal antibody, or a variable domain comprising an immunoglobulin fragment that competes with the intact immunoglobulin for binding to an antigen, for example, monomeric HA or trimeric HA of the influenza A virus. Regardless of structure, the antigen-binding fragment binds to the same antigen recognized by the intact immunoglobulin. The antigen-binding fragment may comprise a peptide or polypeptide comprising an amino acid sequence of two or more consecutive amino acid residues, 20 or more consecutive amino acid residues, 25 or more consecutive amino acid residues, 30 or more consecutive amino acid residues, 35 or more consecutive amino acid residues, 40 or more consecutive amino acid residues, 50 or more consecutive amino acid residues, 60 or more consecutive amino acid residues, 70 or more consecutive amino acid residues, 80 or more consecutive amino acid residues, 90 or more consecutive amino acid residues, 100 or more consecutive amino acid residues, 125 or more consecutive amino acid residues, 150 or more consecutive amino acid residues, 175 or more consecutive amino acid residues, 200 or more consecutive amino acid residues, or 250 or more consecutive amino acid residues.

[0054] In the present invention, the term “antigen-binding fragment” includes, in particular, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragment, single-strand antibody (scFv), bivalent single-strand antibody, single-strand phage antibody, diabody, triabody, tetrabody, polypeptide containing one or more fragments of immunoglobulin sufficient to bind to a specific antigen. The fragment may be produced synthetically, by enzymatic or chemical degradation of complete immunoglobulin, or genetically engineered using recombinant DNA technology. Methods of production are well known in the art.

[0055] The CDRs of the antibodies provided in this invention possess common conserved sequences (motifs). These common conserved sequences are presumed to be strongly structurally conserved CDRs forming the paratope backbone, and are highly likely to be direct antigen contact residues. In the sequence list of this invention, excluding the aforementioned conserved sequences, the remaining parts are indicated by X to specify that they are subject to change.

[0056] In the present invention, the binding molecule may further include an Fc region (Fragment crystallization region) or a constant region. In this case, the Fc region may be the Fc region of an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody, or may be derived therefrom, or may be a hybrid Fc region.

[0057] In the present invention, the Fc region may be the Fc region of a mammalian-derived IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody, and preferably may be the Fc region of a human-derived IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody, but is not limited thereto.

[0058] As an example of the present invention, the Fc region may be a human-derived immunoglobulin lambda constant region, but is not limited thereto.

[0059] In the present invention, the "hybrid Fc" can be derived from a combination of human IgG subclasses or a combination of human IgD and IgG. When the hybrid Fc binds to a biologically active molecule, polypeptide, etc., it not only increases the serum half-life of the biologically active molecule but also has the effect of increasing the expression level of the polypeptide when a nucleotide encoding an Fc-polypeptide fusion protein is expressed.

[0060] In the bonding molecule of the present invention, the Fc or invariant region may be connected to the variable region by a linker. In this case, the linker is connected to the C-terminus of the Fc or invariant region, and the N-terminus of the bonding molecule of the present invention may be connected to the linker, but is not limited thereto.

[0061] In the present invention, the "linker" may include a sequence that can be cleaved by an enzyme that is overexpressed within the tissue or cell of the target disease. In the case where it can be cleaved by an overexpressed enzyme as described above, the reduction of polypeptide activity due to Fc or the constant region can be effectively prevented. In the present invention, a preferred example of the linker may be a peptide linker composed of 33 amino acids located at positions 282 to 314 of human albumin, which is most abundant in blood, and more preferably a peptide linker composed of 13 amino acids located at positions 292 to 304. These parts are mostly exposed to the outside in terms of the three-dimensional structure and are parts where the possibility of inducing an immune response in the body is minimized. However, the invention is not limited thereto.

[0062] The binding molecule of the present invention is characterized as being an antibody or an antigen-binding fragment thereof, but is not limited thereto. The antibody includes all of the following: a monoclonal antibody, a full-length antibody, or an antibody fragment having the ability to bind to the Dkk-1 protein as a part of an antibody and capable of binding to the Dkk-1 antigenic determinant site competitively with the binding molecule of the present invention.

[0063] In the present invention, the "antibody" refers to a protein molecule that acts as a receptor specifically recognizing an antigen, comprising an immunoglobulin molecule having immunological reactivity with a specific antigen. For the purposes of the present invention, the antigen may be the Dkk-1 protein present on the surface of a regulatory T cell.

[0064] In the present invention, the "immunoglobulin" has a heavy chain and a light chain, and each heavy chain and light chain includes an invariant region and a variable region. The variable regions of the light chain and heavy chain include three variable regions and four framework regions called complementarity determining regions (hereinafter referred to as "CDRs"). The CDRs primarily serve to bind to epitopes of antigens. The CDRs of each chain are typically designated sequentially as CDR1, CDR2, and CDR3 starting from the N-terminus, and are also identified by the chain in which a specific CDR is located.

[0065] In addition, in the present invention, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same group of antibodies, which exhibits single-binding specificity and affinity for a specific epitope.

[0066] In the present invention, the "full-length antibody" has a structure having two full-length light chains and two full-length heavy chains, each light chain being connected to the heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes.

[0067] Furthermore, in the present invention, the "antibody fragment" refers to a fragment possessing an antigen-binding function and includes Fab, Fab', F(ab')2, and Fv, etc. Fab has a structure having variable regions of the light and heavy chains, a constant region of the light chain, and a first constant region of the heavy chain (CH1 domain), and has one antigen-binding site. Additionally, Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv (Variable fragment) refers to a minimal antibody fragment possessing only a heavy chain variable region and a light chain variable region. In double-stranded Fv (dsFv), the heavy chain variable region and the light chain variable region are connected by a disulfide bond, and in single-stranded Fv (scFv), the heavy chain variable region and the light chain variable region are generally connected by a covalent bond through a peptide linker. When using a proteolytic enzyme, such as papain or pepsin, the antibody fragment can be a Fab or F(ab')2 fragment, and can be produced through genetic recombination technology.

[0068] In addition, the antibody in the present invention may be a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimic, a unibody, a diabody, a triabody, a tetrabody, or a fragment thereof, but is not limited thereto.

[0069] In the present invention, the "chimeric antibody" is an antibody formed by recombining the variable region of a mouse antibody and the constant region of a human antibody, and is an antibody in which the immune response is significantly improved compared to the mouse antibody.

[0070] In addition, in the present invention, the "humanized antibody" refers to an antibody in which the protein sequence of an antibody derived from a non-human species is modified to be similar to a naturally produced antibody variant in humans. For example, the humanized antibody can be produced by recombining a mouse-derived CDR with a human antibody-derived FR to produce a humanized variable region, and then recombining this with a preferred human antibody constant region to produce the humanized antibody.

[0071] In the present invention, the binding molecule may also be provided as a bispecific antibody or a bispecific antigen binding fragment that can bind to the Dkk-1 protein and also bind to other proteins (e.g., Lrig-1 protein).

[0072] In the present invention, the bispecific antibody and the bispecific antigen-binding fragment may comprise a binding molecule according to the present invention. In one example of the present invention, the bispecific antibody and the bispecific antigen-binding fragment comprise an antigen-binding domain capable of binding to a Dkk-1 protein, wherein the antigen-binding domain capable of binding to the Dkk-1 protein may comprise or be composed of a binding molecule according to the present invention.

[0073] The bispecific antibody and bispecific antigen-binding fragment provided in the present invention comprise an antigen-binding domain, which is a binding molecule capable of binding to the Dkk-1 protein according to the present invention, and an antigen-binding domain capable of binding to another target protein. Here, the antigen-binding domain capable of binding to another target protein may be a protein other than the Dkk-1 protein, and, although not limited thereto, may be, for example, an antigen-binding domain capable of binding to Lrig-1 or PD-1 or a cell surface receptor.

[0074] The bispecific antibody and bispecific antigen binding fragment according to the present invention may be provided in any suitable format, for example, in the format described in the literature in which the full text is cited herein by reference. For example, bispecific antibodies or bispecific antigen-binding fragments include bispecific antibody conjugates (e.g., IgG2, F(ab')2, or CovX-bodies), bispecific IgG or IgG-type molecules (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, or 2-in 1-IgG, mAb2, or Tandemab common LC), asymmetric bispecific IgG or IgG-type molecules (e.g., kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pairs, or SEED-bodies), and small bispecific antibody molecules (e.g., diabody (Db), dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAb / VHH, triple It may be a body, triple head, Fab-scFv, or F(ab')2-scFv2), a bispecific Fc and CH3 fusion protein (e.g., taFv-Fc, di-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or a bispecific fusion protein (e.g., scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). Those skilled in the art can design and manufacture a bispecific antibody and a bispecific antigen-binding fragment according to the present invention.

[0075] The method for producing the bispecific antibody in the present invention comprises chemically crosslinking an antibody or antibody fragment with a reducing disulfide or non-reducing thioether bond. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) may be used to chemically crosslink a Fab fragment, for example, through a hinge region SH- group, to produce a disulfide-linked bispecific F(ab)2 heterodimer.

[0076] In addition, another method for producing the bispecific antibody in the present invention comprises fusing an antibody-producing hybridoma, for example, with polyethylene glycol to produce quadroma cells capable of secreting the bispecific antibody.

[0077] The bispecific antibody and bispecific antigen-binding fragment according to the present invention can be produced by recombination, for example, by expression from a nucleic acid construct encoding a polypeptide for an antigen-binding molecule.

[0078] For example, a DNA construct comprising a sequence encoding light chain and heavy chain variable domains for two antigen-binding domains (i.e., a light chain and heavy chain variable domain for an antigen-binding domain capable of binding to PD-1, etc., and a light chain and heavy chain variable domain for an antigen-binding domain capable of binding to other target proteins) and encoding a suitable linker or dimerization domain between the antigen-binding domains can be produced by molecular cloning technology. The recombinant bispecific antibody can then be produced by expression of the construct (e.g., in vitro) in a suitable host cell (e.g., mammalian host cell), and the expressed recombinant bispecific antibody can then be optionally purified.

[0079] Antibodies can be produced by an affinity maturation process in which modified antibodies are produced in which the affinity of the antibody for the antigen is improved compared to unmodified parent antibodies. Affinity-matured antibodies can be produced by procedures known in the art.

[0080] In addition, the binding molecule provided in the present invention may include variants of the amino acid sequence as long as they can specifically bind to the Dkk-1 protein. For example, changes may be made to the amino acid sequence of the antibody to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody.

[0081] These amino acid variations are based on the relative similarities of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0082] In introducing mutations, the hydropathic index of the amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The hydrophobic amino acid index is very important in conferring interactive biological functions of proteins. It is a known fact that similar biological activity can be achieved by substituting with amino acids having similar hydrophobic indices. When introducing a variation based on the hydrophobic index, the substitution is preferably made between amino acids exhibiting a difference in hydrophobic index within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0083] Meanwhile, it is also well known that substitution between amino acids having similar hydrophilicity values ​​results in proteins having uniform biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspalate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); tryptophan (-3.4). When introducing a variation by referring to the hydrophilicity value, substitution can be performed between amino acids that exhibit a difference in hydrophilicity value within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0084] Amino acid exchanges in proteins that do not alter the overall activity of the molecule are known in the art. The most common exchanges are those between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, and Gln / Glu.

[0085] Considering the variant having the aforementioned biological equivalent activity, the binding molecule of the present invention is interpreted to include a sequence that exhibits substantial identity with the sequence listed in the sequence list.

[0086] In the present invention, the term "substantial identity" refers to a sequence in which, when the sequence of the present invention is paralleled with any other sequence to correspond as much as possible and the parallel sequence is analyzed using an algorithm commonly used in the art, it exhibits at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are accessible from the NCBI Basic Local Alignment Search Tool (BLAST), NBCI (National Center for Biological Information), etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the internet. BLAST is accessible at this address (ncbi.nlm.nih.gov / BLAST / ). Methods for comparing sequence homology using this program can be found online (ncbi.nlm.nih.gov / BLAST / blast_help.html).

[0087] In the present invention, the binding molecule, preferably the antibody, can be produced by a conventional method of producing antibodies, but can also be produced by affinity maturation.

[0088] In the present invention, "affinity maturation" refers to the process in which activated B cells produce antibodies with increased affinity for an antigen during an immune response, that is, antibodies with enhanced binding ability to the antigen. For the purposes of the present invention, the affinity maturation can produce antibodies or antibody fragments generated by affinity maturation based on the principles of mutation and selection, just as processes occur in nature.

[0089] Generally, the effect of an antibody or antigen-binding fragment is determined by which antigen the antibody or antigen-binding fragment binds to. Therefore, it is already widely known in the relevant technical field that an antibody having a superior binding affinity for the same antigen compared to an antibody with a known pharmacological effect will have the same pharmacological effect, and will have an effect equal to or superior to that of the previously known antibody.

[0090] According to another embodiment of the present invention, an antibody-drug conjugate (ADC) comprising an antibody and a drug provided in the present invention is provided.

[0091] In the present invention, the term "Antibody-Drug Conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked without reducing the biological activity of the antibody and the drug. In the present invention, the antibody-drug conjugate refers to a form in which a drug is bound to an amino acid residue at the N-terminus of the heavy chain and / or light chain of an antibody, specifically, a form in which a drug is bound to an α-amine group at the N-terminus of the heavy chain and / or light chain of an antibody.

[0092] In the present invention, the term "drug" may refer to any substance having specific biological activity in cells, and this concept includes DNA, RNA, or peptides. The drug may be in a form containing a reactive group capable of reacting with an α-amine group to form a crosslink, and may also be in a form in which a linker containing a reactive group capable of reacting with an α-amine group to form a crosslink is connected.

[0093] Examples of reaction groups capable of reacting with the α-amine group to crosslink in the present invention include, as long as they can react with the α-amine group at the N-terminus of the heavy or light chain of an antibody to crosslink, the type thereof is not particularly limited and includes all types known in the art that react with amine groups. Examples may be any one of isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester, but are not limited thereto.

[0094] In the present invention, the drug may be included regardless of its type, as long as it is a drug capable of treating metabolic diseases, neurological diseases, or bone diseases.

[0095] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, and bone diseases is provided, comprising a chimeric antigen receptor (CAR) as an active ingredient, the chimeric antigen receptor comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain.

[0096] In the present invention, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor comprising an extracellular antigen binding domain and an intracellular signaling domain. While the most common type of CAR includes a short-chain variable fragment (scFv) derived from a monoclonal antibody fused to a transmembrane and intracellular domain of a T cell co-receptor, such as the CD3 zeta (ζ) chain, the present invention as described herein is not limited to these domains. Rather, "chimeric antigen receptor" or "CAR" as used herein refers to any receptor engineered to express any intracellular signaling molecule and an extracellular antigen binding domain fused to or linked thereto. In the present invention, the binding domain may comprise a short-chain variable fragment (scFv) capable of specifically recognizing the Lrig-1 protein. In the present invention, "short-chain variable fragment" or "scFv" refers to a fusion protein of the variable heavy chain (VH) and variable light chain (VL) of an antibody formed by a peptide linker between VL and VH.

[0097] In addition, in the present invention, the VH domain and the VL domain may be connected through a flexible linker. In the present invention, the flexible linker may be a glycine / serine linker of about 10 to 30 amino acids (e.g., 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids), and preferably may be 15 amino acid lengths. In the present invention, the linker length can act as an important determining site for the chimeric antigen receptor, so a linker shorter than the above range can increase affinity but can also impair CAR expression by causing intracellular multimer formation, whereas a linker longer than the above range can decrease antigen affinity by moving VL and VH CDR further in space.

[0098] The chimeric antigen receptor of the present invention may further include at least one of a hinge region (or spacer) and a signal transduction domain. In the present invention, the hinge region is a part connecting the antigen binding domain and the transmembrane domain, also called a 'spacer,' and is intended to extend the antigen binding domain from the T cell membrane or NK cell membrane. In the present invention, the hinge region may be obtained from any suitable sequence from any genus including, for example, human or part thereof, or may include, but is not limited to, a hinge region of a human protein including, but not limited to, CD8, CD28, 4-1BB, OX40, all or part of the CD3 zeta (ζ) chain, T cell receptor α or β chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof. Additionally, in the present invention, the hinge region may include one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD) without being limited to, but is not limited to.

[0099] In the present invention, the signaling domain refers to a portion of a chimeric antigen receptor that is found or engineered to be found inside a T cell. In the present invention, the signaling domain may or may not include a transmembrane domain that serves to anchor the chimeric antigen receptor to the plasma membrane of the T cell. In the present invention, the transmembrane domain and the signaling domain may be derived from the same protein (e.g., CD3 zeta(ζ) molecule), or the transmembrane domain and the signaling domain may be derived from different proteins (e.g., the transmembrane domain of CD28 and the intracellular signaling domain of the CD3 zeta(ζ) molecule, or vice versa).

[0100] In the present invention, the transmembrane domain may include, for example, a T cell receptor α or β chain, all or part of a CD3 zeta (ζ) chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof, but is not limited thereto. In the present invention, the co-stimulatory domain is 4-1BB (CD137); OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; Natural Killer Group 2 member C (NKG2C); Natural Killer Group 2 member (NKG2D); B7-H3; CD83; ICAM-1; It may include, but is not limited to, a functional signaling domain derived from a polypeptide comprising a ligand that binds to LFA-1 (CD11a / CD18) or ICOS; an active fragment thereof; a functional derivative thereof; or a combination thereof.

[0101] In the present invention, the signal transduction domain may include, but is not limited to, a functional signal transduction domain derived from a polypeptide comprising all or part of CD3 zeta (ζ), common FcR gamma (FcER1G), Fc gamma RIIIa, Fc R beta (Fc epsilon rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX-activated protein 10 (DAP10), DNAX-activated protein 12 (DAP12), an active fragment thereof, a functional derivative thereof, or a combination thereof, and such signal transduction domains are known in the art.

[0102] According to another embodiment of the present invention, the invention relates to a composition for the prevention, improvement, or treatment of metabolic diseases, neurological diseases, or bone diseases, comprising as an active ingredient a binding molecule of the present invention; a nucleic acid molecule; an expression vector; a host cell line; or an antibody-drug conjugate (ADC).

[0103] In the present invention, the term "epitope" refers to a region of an antigen molecule that binds to an antibody, meaning a portion that the antibody can recognize. Generally, antibodies do not recognize the entire antigen molecule but only specific parts; furthermore, even for the same antigen molecule, different types of antibodies may recognize different epitope regions. For the purposes of the present invention, the epitope comprises an antibody epitope capable of effectively binding to a remaining portion of the domain even when a portion of the DKK1 protein exposed outside the cell is partially cleaved and released outside the cell (secretion).

[0104] In the present invention, the polypeptide serving as the antigenic determinant may include both continuous and discontinuous sequences of the portion to which an antibody can bind to the DKK-1 protein according to the present invention.

[0105] In addition, the polypeptide fragment, which is an antigenic determinant of the DKK-1 protein provided in the present invention, may interact with a ligand present in effector T cells to reduce the activity of regulatory T cells.

[0106] According to another embodiment of the present invention, an expression vector having a polynucleotide inserted as provided in the present invention is provided.

[0107] In the present invention, the "vector" is a nucleic acid molecule capable of transporting another nucleic acid to which a certain nucleic acid molecule is connected. One type of vector is a "plasmid," which refers to circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector to which additional DNA segments can be ligated to the viral genome. Some vectors can replicate autonomously in the host cell to which they are introduced (e.g., bacterial vectors are episomal mammalian vectors with a bacterial replication origin). Other vectors (e.g., non-episosomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are connected at the operational level. Such vectors are referred herein as "recombinant expression vectors" or simply "expression vectors." Generally, expression vectors useful in recombinant DNA techniques often exist in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably because plasmid is the most commonly used form of vector.

[0108] Specific examples of the expression vector in the present invention may be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, and T7; and plant viruses, but are not limited thereto. Any expression vector known to those skilled in the art as an expression vector may be used in the present invention, and the selection of the expression vector depends on the properties of the target host cell. When introducing the vector into the host cell, it may be performed by calcium phosphate transfection, viral infection, DEAE-dextran regulated transfection, lipofectamine transfection, or electroporation, but is not limited thereto. Those skilled in the art may select and use an introduction method suitable for the expression vector and the host cell to be used. Preferably, the vector contains one or more screening markers, but is not limited thereto, and screening is possible based on whether a product is produced using a vector that does not contain screening markers. The selection of screening markers is performed by the target host cells, and since this utilizes methods already known to those skilled in the art, the present invention is not limited thereto.

[0109] To facilitate the purification of the nucleic acid molecule of the present invention, a tag sequence may be inserted into an expression vector and fused. The tag includes, but is not limited to, a hexahistidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag known to those skilled in the art that facilitates purification may be used in the present invention.

[0110] According to another embodiment of the present invention, the present invention provides a host cell line transfected with the expression vector provided.

[0111] In the present invention, the "host cell" includes an individual cell or cell culture that may or was a recipient of a vector(s) for the incorporation of a polypeptide insert. The host cell includes progeny of a single host cell, and said progeny may not necessarily be completely identical to the original parent cell (morphologically or in genomic DNA complements) due to natural, accidental, or intentional mutations. The host cell includes a cell transfected in vivo with the polypeptide(s) of the present invention.

[0112] In the present invention, the host cell may include cells of mammalian, plant, insect, fungal, or cellular origin, such as, for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells such as yeast cells and Pichia pasteoris; insect cells such as Drozophylla and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mice myeloma), human lymphoblastoid, COS, NSO (mice myeloma), 293T, Bow melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells) or PERC.6 (human retinal cells); or plant cells, but is not limited thereto, and any cell that can be used as a host cell line known to those skilled in the art may be used.

[0113] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, or bone diseases is provided, comprising as active ingredients antibodies that specifically bind to DKK-1 or antibodies that specifically bind to an epitope of DKK-1.

[0114] The antibodies of the present invention that specifically bind to the DKK-1 or the antibodies that specifically bind to the epitope of the DKK-1 inhibit the cell activity of effector T cells through interaction and can inhibit the immune response.

[0115] In the present invention, antibodies that specifically bind to DKK-1 or antibodies that specifically bind to the epitope of DKK-1 can effectively prevent, improve, or treat immune-related diseases, such as metabolic diseases, neurological diseases, or bone diseases.

[0116] The antibodies provided in the present invention that specifically bind to DKK-1 or antibodies that specifically bind to the epitope of DKK-1 interact with ligands present in effector T cells to suppress the activity of regulatory T cells, thereby effectively preventing, improving, or treating immune-related diseases, such as metabolic diseases, neurological diseases, or bone diseases.

[0117] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, or bone diseases is provided, comprising as an active ingredient an antibody specific to the DKK1 protein or the antigenic determinant of the present invention.

[0118] In addition, in the present invention, the DKK1 may be present on the surface of a regulatory T cell.

[0119] In the present invention, the antibody can effectively prevent, improve, or treat metabolic diseases, neurological diseases, or bone diseases by inhibiting the activity of regulatory T cells.

[0120] In the present invention, the term "antibody" refers to a specific protein molecule indicated for an antigenic site, as is known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the protein of the present invention. Such an antibody can be prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by said marker gene, and then preparing the obtained protein by a conventional method. This includes a partial peptide that can be produced from said protein, and the partial peptide of the present invention comprises at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids.

[0121] The form of the antibody of the present invention is not particularly limited, and polyclonal antibodies, monoclonal antibodies, or parts thereof having antigen-binding ability are included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention includes special antibodies such as humanized antibodies.

[0122] The antibody of the present invention comprises not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. A functional fragment of the antibody molecule refers to a fragment that possesses at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2, and Fv.

[0123] Immunoglobulin comprises four polypeptide chains, namely two heavy chains and two light chains associated via interchain disulfide bonds. Each light chain has two domains, namely a variable light chain domain (VL) and a constant light chain domain (CL), and each heavy chain has two regions, namely a variable heavy chain region (VH) and a constant heavy chain region (CH). The constant heavy chain region (CH) consists of constant heavy chain regions designated by numbers (e.g., CH1, CH2, CH3, etc.). Immunoglobulins are classified into different isoforms (i.e., IgG, IgM, IgA, IgD, and IgE) based on their biological properties, location within the organism, and ability to process different antigens. Depending on the immunoglobulin isoform, the constant heavy chain region (CH) may have three or four CH domains. In addition, in some isoforms (IgA, IgD, and IgG), the heavy chain contains a hinge region that adds flexibility to the molecule.

[0124] Humans have four IgG subclasses (IgG1, 2, 3, and 4), named according to their abundance in serum (IgG1 is the most abundant). IgG isoforms consist of two light chains and two heavy chains, each containing three constant heavy chain domains (CH1, CH2, and CH3). The two heavy chains of IgG are connected to each other and to the light chains by disulfide bonds (-SS-). The antigen-binding site of IgG is located in the fragment antigen-binding region (Fab region), which includes the variable light chain (VL) and variable heavy chain (VH) domains, as well as the constant light chain (CL) and constant heavy chain (CH1) domains. The fragment crystallizable region (Fc region) of IgG is the part of the heavy chain containing CH2 and CH3 domains that binds to Fc receptors found on the surface of specific cells, including the neonatal Fc receptor (FcRn). The heavy chains of IgG also have a hinge region (hinge) between CH1 and CH2 that participates in separating the Fab region from the Fc region and connecting the two heavy chains together via disulfide bonds. The structure of the hinge region contributes to the unique biological properties of each of the four IgG subclasses.

[0125] IgG is secreted as a small monomer that allows it to easily perfuse tissues. It is the only isoform that possesses a receptor (neonatal Fc receptor (FcRn)) that facilitates its passage through the human placenta to protect the fetus within the womb. IgG absorbed through the placenta provides humoral immunity to the newborn before its own immune system develops.

[0126] The IgG neonatal Fc receptor (FcRn) binding site is located in the Fc region of the antibody. FcRn is generally expressed in human placental and epithelial cells and participates in the endocytic salvage pathway that prevents the degradation of IgG. This salvage pathway is mediated by the high pH-dependent binding affinity of IgG to FcRn at acidic pH. The high affinity of IgG to FcRn at acidic pH is thought to induce the binding of internalized IgG to FcRn following uptake into acidic endosomes. While most soluble proteins proceed to lysosomes after internalization, internalized FcRn-bound IgG returns to the plasma membrane and is effectively rescued from the primary degradation pathway. Upon exposure to the neutral pH of the extracellular space, IgG can dissociate from FcRn and return to the circulation. Therefore, the extended serum half-life characteristics of the antibody are maintained in the Fc fragment.

[0127] In the present invention, "immunoglobulin Fc region" refers to a region comprising a heavy chain constant region 2 (CH2) and / or a heavy chain constant region 3 (CH3), excluding the heavy chain and light chain variable regions of the immunoglobulin. The immunoglobulin Fc region may be a component forming a moiety of the protein conjugate of the present invention.

[0128] In the present invention, the immunoglobulin Fc region may affect the structural flexibility of the final fusion protein to be manufactured and may further enhance the productivity and stability of the fusion protein, but is not limited thereto.

[0129] In the present invention, the immunoglobulin Fc region may further include a hinge portion in the heavy chain constant region to influence the structural flexibility of the final fusion protein to be manufactured and to further increase the productivity and stability of the fusion protein, but is not limited thereto.

[0130] In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region comprising some or all of the heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), excluding only the heavy chain and light chain variable regions of the immunoglobulin, insofar as it has substantially equivalent or enhanced effects to the natural form. In addition, it may be a region in which some significantly long amino acid sequences corresponding to CH2 and / or CH3 have been removed.

[0131] For example, the immunoglobulin Fc region of the present invention may be 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains among the CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain and an immunoglobulin hinge region (or a part of a hinge region), and 6) a dimer of each domain of the heavy chain constant region and the light chain constant region. However, it is not limited thereto.

[0132] In addition, the immunoglobulin Fc region of the present invention includes not only natural amino acid sequences but also sequence derivatives thereof. An amino acid sequence derivative means having a different sequence in which one or more amino acid residues of the natural amino acid sequence are deleted, inserted, non-conservative or conservatively substituted, or a combination thereof.

[0133] For example, in the case of IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, can be used as suitable sites for modification.

[0134] In addition, various types of derivatives are possible, such as by removing a site capable of forming disulfide bonds, removing some amino acids from the N-terminus of the natural form Fc, or adding a methionine residue to the N-terminus of the natural form Fc. Furthermore, to eliminate effector function, complement binding sites, such as the C1q binding site, may be removed, or the ADCC (antibody dependent cell mediated cytotoxicity) site may be removed. Techniques for manufacturing sequence derivatives of such immunoglobulin Fc regions are disclosed in International Patent Publication No. WO 97 / 34631, International Patent Publication No. 96 / 32478, etc.

[0135] Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are known in the art (H. Neuras, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications may be made through phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0136] The Fc derivative described above may exhibit biological activity equivalent to the Fc region of the present invention and may have increased structural stability against heat, pH, etc. of the Fc region.

[0137] Additionally, this Fc region may be obtained from a natural form isolated in vivo from animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or it may be a recombinant form or a derivative thereof obtained from transformed animal cells or microorganisms. Here, the method of obtaining from the natural form may be a method of obtaining the total immunoglobulin by isolating it from the body of a human or animal and then treating it with a protease. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c may be separated using size-exclusion chromatography, etc. In a more specific embodiment, the Fc region derived from humans or mice is a recombinant immunoglobulin Fc region obtained from microorganisms.

[0138] Furthermore, the immunoglobulin Fc region may be in the form of a natural glycosylation, an increased glycosylation compared to the natural form, a decreased glycosylation compared to the natural form, or a form with the glycosylation removed. Conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms, can be utilized for increasing, decreasing, or removing these immunoglobulin Fc glycosylations. Here, the immunoglobulin Fc region with the glycosylation removed exhibits significantly reduced binding affinity to complement (c1q), and since antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, it does not induce unnecessary immune responses in vivo. In this regard, the form with the glycosylation removed or deglycosylated immunoglobulin Fc region is considered to be more suitable for its original purpose as a drug carrier.

[0139] In the present invention, "deglycosylation" refers to the Fc region from which sugar has been removed by an enzyme, and "aglycosylation" refers to the Fc region that has not been glycosylated, produced in prokaryotes, or in a more specific embodiment, in E. coli.

[0140] Meanwhile, the immunoglobulin Fc region may be derived from humans or animals such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and preferably may be derived from humans or mice.

[0141] Additionally, the immunoglobulin Fc region of the present invention may be an IgG, IgA, IgD, IgE, IgM-derived Fc region, a heavy chain constant region 2 (CH2), a heavy chain constant region 3 (CH3), a hinge, a fragment thereof, or a combination thereof, or a hybrid Fc comprising a combination thereof.

[0142] Meanwhile, in the present invention, "combination" means that when forming a dimer or a multimer, a polypeptide encoding a short-chain immunoglobulin Fc region, a heavy-chain constant region 2 (CH2), or a heavy-chain constant region 3 (CH3) of the same origin forms a combination with a short-chain polypeptide of a different origin. That is, it is possible to prepare a dimer or a multimer from two or more fragments selected from the Fc region, heavy-chain constant region 2 (CH2), or heavy-chain constant region 3 (CH3) derived from IgG, IgA, IgM, IgD, or IgE.

[0143] In the present invention, the "hybrid Fc" may be derived from a combination of human IgG subclasses or a combination of human IgD and IgG. In one embodiment, the hybrid Fc may include, for example, an IgD hinge region and a CH2 N-terminal region + IgG4 CH2 and CH3 regions, and, for example, the hybrid Fc form disclosed in Korean Registered Patent No. 0897938 may be adopted and used in the same manner and is incorporated herein by reference. In the present invention, when the hybrid Fc binds to a biologically active molecule, polypeptide, etc., it not only increases the serum half-life of the biologically active molecule but also has the effect of increasing the expression level of the polypeptide when the nucleotide encoding the Fc-polypeptide fusion protein is expressed.

[0144] In one example of the present invention, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgM, IgD, or IgE, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG, IgA, IgM, IgD, or IgE, but is not limited thereto.

[0145] In one example of the present invention, the immunoglobulin Fc region may be an IgG or IgM-derived Fc region most abundant in human blood, or may include an IgG or IgM-derived heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3); in another example, it may be an IgG-derived Fc region known to enhance the half-life of a ligand-binding protein, or may include an IgG-derived heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3); in yet another example, it may be an IgG1, IgG2, IgG3, or IgG4-derived Fc region, or may include an IgG1, IgG2, IgG3, or IgG4-derived heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3); and in yet another example, it may include an IgG1 or IgG2-derived heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3).

[0146] In one example of the present invention, the immunoglobulin Fc region may include a hinge region derived from IgG, IgA, IgM, IgD, IgE, or abatacept, and in another example, may include a hinge region derived from IgG, IgD, or abatacept, or may include a hinge region derived from IgG1, IgG2, IgG3, IgG4, IgD, or abatacept, but is not limited thereto.

[0147] In the present invention, "prevention" may include, without limitation, any act of blocking, suppressing, or delaying the symptoms of a disease using the pharmaceutical composition of the present invention.

[0148] In addition, in the present invention, "treatment" may be included without limitation as long as it is any act that improves or benefits the symptoms of a disease using the pharmaceutical composition of the present invention.

[0149] In the present invention, the pharmaceutical composition may be characterized in that it is in the form of a capsule, tablet, granule, injectable, ointment, powder, or beverage, and the pharmaceutical composition may be characterized in that it is intended for humans. Preferably, the pharmaceutical composition according to the present invention may be prepared in the form of an injectable and injected directly into a site where a metabolic disease, neurological disease, or bone disease has occurred, but is not limited thereto.

[0150] The pharmaceutical composition of the present invention is not limited to these but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. For injectable preparations, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., in combination; and for topical administration, a base, excipients, lubricants, preservatives, etc. may be used. The formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc.

[0151] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0152] Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal. Oral or parenteral administration is preferred. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0153] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, form of medication, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0154] The pharmaceutical composition of the present invention may be additionally administered in combination with other therapeutic agents, and thereby may be used to treat metabolic diseases, neurological diseases, or bone diseases.

[0155] The pharmaceutical composition of the present invention can be additionally administered in combination with other immunosuppressants, thereby effectively suppressing diseases caused by general hyperimmune responses.

[0156] In the present invention, one or more immunosuppressants selected from the group consisting of glucocorticoids, cyclophosphamid, cyclosporin, tacrolimu, rapamycin, type IV PDE inhibitors, p38 kinase inhibitors, azathioprine, mycophenolate mofetil, mizoribin, methotrexate, leflunomid, and breequina may be used as the immunosuppressants, but are not limited thereto.

[0157] In one embodiment of the present invention, administration means introducing the composition of the present invention to a patient by any appropriate method, and the route of administration of the composition of the present invention may be administered through any general route as long as it can reach the target tissue. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intraluminal administration, intraperitoneal administration, and intrathecal administration may be performed, but are not limited thereto. In the present invention, the effective dose may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, weight, general health condition, gender and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and concurrently used drugs. In adults, when an expression inhibitor of the above gene or an activity inhibitor of the above protein is administered once or several times a day, it may be administered at a dose of 0.01 ng / kg to 10 mg / kg in the case of siRNA, 0.01 ng / kg to 10 mg / kg in the case of an antisense oligonucleotide against the mRNA of the above gene, 0.1 ng / kg to 10 mg / kg in the case of a compound, or 0.1 ng / kg to 10 mg / kg in the case of a monoclonal antibody against the above protein.

[0158] In the present invention, the term "intended individual" refers to an individual that has developed or is highly likely to develop a metabolic disease, a neurological disease, or a bone disease, and may be a mammal including humans, and may be selected from the group consisting of, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats, and preferably may be a human, but is not limited thereto.

[0159] In the above method of the present invention, descriptions regarding DKK-1, metabolic diseases, neurological diseases or bone diseases, prognosis, target subjects, biological samples, control groups, etc., overlap with those previously described, and in order to avoid excessive complexity of the specification, detailed descriptions thereof are omitted below.

[0160] Antibodies that specifically bind to DKK-1 (Dickkopf-1) can be utilized as an effective means for the prevention and / or treatment of various bone diseases, including osteoporosis. DKK-1 is known to play a key role in regulating osteoblastogenesis and bone metabolism as a representative antagonist of the Wnt / β-catenin signaling pathway. In particular, by binding to LRP5 / 6 receptors and inhibiting Wnt ligand signaling, DKK-1 can impede the differentiation and activity of osteoblasts, thereby reducing bone formation and creating an environment where bone resorption by osteoclasts becomes relatively dominant. In some embodiments, antibodies that specifically bind to DKK-1 can restore or enhance Wnt / β-catenin signaling by blocking the interaction between DKK-1 and LRP5 / 6 or by neutralizing the biological activity of DKK-1. Accordingly, the differentiation and proliferation of osteoblasts are promoted, the production of bone matrix increases, and bone mineral density may be improved. In addition, activation of Wnt signaling can increase the expression of osteoprotegerin (OPG) and decrease the expression of receptor activator of nuclear factor kappa-B ligand (RANKL), thereby indirectly inhibiting the formation and activity of osteoclasts and exhibiting an effect of reducing bone resorption. In another embodiment, administration of the antibody can inhibit the decrease in bone density observed in patients with osteoporosis, reduce the risk of fracture, and induce improvement of the microarchitecture. Furthermore, it can be applied to pathological bone loss occurring in rheumatoid arthritis, bone metastasis, multiple myeloma, etc., and can alleviate bone damage by inhibiting the overexpression of DKK-1 induced by inflammation or the tumor microenvironment.The above antibody may be used alone or in combination with bisphosphonates, selective estrogen receptor modulators (SERMs), RANKL inhibitors, or other bone metabolism-related therapeutic agents, and may be administered via various routes such as intravenous, subcutaneous, or intramuscular injection. Additionally, the above antibody may be provided in the form of humanized antibodies, chimeric antibodies, or antibody fragments such as Fab, scFv, etc. Accordingly, antibodies that specifically bind to DKK-1 can be usefully applied to the prevention and treatment of various bone diseases, including osteoporosis, through a novel mechanism of action that simultaneously induces the promotion of bone formation and the inhibition of bone resorption.

[0161] In the present invention, the bone disease may be, but is not limited to, fracture, osteoporosis, periodontitis, Paget's disease, osteomalacia, osteopenia, bone atrophy or avascular necrosis of the femur, bone defect, osteoporotic fracture, diabetic fracture, nonunion fracture, osteogenesis imperfecta, osteomalaciatic fracture, osteoplastic disorder, degenerative bone disease, malocclusion, bone union disorder, pseudoarthrosis, bone necrosis, bone tumor, bone cancer, etc.

[0162] The composition of the present invention may be used in combination with other drugs for treating the bone disease. For example, it may be, but is not limited to, bisphosphonates such as alendronate, risedronate, ibandronate, pamidronate, zoledronate, etc., female hormones, selective estrogen receptor modulators (SERMs), RANKL inhibitors such as denosumab, bone formation promoters such as parathyroid hormone (PTH) such as teriparatide, active vitamin D which can be classified as others, tissue-selective estrogen complexes such as bazedoxifene / conjugated estrogen, or other substances such as calcitonin and strontium.

[0163] In the present invention, 'metabolic disease' refers to a condition or disease that is closely associated with or caused by obesity, and specifically, it may be one or more selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, and arteriosclerosis.

[0164] Antibodies that specifically bind to DKK-1 (Dickkopf-1) can be utilized as an effective means for the prevention and / or treatment of various metabolic diseases, including obesity. DKK-1 is known to act as a representative antagonist of the Wnt / β-catenin signaling pathway, playing a crucial role in adipogenesis, energy homeostasis, and metabolic regulation. In particular, increased expression of DKK-1 inhibits Wnt / β-catenin signaling, thereby promoting the differentiation of preadipocytes into mature adipocytes, which can consequently induce fat accumulation and an increase in body fat. In some embodiments, antibodies that specifically bind to DKK-1 can restore the inhibited Wnt / β-catenin signaling pathway by blocking the interaction between DKK-1 and its receptors (e.g., LRP5 / 6) or by neutralizing the biological activity of DKK-1. Consequently, this can lead to the inhibition of adipocyte differentiation, a reduction in lipid accumulation in existing adipocytes, and the promotion of energy consumption. In addition, metabolic diseases are closely associated with chronic low-grade inflammation, and DKK-1 is known as a factor associated with inflammatory responses. In some embodiments, the antibody can improve insulin sensitivity by reducing the expression of inflammatory cytokines such as TNF-α and IL-6 by inhibiting DKK-1-mediated inflammatory signals and alleviating inhibition of the insulin signaling pathway. As a result, blood glucose control is improved, and the progression of metabolic diseases such as type 2 diabetes can be suppressed. In another embodiment, administration of the antibody can induce improvements in various metabolic indicators, such as inhibition of weight gain, reduction of body fat mass, reduction of fasting blood glucose, improvement of glycated hemoglobin (HbA1c) levels, and improvement of blood lipid profiles (e.g., triglycerides, LDL cholesterol). Additionally, it can contribute to the improvement of non-alcoholic fatty liver disease (NAFLD) by reducing fat accumulation in the liver.The above-mentioned antibody may be used alone or in combination with existing anti-obesity agents, anti-diabetic agents, or lipid-lowering agents, and may be administered via various routes such as intravenous, subcutaneous, or intramuscular injection. Additionally, the above-mentioned antibody may be provided in the form of a humanized antibody, a chimeric antibody, or an antibody fragment (Fab, scFv, etc.). Accordingly, antibodies that specifically bind to DKK-1 can be usefully applied to the prevention and treatment of various metabolic diseases, including obesity, based on a novel mechanism of action that simultaneously regulates adipocyte differentiation, inflammatory responses, and energy metabolism.

[0165] In this invention, "obesity" refers not merely to having a high body weight, but to a state in which body fat is excessively accumulated. This means that even if a person appears to have a normal weight on the outside, they can be classified as obese if their body fat percentage is high. Obesity is typically assessed using the Body Mass Index (BMI); a BMI of 23–24.9 is classified as overweight, 25–29.9 as mild obesity, 30–34.9 as moderate obesity, and 35 or higher as severe obesity. Obesity occurs due to the complex interplay of multiple factors rather than a single cause, including poor dietary habits (including Westernized eating habits), reduced physical activity, emotional factors, and genetic factors. Consequently, obesity increases the risk of developing chronic diseases such as hyperlipidemia, diabetes, and hypertension.

[0166] In this invention, fatty liver refers to a condition or disease in which fat accumulates in excessive amounts in liver cells due to a disorder of lipid metabolism in the liver.

[0167] In this invention, hyperlipidemia refers to a condition or disease in which the concentration of lipid components in the blood, particularly cholesterol and triglycerides, is higher than normal levels, and is used in a broad sense to include all conditions requiring a reduction in blood lipid concentrations.

[0168] In this specification, arteriosclerosis refers to a condition or disease in which blood circulation to organs and tissues in the body is reduced due to the thickening of artery walls and a decrease in elasticity, and includes the meaning of "atherosclerosis," which refers to a condition or disease in which blood circulation is reduced as the lumen narrows due to the deposition of fat, cholesterol, and other substances on the inner wall of the artery forming plaque. Arteriosclerosis can occur in any part of the body; if it occurs in the blood vessels of the heart, it can cause coronary artery diseases such as angina pectoris and myocardial infarction; if it occurs in the brain, it can cause cerebral infarction; and if it occurs in the kidneys, it can cause renal failure.

[0169] The neurological disease to be prevented, improved, or treated by the composition provided in the present invention may be a neurodegenerative disease or a neuroinflammatory disease.

[0170] Antibodies that specifically bind to DKK-1 (Dickkopf-1) can be utilized as an effective means for the prevention and / or treatment of various neurodegenerative diseases, including Alzheimer's disease. DKK-1 is a representative antagonist of the Wnt / β-catenin signaling pathway and is known to play a crucial role in neuronal survival, synapse formation and maintenance, and neuronal plasticity. In particular, increased expression of DKK-1 inhibits Wnt / β-catenin signaling, inducing synaptic loss and neuronal death, which is closely associated with the pathological progression of neurodegenerative diseases. In neurodegenerative diseases such as Alzheimer's disease, the accumulation of amyloid-β has been reported to induce DKK-1 expression, and increased DKK-1 levels can accelerate synaptic breakdown and neuronal damage by inhibiting Wnt signaling. Furthermore, DKK-1 is associated with the activation of microglia and astrocytes, amplifying neuroinflammatory responses, which can lead to neuronal dysfunction and death. In some embodiments, an antibody that specifically binds to DKK-1 can restore the Wnt / β-catenin signaling pathway by neutralizing the activity of DKK-1 or blocking the binding between DKK-1 and LRP5 / 6 receptors. Consequently, the stability of synaptic structure is maintained or restored, neuronal viability is increased, and neuroplasticity can be enhanced. Additionally, the antibody can contribute to reducing synaptic loss and alleviating cognitive decline by inhibiting DKK-1-mediated signals induced by amyloid-beta. In another embodiment, the antibody can suppress the neuroinflammatory response to reduce the overactivation of microglia and astrocytes and reduce the secretion of inflammatory cytokines (e.g., IL-1β, TNF-α, etc.). This prevents secondary damage to neurons and can delay the progression of the disease.In addition, it may indirectly affect signaling pathways associated with the abnormal phosphorylation and aggregation of tau proteins, thereby exhibiting an effect that inhibits the formation of neurofibrillary tangles. The antibody may be designed to cross the blood-brain barrier (BBB) ​​or may be combined with a delivery system that facilitates BBB penetration, and may be administered via intravenous injection, subcutaneous injection, or other appropriate routes. Additionally, it may be provided in the form of humanized antibodies, chimeric antibodies, or antibody fragments such as Fab or scFv. Therefore, antibodies that specifically bind to DKK-1 can be usefully applied to the prevention and treatment of various neurodegenerative diseases, including Alzheimer's disease, through multiple mechanisms of action such as synaptic protection, promotion of neuronal survival, and inhibition of neuroinflammation.

[0171] In the present invention, the "neurodegenerative disease" may refer to a disease caused by a decrease or loss of function of nerve cells, and the "neuroinflammatory disease" may refer to a disease caused by an excessive inflammatory response of the nervous system. Specific examples of the neurodegenerative disease or neuroinflammatory disease in the present invention may be selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, neurological autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy, but It is not limited.

[0172] In the present invention, the term “aptamer” refers to a single-stranded nucleic acid molecule selected to bind specifically to a target molecule with high affinity, and is generally composed of DNA, RNA, or modified nucleotides thereof. Aptamers can selectively bind to proteins, peptides, small molecule compounds, sugars, cell surface markers, and various other targets by forming a three-dimensional structure, and exhibit binding characteristics similar to antibodies.

[0173] Aptamers are typically prepared via the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) method. This method is a technique that selects sequences binding to target molecules from a nucleic acid library containing random sequences through a repetitive process of binding, separation, and amplification. Through this process, aptamers with high binding affinity and specificity can be obtained. In some embodiments, aptamers may include various chemical modifications to improve chemical stability and in vivo half-life. For example, ribose modification at the 2'-position (2'-fluoro, 2'-O-methyl), phosphorothioate binding, end capping, or polyethylene glycol (PEG) conjugation (PEGylation) may be applied. Additionally, aptamers may be conjugated with fluorescent labels, radioisotopes, drugs, or other functional molecules as needed.

[0174] In another embodiment, aptamers can be utilized as therapeutic agents, diagnostic agents, or targeted delivery systems. For example, they can treat diseases by inhibiting or activating the function of specific proteins, and can be used to deliver drugs by selectively binding to specific cells or tissues. Additionally, due to their high specificity and low immunogenicity, aptamers can be usefully employed as a means to replace or complement antibodies. Therefore, based on the advantages of having high selectivity and affinity for various targets, being easy to chemically synthesize, and being modifiable, aptamers are useful molecules that can be widely utilized in the fields of medicine, diagnostics, and biotechnology.

[0175] In addition, the composition provided in the present invention may be used as a pharmaceutical composition or a food composition, but is not limited thereto.

[0176] A food composition containing the composition of the present invention as an active ingredient can be manufactured in the form of various food products, such as beverages, chewing gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts that have almost no toxicity or side effects, it can be used safely even when taken for a long period for preventive purposes.

[0177] When the composition of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight.

[0178] Here, when the above food composition is prepared in the form of a beverage, there are no special limitations other than containing the above food composition in the indicated proportions, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in ordinary beverages. That is, as natural carbohydrates, it may include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, conventional sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Examples of the above flavoring agents include natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

[0179] In addition, the food composition of the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0180] These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0181]

[0182] In the present invention, since the DKK1 protein is specifically present on the surface of immune cells, particularly regulatory T cells, it can be used as a novel target for therapeutic agents for various metabolic diseases, neurological diseases, and bone diseases. The DKK-1 antibodies provided in the present invention can prevent or treat various metabolic diseases, neurological diseases, and bone diseases by specifically binding to the DKK-1 protein and regulating Wnt signaling.

[0183]

[0184] Figure 1 shows that the DKK-1 expression level is elevated in human idiopathic pulmonary fibrosis patients.

[0185] Figure 2 shows that the expression of DKK-1 was increased in lungs stimulated by BLM.

[0186] Figure 3 shows the hypomorphic DKK-1. d / d This indicates that mice are protected from BLM-mediated lung injury.

[0187] Figure 4 shows that platelets are a major source of DKK-1 histological observations.

[0188] Figure 5 shows the expression of DKK-1-mediated alternatively activated macrophage (AAM) markers in bone marrow-derived macrophages (BMDM).

[0189] Figure 6 shows that the removal of DKK1 receptor LRP6 protects mice from BLM-induced injury.

[0190] Figure 7 shows that delayed DKK1 antibody treatment inhibits collagen deposition in a BLM-induced lung injury model.

[0191] Figure 8 shows the results confirming the induction of Wnt3a-induced reporter activity by anti-DKK1 in TCF / LEF reporter cells.

[0192] Figure 9 shows the results of confirming the obesity treatment effects of various clones or antibodies binding to the DKK-1 epitope.

[0193] Figure 10 shows the results of confirming the therapeutic effects of various clones or antibodies binding to the DKK-1 epitope on dementia.

[0194] Figure 11 shows the results of confirming the therapeutic effects of various clones or antibodies binding to the DKK-1 epitope on bone diseases.

[0195] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0196]

[0197] [Example]

[0198]

[0199] [Example 1] Confirmed that the DKK-1 protein expression level is elevated in lung tissue of a human idiopathic pulmonary fibrosis (IPB) patient.

[0200] To confirm DKK-1 expression in pulmonary fibrosis tissue in human tissue, a paraffin-block immobilized human lung tissue array kit was purchased (Pulmonary interstitial fibrosis tissue array, LC561; Biomax). Each lung tissue was deparaffinized with Histoclear and then dehydrated. Antigens were removed using sodium citrate buffer (10 mM, pH 6.0). Blocking was performed with 3% H2O2 to inhibit endogenous peroxidase activity. Each section was blocked in 5% BSA, treated with a primary antibody (Goat polyclonal anti-m / hDKK1, R&D systems; AF1096), and stained with a secondary antibody at a concentration of 5 µg / mL. Antibody color development was performed using a DAB substrate kit (SK-4100; Vector Laboratories), and images were observed by simultaneously performing H&E staining. The results are shown in Figure 1.

[0201] As shown in Figure 1, it was confirmed that the expression of DKK1 is much higher in patients with human idiopathic pulmonary fibrosis (IPB) than in healthy people.

[0202]

[0203] [Example 2] It was confirmed that DKK-1 expression increased when treated with bleomycin (BLM).

[0204] C57BL / 6 mice were treated intranasally with BLM 4 U / kg dissolved in 40 uL PBS. Mice aged 8–10 weeks were treated with BLM, and after 2 weeks, the mice were sacrificed and their lungs were excised. The excised lungs were perfused, fixed in 10% formalin for at least 48 hours, and paraffin-blocked for histological analysis. This was sectioned using a microtome. The paraffin-blocked lung tissue was subjected to immunostaining in the same manner as described in [Example 1]. The results are shown in Figure 2.

[0205] As shown in Figure 2, it was found that the expression of DKK-1 increased when fibrosis was induced by bleomycin (BLM).

[0206]

[0207] [Example 3] Hypomorphic Dkk-1 d / d It was confirmed that mice are protected from BLM-mediated lung injury.

[0208] WT and DKK-1 d / dMice (8–10 weeks old) were randomly assigned to either a BLM-induced pulmonary fibrosis mouse group or a saline group. The BLM-induced pulmonary fibrosis mouse group was anesthetized with 1% pentobarbital sodium and then administered 4 U / kg BLM (BLM, S121415; Selleckchem, Houston, TX) in 40 μl physiological saline via the endotracheal route as reported. Mice administered the same amount of physiological saline were used as a control group, and mice were sacrificed 14 days after BLM administration for pulmonary fibrosis analysis. Lungs were excised from the mice, perfused, fixed in 10% formalin for at least 48 hours, and paraffin-blocked for histological analysis. These were dissected with a microtome. The paraffin-blocked lung tissues were subjected to immunostaining in the same manner as described in [Example 1]. The results are shown in Figure 3.

[0209] As shown in Figure 3, it was found that the low DKK-1 type was relatively protected from lung damage induced by bleomycin (BLM).

[0210]

[0211] [Example 4] Confirmed that platelets are the primary source of DKK-1 histological observation.

[0212] Platelets were isolated from mice after partial modification as previously described. Blood was collected via cardiac puncture in 3.8% sodium citrate, pH 7.4 (2:1 by volume) and centrifuged at 250g for 10 minutes to obtain platelet-rich plasma (PRP). For platelet stimulation, the PRP was treated with 100 μM thrombin receptor activating peptide (SFLLRN; Sigma-Aldrich) for the indicated times, followed by centrifugation at 657g for 7 minutes. The supernatant was collected as plasma, and the platelet pellet was also collected. To collect the platelet efflux, the PRP was centrifuged at 657g for 7 minutes to pellet the platelets. Then, platelets were resuspended in Tyrode-HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer and stimulated with 0.1 U / mL thrombin (Sigma-Aldrich) for 90 minutes. Afterward, the platelet release was collected by centrifugation at 2500g for 10 minutes at 4°C.

[0213] As a result, as shown in Figure 4, it was found that platelets are the main source of DKK-1 histological observation.

[0214]

[0215] [Example 5] Confirmation of DKK-1-mediated Alternatively Activated Macrophage (AAM) Marker Expression in Bone Marrow-Derived Macrophages (BMDM)

[0216] To determine whether DKK-1 regulates the expression of alternative activated macrophages, changes in the expression levels of Arg1 and Cd206 in bone marrow-derived macrophages (BMDMs) with and without DKK-1 treatment were observed via flow cytometry. Mice were sacrificed for BMDM differentiation, and bone marrow cells were extracted on day 0 and cultured in DMEM medium containing 20% ​​FBS, GlutaMAX, MEM-NEAA, sodium pyruvate, 1x penicillin & streptomycin, and 20 ng / mL MCSF (macrophage colony-stimulating factor). The medium was changed every two days, and differentiation into M2-like macrophages was induced by treating with IL-4 and IL-13 on day 6.

[0217] As shown in Fig. 5, Arg + and CD206 + It was found that the expression of DKK-1 was high in macrophages.

[0218]

[0219] [Example 6] Confirmed that removal of DKK1 receptor LRP6 protects mice from bone marrow-derived macrophage (BMDM)-induced injury.

[0220] We investigated the effects of LRP6, known as a binding molecule for DKK-1 through prior research, on DKK-1-mediated BLM-induced lung injury. LRP6 fl / fl and, LysM-Cre LRP6 fl / fl Lung damage was induced in conditional KO mice using BLM, and the degree of lung damage was compared through collagen mRNA and immunohistochemical staining.

[0221] As shown in Figure 6, it was found that the removal of DKK1 receptor LRP6 protected mice from BLM-induced injury.

[0222]

[0223] [Example 7] Delayed DKK1 antibody treatment was confirmed to inhibit collagen deposition in a BLM-induced lung injury model.

[0224] C57BL6 mice were treated with BLM on day 0, and 150 μg / mouse of DKK-1 antibody was introduced using two different administration methods to confirm the therapeutic effect of the DKK-1 antibody. The first administration group was treated with the antibody on days 1, 1, 3, 7, 9, and 11, and the second group was treated on days 7, 9, and 11.

[0225] As shown in Figure 7, both treatment groups reduced the expression of DKK-1 and consequently inhibited collagen deposition in the lungs. Delayed DKK1 antibody treatment also showed a sufficient therapeutic effect, inhibiting collagen deposition to a similar degree as the group administered earlier.

[0226] In other words, reduced DKK-1 expression in DKK-1 hypomorphic doubleridge mice protects against inflammation and fibrosis induced by BLM. Also, DKK-1 PKO Platelet-specific deletion of DKK-1 in mice is Arg1 + and CD206 + DKK-1 reduces AAM markers such as those mentioned above, and induces various pro-inflammatory and fibrosis-promoting markers without type 2 cytokines. Furthermore, DKK-1 enhances the IL-13-mediated AAM phenotype, and the co-inhibition of STAT6 and JNK is sufficient to eliminate excessive AAM-like macrophage gene expression. In addition, Lrp6 MKO Deletion of the DKK-1 receptor in mouse myeloid lineage cells protects against BLM-induced inflammation and fibrosis. Delayed treatment with DKK-1 antibodies suppresses BLM-induced inflammation and fibrosis.

[0227]

[0228] [Example 8] Luciferase assay (confirmation of β-catenin signaling pathway)

[0229] First, 293T_TCF / LEF_Luc cells, DMEM with 10% FBS, 1% Pen / Strep, 8M LiCl (Sigma, Cat# L7026), hWnt3a (reacted in both human and mouse), hDKK1, the previously prepared anti-DKK1 antibodies, Luciferase assay system (Promega, Cat# E1500), 96-well cell culture plate (TC-treated), and 96-well white bottom flat plate (Corning) were prepared.

[0230] On the first day, 5 x 10⁴ cells / 100 μL were dispensed into a 96-well cell culture plate (+10 mM LiCl, O / N). Additionally, wells not treated with LiCl were dispensed as a negative control. On the second day, the media was replaced with fresh media (+10 mM LiCl), and Wnt3a, DKK1, and antibodies were added at the following concentrations (Final 100 μL).

[0231] Group(-)C1C2AbLiCl (10mM)-+++Wnt3a (50ng / mL)-+++DKK-1 (500ng / mL)--++Antibody (1-100nM)---+

[0232] Next, 5X cell culture lysis reagent (CCLR) was prepared as 1X by diluting it with distilled water at RT (30 μL required for a 96-well plate), and after 6 hours, the wells were washed with PBS (200 μL aliquots, x2). Subsequently, 30 μL of 1X CCLR was added and pipetted (empty wells were also treated for blanking). Additionally, 100 μL of Luciferase assay substrate (stored at -20 ℃, thawed at RT before use) was dispensed into white bottom flat plates, 20 μL of cell lysate was added, pipetted, and measured using a luminometer. (Promega instrument used. Protocol: Luciferase assay system-non-injector)

[0233]

[0234] [Example 9] Confirmation of therapeutic effects for obesity, dementia, and osteoporosis

[0235] 1. Preparation of active ingredients such as anti-DKK-1 antibodies

[0236] Previously known anti-DKK-1 antibodies, aptamers, or antibodies that bind to DKK-1 ligands and consequently inhibit DKK-1, were primarily known for use in cancer treatment. Accordingly, we sought to verify the therapeutic effects of the above active ingredients for obesity, dementia, and bone diseases as new applications. Therefore, active ingredients including anti-DKK-1 antibodies were prepared according to methods described in existing patent literature and papers. Among the numerous active ingredients that inhibit DKK-1, active ingredients with confirmed pharmacological effects for obesity, dementia, or bone diseases were selected and are shown in Table 4. Furthermore, to avoid unnecessary duplication, the specific manufacturing methods of the active ingredients have been omitted as they are described in detail in the patent literature listed in Table 4, and the entirety of the patent literature listed in Table 4 is cited by reference in this invention.

[0237] Number Patent Document Number Effective Ingredient (Clone Name) 1CN 117916377 AAptdkk-52US 11807679 B2From hybridoma 3F11-2B103US 8586721 B2huMabJC184US 9879072 B25.80.15US 2012-0052070 A1MOR049456US 8148498 B2Antibody II7US 8101184 B211H108US 8715941 B2anti-LRP6 mAb 135.169US 7994293 B2RH2-1810US 7700101 B2DKK-1 epitope11US 8673306 B2MOR0491012US 8252267 B2DKK-1 epitope13US 7446181 B2DKK-1 epitope14US 2003-0165501 A1From ATCC No: PTA-3086

[0238] 2. Confirmation of obesity treatment effect – Inhibition of lipid droplet formation during adipocyte differentiation

[0239] To evaluate the anti-obesity effect by inhibiting lipid droplet formation during adipocyte differentiation, 3T3-L1 preadipocytes or human adipose-derived stem cell / hMSC-derived adipocytes were used. First, cells were cultured to a confluent state, and adipogenesis was initiated using differentiation induction medium (usually containing IBMX, dexamethasone, insulin, and, if necessary, rosiglitazone). The test compounds were either administered simultaneously from the start of differentiation induction or divided among specific differentiation stages (early 0–2 days, mid-2 days, late 4–8 days). The differentiation period was typically about 6–10 days for 3T3-L1 cells, during which the compounds were repeatedly administered while the medium was replaced every two days. At the end of the experiment, intracellular triglyceride accumulation and lipid droplet formation were observed using Oil Red O staining or BODIPY / Nile Red fluorescent staining, and the number of droplets, droplet size, and lipid-positive area were quantified through microscopic image analysis.

[0240] As a result, as shown in Figure 9, it was confirmed that active ingredients such as antibodies and aptamers that bind to DKK-1 can treat metabolic diseases including obesity by inhibiting lipid droplet formation during lipid cell differentiation.

[0241]

[0242] 3. Confirmation of Dementia Treatment Effect – Inhibition of Tau Protein Aggregation

[0243] To confirm the therapeutic effect of antibodies specifically binding to DKK-1 on dementia, tau protein aggregation and seeding assays were performed. Aggregation was induced in tau RD-P301S biosensor cells or full-length tau-expressing cells by treating them with preformed tau fibril or patient-derived tau seeds. Subsequently, whether the antibody specifically binding to DKK-1 provided in this invention inhibited aggregation was analyzed using Thioflavin S / T staining, filter trap assay, sarkosyl-insoluble tau fraction, and FRET-based aggregation assay. In immunohistochemistry, abnormal tau aggregate formation was quantified using AT8, MC1, and T22 oligomeric tau antibodies, and intracellular aggregate burden was measured using a confocal microscope.

[0244] As a result, as shown in Figure 10, it was confirmed that active ingredients such as antibodies and aptamers that bind to DKK-1 can treat neurological diseases including dementia by inhibiting tau protein aggregation.

[0245]

[0246] 4. Confirmation of osteoporosis treatment efficacy

[0247] To confirm the therapeutic effects of the above antibodies on osteoporosis, an osteoblast assay was performed using MC3T3-E1 cells, primary calvarial osteoblasts, or human BMSCs. After culturing cells under α-MEM or DMEM + 10% FBS conditions, differentiation media containing ascorbic acid (50 μg / mL) and β-glycerophosphate (5–10 mM) were used to induce osteogenic differentiation, and test compounds were treated at various concentrations. Initial differentiation was evaluated at 5–7 days using an ALP activity assay (color change-based pNPP assay) and ALP staining, while later differentiation and mineralization were assessed by quantifying calcium deposition using Alizarin Red S or von Kossa staining after 14–21 days of culture. At the same time, RNA and proteins were extracted and osteogenic markers such as RUNX2, ALPL, COL1A1, SP7 (Osterix), BGLAP (osteocalcin), and SPP1 (OPN) were analyzed by qPCR and Western blot, and the pure bone formation-promoting effect of the compound was verified by performing a CCK-8 or MTT assay in parallel to exclude cytotoxicity.

[0248] As a result, as shown in Figure 11, it was confirmed that active ingredients such as antibodies and aptamers that bind to DKK-1 have a promoting effect on osteoblasts.

[0249]

[0250] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0251]

Claims

1. A pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, or bone diseases, comprising as an active ingredient a binding molecule that specifically binds to DKK-1 protein.

2. In paragraph 1, the bonding molecule is, Heavy chain complementarity-determining region 1 (CDR-H1) comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 2, 8, 14, 20, 26, 32, 38, and 45; Heavy chain complementarity-determining region 2 (CDR-H2) comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 3, 9, 15, 21, 27, 33, 39, and 46; and A heavy chain variable region comprising: a heavy chain complementarity-determining region 3 (CDR-H3) comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 4, 10, 16, 22, 28, 34, 40, and 47; and Light chain complementarity-determining region 1 (CDR-L1) comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 5, 11, 17, 23, 29, 35, 41, and 48; Light chain complementarity-determining region 2 (CDR-L2) comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 6, 12, 18, 24, 30, 36, 42, and 49; and A pharmaceutical composition comprising a binding molecule comprising: a light chain complementarity-determining region 3 (CDR-L3) comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 7, 13, 19, 25, 31, 37, 43, and 50; and a light chain variable region comprising a light chain variable region.

3. In Paragraph 2, A pharmaceutical composition in which the above-mentioned binding molecule is selected from the following groups. (1) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 2; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 3; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 4; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 5; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 6; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 7; (2) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 8; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 9; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 10; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 11; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 12; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 13; (3) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 14; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 15; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 16; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 17; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 18; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 19; (4) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 20; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 21; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 22; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 23; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 24; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 25; (5) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 26; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 27; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 28; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 29; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 30; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 31; (6) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 32; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 33; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 34; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 35; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 36; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 37; (7) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 38; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 39; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 40; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 41; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 42; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 43; (8) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 45; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 46; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 47; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 48; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 49; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO.

50.

4. In any one of paragraphs 1 through 3, A pharmaceutical composition in which the above-mentioned binding molecule further comprises an Fc region (Fragment crystallization region) or a constant region.

5. In Paragraph 4, A pharmaceutical composition wherein the above Fc region is the Fc region of an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody, or a hybrid Fc region.

6. In Paragraph 1, A pharmaceutical composition in which the above-mentioned binding molecule is an antibody or a binding fragment thereof.

7. In Paragraph 6, A pharmaceutical composition wherein the antibody or its binding fragment is a chimeric antibody, humanized antibody, bivalent, amphoteric molecule, minibody, domain antibody, bispecific antibody, antibody mimic, unibody, diabody, triabody, tetrabody, or a fragment thereof.

8. A pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, or bone diseases, comprising as an active ingredient a nucleic acid molecule encoding a binding molecule of any one of claims 1 to 3.

9. A pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, or bone diseases, comprising as an active ingredient an expression vector into which a nucleic acid molecule encoding a binding molecule of any one of claims 1 to 3 has been inserted.

10. (1) A heavy chain variable region comprising: a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 2; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 3; and a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 4; and A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 5; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 6; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 7; (2) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 8; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 9; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 10; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 11; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 12; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 13; (3) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 14; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 15; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 16; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 17; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 18; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 19; (4) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 20; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 21; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 22; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 23; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 24; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 25; (5) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 26; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 27; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 28; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 29; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 30; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 31; (6) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 32; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 33; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 34; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 35; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 36; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 37; (7) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 38; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 39; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 40; and a heavy chain variable region comprising A binding molecule comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 41; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 42; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 43; (8) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 45; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 46; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 47; and a heavy chain variable region comprising An antibody-drug conjugate (ADC) comprising: a binding molecule comprising a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 48; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 49; a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 50; and a drug.

11. In a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linkage domain, and a CD3 zeta (ζ) signaling domain, The above antigen-specific binding domain is a chimeric antigen receptor selected from the following group: (1) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 2; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 3; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 4; and a heavy chain variable region comprising An antigen-specific binding domain comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 5; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 6; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 7; (2) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 8; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 9; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 10; and a heavy chain variable region comprising An antigen-specific binding domain comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 11; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 12; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 13; (3) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 14; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 15; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 16; and a heavy chain variable region comprising An antigen-specific binding domain comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 17; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 18; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 19; (4) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 20; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 21; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 22; and a heavy chain variable region comprising An antigen-specific binding domain comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 23; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 24; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 25; (5) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 26; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 27; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 28; and a heavy chain variable region comprising An antigen-specific binding domain comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 29; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 30; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 31; (6) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 32; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 33; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 34; and a heavy chain variable region comprising An antigen-specific binding domain comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 35; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 36; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 37; (7) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 38; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 39; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 40; and a heavy chain variable region comprising An antigen-specific binding domain comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO. 41; a light chain complementarity-determining region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO. 42; and a light chain complementarity-determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO. 43; (8) a heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO. 45; a heavy chain complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO. 46; a heavy chain complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO. 47; and a heavy chain variable region comprising An antigen-specific binding domain comprising a heavy chain variable region including a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO. 48; a light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO. 49; and a light chain complementarity-determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO.

50.

12. A pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, or bone diseases, comprising as an active ingredient an aptamer that specifically binds to DKK-1 containing the nucleotide sequence of SEQ ID NO.

1.

13. A pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases or bone diseases, comprising as an active ingredient an antibody that specifically binds to the DKK-1 epitope of SEQ ID NO. 44, 51, or 52.

14. A pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, or bone diseases comprising, as an active ingredient, an anti-CKAP4 monoclonal antibody produced from hybridoma 3F11-2B10, deposited as NITE BP-03885 on April 18, 2023, or an antigen-binding fragment thereof.

15. A pharmaceutical composition for the prevention or treatment of metabolic diseases, neurological diseases, or bone diseases, comprising as an active ingredient an antibody or antigen-binding fragment that specifically binds to the DKK-1 protein produced from the hybridoma of ATCC accession number PTA-3086.

16. A method for preventing or treating metabolic diseases, neurological diseases, or bone diseases, comprising the step of administering a binding molecule that specifically binds to DKK-1 protein to a subject.

17. A method for preventing or treating metabolic diseases, neurological diseases, or bone diseases, comprising the step of administering to a subject an aptamer that specifically binds to DKK-1 containing the nucleotide sequence of SEQ ID NO.

1.

18. A method for preventing or treating metabolic diseases, neurological diseases or bone diseases, comprising the step of administering to a subject an antibody that specifically binds to the DKK-1 epitope of sequence number 44, 51, or 52.

19. A method for preventing or treating metabolic diseases, neurological diseases, or bone diseases, comprising the step of administering to a subject an anti-CKAP4 monoclonal antibody produced from hybridoma 3F11-2B10, deposited as NITE BP-03885 on April 18, 2023, or an antigen-binding fragment thereof.

20. A method for preventing or treating metabolic diseases, neurological diseases, or bone diseases, comprising the step of administering to a subject an antibody or antigen-binding fragment that specifically binds to the DKK-1 protein produced from the hybridoma of ATCC accession number PTA-3086.

21. In any one of paragraphs 12 through 15, A pharmaceutical composition wherein the above-mentioned bone disease is one or more selected from the group consisting of fracture, osteoporosis, periodontitis, Paget's disease, osteomalacia, osteopenia, bone atrophy or avascular necrosis of the femoral bone, bone defect, fracture, osteoporotic fracture, diabetic fracture, nonunion fracture, osteogenesis imperfecta, osteomalacia fracture, osteoplastic disorder, degenerative bone disease, malocclusion, bone union disorder, pseudoarthrosis, bone necrosis, bone tumor, and bone cancer.

22. In any one of paragraphs 12 through 15, A pharmaceutical composition wherein the above metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes mellitus, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

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