Synthetic protein, complex, composition, polynucleotide, expression vector, transformant, osteogenesis promoter, cartilage proliferation promoter, angiogenesis promoter, and pharmaceutical composition

Abstract

A synthetic protein comprising: an N-terminal collagen-binding domain (CBD); a C-terminal C-type natriuretic peptide domain (CNP); and optionally, a spacer linking the CBD and the CNP.

Description

Synthetic proteins, complexes, compositions, polynucleotides, expression vectors, transformants, bone formation promoters, cartilage growth promoters, angiogenesis promoters, and pharmaceutical compositions

[0001] The present invention relates to a synthetic protein, a complex, a composition, a polynucleotide, an expression vector, a transformant, an agent for promoting bone formation, an agent for promoting cartilage growth, an agent for promoting angiogenesis, and a pharmaceutical composition.

[0002] In this specification, the synthetic protein of the present invention may be referred to as "CBD-CNP."

[0003] Fractures with delayed union, malunion, or nonunion require bone grafts or bone graft substitutes such as allogeneic demineralized bone matrix. However, the effectiveness of allogeneic demineralized bone matrix alone in repairing bone defects is limited, necessitating the use of osteoinductive growth factors. Growth factors such as BMP (bone morphogenetic protein) 2, BMP7, and FGF2 (fibroblast growth factor 2) have been investigated or used as treatments for fractures, bone defects, and nonunions. However, because these agents rapidly diffuse within the body, they require high doses or repeated administration to maintain therapeutic effects at the fracture site. However, these administration regimens are likely to result in systemic side effects and are expensive.

[0004] Knee osteoarthritis, a typical example of articular cartilage damage, is the most common musculoskeletal disease, affecting over 300 million people worldwide, with the number of patients expected to increase further as society ages. Age-related wear of the articular cartilage causes pain and functional impairment, significantly reducing patients' quality of life and healthy life expectancy, but treatment options are limited to symptomatic treatment such as painkillers and artificial joint replacement surgery.

[0005] Coronary artery disease and peripheral artery disease are also diseases for which promoting angiogenesis is therapeutically beneficial. In coronary artery disease, myocardial ischemia can lead to chest pain and cardiac dysfunction, which can be fatal. In peripheral artery disease of the lower limbs, symptoms such as intermittent claudication and ulcers can occur, requiring lower limb amputation. In patients with the above diseases who cannot undergo surgical bypass surgery or percutaneous intervention, the ischemic tissue becomes necrotic and its function does not return.

[0006] As mentioned above, in all of these areas, there is a need for the development of novel therapeutic agents that improve function and symptoms by promoting bone formation, cartilage proliferation, and angiogenesis in the local affected area.

[0007] C-type natriuretic peptide (CNP) is produced in the body in the central nervous system, reproductive organs, vascular endothelium, etc. (Non-Patent Document 1) and has the effect of promoting osteoblast formation and differentiation (Non-Patent Document 2), promoting chondrocyte proliferation (Non-Patent Document 3), and promoting angiogenesis (Non-Patent Document 4). However, the problem with the naturally occurring CNPs expressed in the body, CNP-22 and CNP-53, is that they have an extremely short half-life; the half-life of CNP-22 in human blood is only 2.6 minutes (Non-Patent Document 5). Therefore, their efficacy cannot be achieved unless they are administered continuously intravascularly (Non-Patent Document 6).

[0008] BMN111 (Patent Document 1, Patent Document 2) is a CNP analogue in which two amino acid residues (Pro-Gly) have been added to the N-terminus of the 37 C-terminal residues of CNP-53. It is designed to be more resistant to neutral endopeptidase degradation than CNP, and promotes endochondral bone growth with a single daily subcutaneous injection (Non-Patent Document 7). However, BMN111 also has a short half-life of approximately 20 minutes in non-clinical trials, and because it diffuses throughout the body, it cannot act locally on target organs.

[0009] Patent Document 3 describes a synthetic protein (collagen-binding protein) in which the collagen-binding domain (CBD), a constituent domain of collagenase produced by Hathewaya histolytica (formerly known as Clostridium histolyticum), is fused with other physiologically active substances as an anchor module, and Patent Document 4 describes a bone graft material kit using a fusion protein of CBD and FGF2. However, there are no disclosures regarding fusion proteins in which CBD is located on the N-terminus, proteins in which CBD is fused with CNP, or drugs that combine the diverse effects of promoting bone formation, cartilage growth, and angiogenesis with a single protein.

[0010] WO2021 / 055497 (PCT / US2020 / 051100) WO2017 / 100400 (PCT / US2016 / 065520) Japanese Patent No. 6697019 Japanese Patent No. 5512887

[0011] Hypertension. 49: 419-426, 2007Am J Physiol. 270: C1311-C1318, 1996J Biol Chem. 269: 10729-10733, 1994Circulation. 139: 1612-1628, 2019J Clin Endocrinol Metab. 78: 1428-1435, 1994Endocrinology. 150: 3138-3144, 2009N Engl J Med. 381: 25-35, 2019

[0012] One object of the present invention is to provide a novel therapeutic agent that improves function and symptoms by promoting osteogenesis, cartilage growth, and angiogenesis.

[0013] The present invention provides the following synthetic proteins, complexes, compositions, polynucleotides, expression vectors, transformants, bone formation promoters, cartilage growth promoters, angiogenesis promoters, and pharmaceutical compositions. [1] A synthetic protein comprising an N-terminal collagen-binding domain (CBD) and a C-terminal C-type natriuretic peptide domain (CNP). [2] The synthetic protein according to [1], further comprising a spacer linking the CBD and CNP. [3] The synthetic protein according to [1] or [2], which is any of (i) to (iii) below. (i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 2 or 3; (ii) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 2 or 3 in which one or more amino acids have been deleted, substituted or added, and having at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity; (iii) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, 2 or 3, and having at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity. [4] A complex formed by binding the synthetic protein according to any of [1] to [3] and collagen. [5] A composition comprising the synthetic protein according to any of [1] to [3] and collagen. [6] A polynucleotide of any of (a) to (e) below.(a) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 2 or 3; (b) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1, 2 or 3, and having at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity; (c) a polynucleotide encoding a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, 2 or 3, and having at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity; (d) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4, 5 or 6; (e) a polynucleotide encoding a protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 4, 5 or 6, and having at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity. [7] An expression vector comprising the polynucleotide according to [6]. [8] A transformant into which the polynucleotide according to [6] or the expression vector according to [7] has been introduced. [9] A osteogenesis promoter comprising, as an active ingredient, the synthetic protein according to any one of [1] to [3], the complex according to [4], or the composition according to [5].

[10] A cartilage growth promoter comprising, as an active ingredient, the synthetic protein according to any one of [1] to [3], the complex according to [4], or the composition according to [5].

[11] An angiogenesis promoter comprising, as an active ingredient, the synthetic protein according to any one of [1] to [3], the complex according to [4], or the composition according to [5].

[12] A pharmaceutical composition comprising, as an active ingredient, the synthetic protein according to any one of [1] to [3], the complex according to [4], or the composition according to [5].

[13] The pharmaceutical composition according to

[12] , which is for promoting osteogenesis, cartilage growth, or angiogenesis.

[14] A method for preventing or treating knee osteoarthritis, achondroplasia, dwarfism, congenital leg length variance, ischemic heart disease, or peripheral arterial disease, comprising administering to a patient the synthetic protein according to any one of [1] to [3], the complex according to [4], or the composition according to [5].

[15] Use of the synthetic protein of any of [1] to [3], the complex of [4], or the composition of [5] for producing an agent for promoting bone formation, cartilage growth, or angiogenesis.

[16] Use of the synthetic protein of any of [1] to [3], the complex of [4], or the composition of [5] for preventing or treating knee osteoarthritis, achondroplasia, dwarfism, congenital leg length variance, ischemic heart disease, or peripheral arterial disease.

[17] Use of the synthetic protein of any of [1] to [3], the complex of [4], or the composition of [5] for preventing or treating vascular disease.

[0014] The present invention makes it possible to achieve long-term local effects by imparting collagen binding ability to CNP, which has the effects of promoting osteoblast formation and differentiation, chondrocyte proliferation, and angiogenesis.

[0015] Natural CNP has a very short half-life of 2.6 minutes, so it is quickly inactivated when administered locally, and systemic administration requires continuous infusion. Furthermore, BMN111, a CNP analog designed to be resistant to neutral endopeptidase degradation, promotes endochondral bone growth with a single daily subcutaneous injection. However, its short half-life of approximately 20 minutes means that it diffuses throughout the body, preventing it from acting locally on the target organ.

[0016] The CBD-CNP of the present invention overcomes the weaknesses of CNP, such as its early inactivation and short half-life, and its collagen-binding ability enables efficient local drug delivery, which is expected to improve local efficacy, reduce the number of administrations and total dosage, and reduce systemic side effects compared to systemic administration.

[0017] Compared to other collagen-binding proteins, the CBD-CNP of the present invention has a smaller molecular weight than FGF2-CBD, thereby offering advantages in terms of improved productivity and reduced antigenicity. Furthermore, while other CBD-containing fusion proteins have a physiologically active substance located at the N-terminus and a CBD located at the C-terminus, the CBD-CNP of the present invention is designed with the physiologically active substance CNP located at the C-terminus and the CBD located at the N-terminus, thereby conferring collagen-binding ability while maintaining the physiological activity of the C-terminus of CNP. The present invention also represents a technological advancement in demonstrating a new fusion scheme in which the CBD is located at the N-terminus of a physiologically active substance located at the C-terminus.

[0018] While bone morphogenetic proteins BMP2 and BMP7 are currently used in Europe and the United States as bone formation promoters, they are known to pose risks of carcinogenicity and heterotopic ossification. CNP has no known carcinogenic side effects, and CBD-CNP is advantageous in that it acts via local administration, reducing the risks associated with systemic administration, such as heterotopic ossification.

[0019] Existing treatments for knee osteoarthritis, a common articular cartilage disorder, are limited to oral nonsteroidal anti-inflammatory drugs (NSAIDs) for pain relief, intra-articular injections of hyaluronic acid or steroids, or artificial joint replacement. Oral NSAIDs are symptomatic treatments, and long-term administration can cause side effects such as gastrointestinal disorders. Intra-articular injections of hyaluronic acid or steroids have limited long-term analgesic effects, and intra-articular injections of mesenchymal stem cells have shown some pain relief, but they do not result in structural restoration of worn cartilage (NPJ Regen Med. 3: 15, 2018). Artificial joint replacement surgery is associated with complications such as bacterial infection and thrombus formation, and is a socioeconomic burden, so it is limited to patients in advanced to terminal stages of the disease. As mentioned above, there are currently no treatments that can regenerate cartilage to improve symptoms. Therefore, the regeneration of worn cartilage proposed by this invention is positioned as an intermediate treatment that bridges the gap between symptomatic treatment and artificial joint replacement, potentially avoiding the need for artificial joint replacement through the proliferation of articular cartilage.

[0020] The CNP analogue BMN111 is used to promote endochondral ossification in achondroplasia and improve short stature. While BMN111 is administered systemically via subcutaneous injection, the CBD-CNP of the present invention can be administered locally via intra-articular injection. Local administration not only enhances efficacy at the affected site and reduces systemic side effects, but also allows for limited action on the affected side in cases such as congenital leg length inequality.

[0021] Existing angiogenesis treatments for coronary artery disease and peripheral arterial disease involve surgical bypass surgery or percutaneous catheter-based interventions for ischemic sites. However, these treatments do not restore function to ischemic necrotic tissue, necessitating heart transplantation or lower limb amputation. Gene therapy using plasmid vectors expressing physiologically active substances such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and FGF2, as well as stem cell transplantation, have been investigated as treatments aimed at promoting angiogenesis in ischemic tissue. Regarding ischemic heart disease, although several clinical studies of gene therapy have been conducted, no consensus has been reached regarding its efficacy. Intracardiac stem cell transplantation also failed to demonstrate significant differences in left ventricular ejection fraction or myocardial fibrosis compared to placebo (Eur Heart J. 41: 3451-3458, 2020), and there is currently no fundamental treatment that can restore ischemic myocardial function. Regarding peripheral arterial disease, HGF plasmid vector preparations and adipose-derived stem cell transplantation therapy have been conditionally approved or are being implemented as advanced medical treatments in Japan, but their safety and efficacy have not yet been established and they have not yet become common treatments. The present invention is a synthetic protein preparation, which is less likely to cause side effects such as tumor formation than vector preparations and cell transplantation and is expected to be safer.

[0022] CBD-CNP is suitable for bone formation promotion and its administration method is to administer it in combination with collagen powder to the fracture or osteotomy site during osteosynthesis surgery for fractures or during osteotomy procedures such as high tibial osteotomy for knee osteoarthritis, promoting early bone union through long-term local action. The administration method imposes minimal burden on patients and surgeons. Examples of collagen include type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, and atelocollagen, with type I collagen being preferred.

[0023] CBD-CNP is suitable for treating conditions such as osteoarthritis, achondroplasia, congenital leg length inequality, and dwarfism. By administering a solution containing CBD-CNP intra-articularly and allowing it to act locally on articular cartilage and growth plates, it promotes articular cartilage proliferation and bone elongation. Intra-articular administration may be repeated periodically.

[0024] CBD-CNP is angiogenically applicable to ischemic heart disease (angina pectoris, myocardial infarction, ischemic cardiomyopathy, etc.) and peripheral arterial disease (including arteriosclerotic diseases and non-arteriosclerotic diseases such as Buerger's disease, Takayasu's arteritis, collagen disease-associated vasculitis, fibromuscular dysplasia, external compression, and thrombotic occlusion). Local administration via subcutaneous or intramuscular injection to the ischemic site promotes angiogenesis. Furthermore, vascular grafts, particularly small-diameter ones, frequently become occluded after replacement surgery. By combining collagen with CBD-CNP to create vascular grafts and using them in vascular bypass surgery and vascular graft replacement surgery for large- to medium-sized aneurysms, arterial dissection, and stenotic lesions, it is anticipated that these grafts could be used to prevent occlusion by promoting endothelial cell proliferation in the vascular graft lumen.

[0025] In any of the above-mentioned effects, the applicable diseases, administration methods, dosage forms, solvents, administration forms, administration times and administration intervals are not limited to those in the examples and can be changed as appropriate.

[0026] This is a schematic diagram of the three-dimensional structure of CBD-CNP. The N-terminal CBD binds to collagen, allowing CBD-CNP to remain in a localized location, while the C-terminal CNP binds to the NPR-B (Natriuretic peptide receptor B) receptor, exerting a localized effect on cells. This is a diagram showing the molecular weight measurement of CBD-CNP using a time-of-flight mass spectrometer (TOF-MS). This is a diagram showing the collagen binding ability and thermal stability of CBD-CNP (Example 1). M indicates a molecular weight marker, B indicates a buffer control, No heat indicates no preheating, 56°C 30 min indicates preheating, (-) indicates no collagen powder addition, and (+) indicates collagen powder addition. This is a diagram showing the binding ability of CBD-CNP to the NPR-B receptor (Example 2). An in vitro protein binding assay was performed using CBD-CNP and His-NPR-B, and CBD-CNP was detected only when both CBD-CNP and His-NPR-B were added. Addition of FGF2 to RCS cells resulted in phosphorylation of ERK1 / 2, but addition of CNP-22 or CBD-CNP inhibited this phosphorylation. Figure 3 shows the bone formation promoting effect of CBD-CNP (Example 3). In a mouse femur fracture model, phosphate-buffered saline (PBS), CNP-22, or CBD-CNP was locally administered to the fracture site along with collagen powder. Evaluation was performed using micro-CT four weeks later. The CBD-CNP group showed significantly increased bone mineral content and bone mass compared with the PBS and CNP-22 groups. Figure 4 shows the chondrocyte activation promoting effect of CBD-CNP (Example 4). Addition of 0.2 μM of CNP-22 or CBD-CNP along with FGF2 to rat chondrosarcoma (RCS) cells, a chondrocyte cell line, promoted chondrocyte activation and restored Alcian blue staining in the CBD-CNP group. FIG. 10 shows DAB staining of ex vivo organ culture of mouse fetal tibia, demonstrating that CBD-CNP binds to articular cartilage and remains there for a long period of time (Example 5).This figure shows that CBD-CNP has a significant bone elongation effect in ex vivo organ culture of mouse fetal tibia (Example 5). This figure shows DAB staining performed on mice 1 minute, 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours after a single intra-articular administration of CNP-22 or CBD-CNP to the knee joint. Compared to CNP-22, CBD-CNP bound to the articular cartilage and remained for a longer period (Example 6). This figure shows that, after intra-articular administration of CNP-22 or CBD-CNP three times a week for a total of 8 weeks to a mouse model of knee osteoarthritis, DAB staining revealed that CBD-CNP bound to the articular cartilage and growth plate and remained for a longer period than CNP-22 (Example 6). This figure shows that intra-articular administration of CBD-CNP inhibited subchondral bone thickening using micro-CT of the knee joint (Example 6).

[0027] As used herein, the singular forms (a, an, the, etc.) include both the singular and the plural unless otherwise specified herein or clearly contradictory in context. As used herein, "comprise" is a concept that also encompasses "consist essentially of" and "consist of."

[0028] The CBD used in the present invention is a constituent domain of collagenase produced by Hathewaya histolytica (formerly known as Clostridium histolyticum). A preferred CBD amino acid sequence is composed of 116 amino acids, from positions 1 to 116, of the proteins set forth in SEQ ID NOS: 1 to 3. However, modified proteins in which one or more amino acids, or one to several amino acids (e.g., 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) have been substituted, added, deleted, or inserted may also be used as CBD, as long as they have collagen-binding ability.

[0029] CNP is a peptide isolated from mammals such as pigs, cows, goats, rabbits, rats, mice, monkeys, and humans. For example, the amino acid sequence of CNP-22 is identical among pigs, rats, and humans, with little difference in the amino acid sequence between species. Preferred CNPs of the present invention include CNP-22, CNP-37, CNP-53, or variants thereof, more preferably CNP-22, CNP-37, or variants thereof, and even more preferably CNP-37 or a variant thereof. For example, CNP-37 consists of 37 amino acids from the C-terminus of SEQ ID NO: 1, CNP-53 consists of 53 amino acids from the C-terminus of SEQ ID NO: 2, and CNP-22 consists of 22 amino acids from the C-terminus of SEQ ID NO: 3. The CNP used in the present invention preferably contains 22 amino acids from the C-terminus of SEQ ID NO: 1, and even more preferably contains 37 amino acids from the C-terminus of SEQ ID NO: 1. The CNP variant may be a variant of CNP-37 consisting of 37 amino acids from the C-terminus of SEQ ID NO: 1, CNP-53 consisting of 53 amino acids from the C-terminus of SEQ ID NO: 2, or CNP-22 consisting of 22 amino acids from the C-terminus of SEQ ID NO: 3, and may be a variant of the above sequence in which one or more or one to several, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid has been substituted, added, deleted, or inserted, as long as it has at least one activity selected from the group consisting of an osteogenesis-promoting activity, a cartilage growth-promoting activity, and angiogenesis-promoting activity.

[0030] Although CBD and CNP may be directly linked, they are preferably linked via a spacer. The spacer is composed of amino acids, and the number of amino acids in the spacer is preferably 1 to 10, more preferably 4 to 6. The presence of a spacer reduces the interaction between CBD and CNP and allows CNP to act on NPR-B receptors present in cells at sites distant from the CBD binding site, making it preferable for maintaining both collagen binding ability and physiological activity (osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity). However, if the spacer contains too many amino acids, it may be rapidly cleaved in vivo and physiological activity may be reduced, so there is a preferred range for the number of amino acids in the spacer. Amino acids used in spacers and for preparing variants include the 20 naturally occurring amino acids that constitute proteins (Gly, Ala, Met, Pro, Cys, Ser, Thr, Leu, Ile, Val, Glu, Gln, Asp, Asn, Lys, Arg, His, Trp, Phe, and Tyr). Amino acid residues are classified into several families based on their side chains, such as basic side chains (e.g., Lys, Arg, His), acidic side chains (Asp, Glu), uncharged polar side chains (Gly, Asn, Gln, Ser, Thr, Tyr, Cys), nonpolar side chains (Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), β-branched side chains (Thr, Val, Ile), and aromatic side chains (Tyr, Phe, Trp, His), and amino acid residues within the same family may be substituted with each other.

[0031] A modified CBD-CNP is one in which one or more or one to several amino acids, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1, have been substituted, added, deleted or inserted in SEQ ID NO: 1, 2 or 3, as long as it has at least one activity selected from the group consisting of osteogenesis promoting activity, cartilage growth promoting activity and angiogenesis promoting activity.

[0032] One preferred embodiment of the synthetic protein CBD-CNP of the present invention is a purified synthetic protein obtained by fusing CBD with CNP-37. It retains both collagen-binding ability and the physiological activities of CNP, such as promoting bone formation, cartilage growth, and angiogenesis. Furthermore, as shown in the Examples, it is resistant to heat treatment and has been confirmed to remain in the body for a long period after local administration. This formulation overcomes the short half-life of natural CNP and BMN111, enabling local administration to collagen-containing sites. To facilitate purification, this synthetic protein may be conjugated with a protein tag such as His tag, GST, or MBP, or with a tag such as HA tag, myc tag, or FLAG tag. Furthermore, synthetic proteins of the present invention also include those further conjugated with any peptide at the N- or C-terminus.

[0033] Mutation of one or more, for example, one or several amino acids, of the synthetic protein of the present invention can be carried out by introducing mutations into the DNA encoding the synthetic protein of the present invention using known techniques such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis, or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York).

[0034] From the viewpoint of maintaining the osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity, it is preferable that the above mutations exist in sites that do not affect the active sites or substrate-binding sites of CBD and CNP.

[0035] If the synthetic protein used in the present invention has the effect of promoting bone formation, cartilage growth, or angiogenesis, it preferably has an identity of 90% or more, more preferably 93% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more, to the amino acid sequence shown in SEQ ID NO: 1, 2, or 3. Synthetic proteins consisting of amino acid sequences with such a certain level of identity can be prepared based on known genetic engineering techniques as described above.

[0036] If the synthetic protein has the activity of promoting bone formation, cartilage growth, or angiogenesis, the polynucleotide encoding the preferred synthetic protein used in the present invention has an identity of 90% or more, more preferably 93% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more, to the polynucleotide sequence set forth in SEQ ID NO: 4, 5, or 6. Polynucleotides having such a certain level of identity or more can be prepared based on known genetic engineering techniques as described above.

[0037] Various methods are known for calculating the identity of amino acid sequences or polynucleotide sequences. For example, it can be calculated using an analytical tool that is commercially available or available via an electronic communication line (Internet). In this specification, the identity of amino acid sequences or polynucleotide sequences is calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / with default (initial setting) parameters.

[0038] A preferred polynucleotide of the present invention can be produced or obtained by chemical DNA synthesis based on the present specification or SEQ ID NOs: 4, 5, and 6, or can be easily prepared using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. (see, for example, Molecular Cloning 3d Ed., Cold Spring Harbor Lab. Press (2001)). An example of a chemical DNA synthesis method is solid-phase synthesis using the phosphoramidite method. An automated synthesizer can be used for this synthesis method.

[0039] The advantage of constructing a full-length polynucleotide by chemical synthesis is that the codons used can be designed throughout the entire length of the polynucleotide to suit the host into which the polynucleotide is introduced. Multiple codons encoding the same amino acid are not uniformly used, and their frequency of use varies depending on the biological species. Generally, codons contained in genes that are highly expressed in a given biological species contain many codons that are frequently used in that species. Conversely, in many cases, genes with low expression levels are bottlenecked by the presence of infrequently used codons, hindering high expression. Numerous examples have been reported in which the expression level of a heterologous protein was increased by substituting codons frequently used in the host organism for the polynucleotide sequence during heterologous polynucleotide expression. Such codon modification is expected to be effective in increasing the expression level of the synthetic protein of the present invention.

[0040] The vector of the present invention is a vector into which DNA encoding the synthetic protein of the present invention has been incorporated. Here, a "vector" refers to a nucleic acid molecule (carrier) capable of transporting an inserted nucleic acid molecule into a target such as a cell. There are no particular limitations on the type or structure of the vector, as long as it is capable of replicating and expressing the DNA of the present invention in a suitable host cell. In other words, the vector of the present invention is an expression vector. The type of vector is selected based on the type of host cell. Specific examples of vectors include plasmid vectors, cosmid vectors, phage vectors, and viral vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, herpes virus vectors, etc.). It is also possible to use vectors suitable for use in filamentous fungi as hosts, or vectors suitable for self-cloning.

[0041] When Escherichia coli is used as the host, for example, M13 phage or modified forms thereof, λ phage or modified forms thereof, pBR322 or modified forms thereof (pB325, pAT153, pUC8, etc.) can be used. When yeast is used as the host, pYepSec1, pMFa, pYES2, etc. can be used. When filamentous fungi are used as the host, pUN1, pUSC, etc. can be used. When insect cells are used as the host, for example, pAc, pVL, etc. can be used, and when mammalian cells are used as the host, for example, pCDM8, pMT2PC, etc. can be used, but are not limited to these.

[0042] Expression vectors usually contain promoter sequences necessary for the expression of inserted nucleic acids, enhancer sequences that promote expression, and the like. Expression vectors containing selection markers can also be used. When such expression vectors are used, the presence or absence (and degree) of introduction of the expression vector can be confirmed using the selection marker. Insertion of the DNA of the present invention into a vector, insertion of a selection marker gene (if necessary), insertion of a promoter, and the like can be carried out using standard recombinant DNA techniques (for example, see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York; well-known methods using restriction enzymes and DNA ligase).

[0043] The present invention relates to a transformant in which DNA encoding the synthetic protein of the present invention has been introduced into a host cell. The means for introducing the DNA of the present invention into a host is not particularly limited, and for example, it may be introduced into the host in a state where it has been incorporated into the vector described above. The host cell is not particularly limited as long as it is capable of expressing the DNA of the present invention to produce the synthetic protein. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, mold, insect cells, cultured plant cells, and mammalian cells can be used.

[0044] When the host is a prokaryotic cell, examples include the genera Escherichia, Bacillus, Brevibacillus, and Corynebacterium, such as Escherichia coli C600, Escherichia coli HB101, Escherichia coli DH5α, Bacillus subtilis, Brevibacillus choshinensis, and Corynebacterium glutamicum, respectively. Examples of vectors include pBR322, pUC19, and pBluescript.

[0045] When the host is a yeast, examples include the genera Saccharomyces, Schizosaccharomyces, Candida, Pichia, and Cryptococcus, respectively, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Pichia pastoris, and Cryptococcus sp. Examples of vectors include pAUR101, pAUR224, and pYE32. When the host is a filamentous fungal cell, examples include the genera Aspergillus, Trichoderma, and Talaromyces, respectively, such as Aspergillus oryzae, Aspergillus niger, Trichoderma reesei, and Talaromyces sp.

[0046] The transformant of the present invention is preferably prepared by transfection or transformation using the above-mentioned expression vector. Transformation may be transient or stable. Transfection and transformation can be carried out using calcium phosphate coprecipitation, electroporation, lipofection, microinjection, Hanahan's method, lithium acetate method, protoplast-polyethylene glycol method, etc.

[0047] The transformant of the present invention has the ability to produce the synthetic protein of the present invention, and therefore, it can be used to efficiently produce the synthetic protein of the present invention.

[0048] The expressed synthetic protein can be purified by one or more of the following known methods used in protein and peptide purification: bacterial cells collected from the culture supernatant by centrifugation or the like are disrupted with ultrasound or glass beads, and solid matter such as cell debris is removed by centrifugation or the like to prepare a crude enzyme solution. The resulting solution can then be purified using one or more of the following known methods: ammonium sulfate salting out, precipitation with organic solvents (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectric focusing chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using a substrate or antibody, reverse phase column chromatography, chromatography such as HPLC, and filtration such as microfiltration, ultrafiltration, and reverse osmosis.

[0049] The present invention encompasses pharmaceutical compositions containing synthetic proteins as active ingredients, preferably pharmaceutical compositions for promoting osteogenesis, cartilage growth, or angiogenesis.

[0050] In a preferred embodiment of the present invention, the synthetic protein of the present invention can be administered to a patient as a complex bound to collagen or as a composition containing the synthetic protein and collagen. Collagen can be derived from any animal, such as bovine, porcine, or chicken.

[0051] The pharmaceutical composition or agent for promoting bone formation can be administered during osteosynthesis surgery for fractures, or during a surgical procedure involving bone resection, such as high tibial osteotomy for knee osteoarthritis.

[0052] The pharmaceutical composition or cartilage growth promoter for promoting cartilage growth can be administered to treat diseases such as knee osteoarthritis, achondroplasia, dwarfism, and congenital leg length variance.

[0053] The pharmaceutical composition for promoting angiogenesis or the angiogenesis promoter can be administered to diseases such as ischemic heart disease (angina pectoris, myocardial infarction, ischemic cardiomyopathy, etc.) and peripheral arterial disease (including arteriosclerotic diseases and non-arteriosclerotic diseases such as Buerger's disease, Takayasu's arteritis, collagen disease-associated vasculitis, fibromuscular dysplasia, external compression, and thrombotic occlusion).

[0054] Preparations containing the synthetic protein of the present invention as an active ingredient can be formulated with a pharmaceutical carrier and produced as pharmaceutical compositions suitable for various administration forms. Examples of such forms include oral preparations, injections, suppositories, ointments, and patches. Each of these administration forms can be prepared by conventional formulation methods known to those skilled in the art.

[0055] The synthetic proteins of the present invention can be administered to patients in the form of pharmaceutically acceptable salts as active ingredients in pharmaceutical compositions, bone formation promoters, cartilage growth promoters, and angiogenesis promoters. Pharmaceutically acceptable salts include base addition salts and acid addition salts. Examples of base addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; and organic amine salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, procaine salts, and N,N'-dibenzylethylenediamine salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, and perchlorate; organic acid salts such as acetate, formate, maleate, fumarate, tartrate, citrate, ascorbate, and trifluoroacetate; and sulfonate salts such as methanesulfonate, isethionate, benzenesulfonate, and p-toluenesulfonate.

[0056] As pharmaceutical carriers, various organic or inorganic carrier substances commonly used as formulation materials are used, and are incorporated as excipients, binders, disintegrants, lubricants, coating agents, etc. in solid formulations, and as solvents, solubilizers, suspending agents, isotonicity agents, pH adjusters / buffers, soothing agents, etc. in liquid formulations. Optionally, formulation additives such as preservatives, antioxidants, colorants, flavorings / odors, stabilizers, etc. can also be used.

[0057] When preparing a solid oral preparation, an excipient, optionally an excipient, a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, an odor masking agent, etc., can be added to the compound of the present invention, and then tablets, coated tablets, granules, powders, capsules, etc. can be produced by conventional methods.

[0058] When preparing oral liquid preparations, pH adjusting agents, buffering agents, stabilizers, flavoring agents, odor masking agents, etc. can be added to the compound of the present invention to produce oral liquid preparations, syrups, elixirs, etc. in a conventional manner.

[0059] When preparing injections, pH adjusting agents, buffering agents, stabilizers, isotonicity agents, local anesthetics, etc. are added to the compound of the present invention, and subcutaneous, intramuscular, and intravenous injections can be produced by conventional methods.

[0060] The amount of synthetic protein to be incorporated into each of the above-mentioned dosage unit forms varies depending on the symptoms of the patient to which it is to be administered or on the dosage form, but generally, it is desirable that the amount per dosage unit form be 0.0001 to 1000 mg for oral preparations, 0.001 to 500 mg for injections, and 0.01 to 1000 mg for suppositories.

[0061] The daily dose of the synthetic protein varies depending on the symptoms, body weight, age, sex, etc. of the patient and cannot be determined in general, but is usually about 0.01 to 1000 mg of the synthetic protein or a pharmaceutically acceptable salt thereof per day for an adult (body weight 60 kg), preferably about 0.1 to 300 mg per day, and more preferably about 0.5 to 100 mg per day.

[0062] The present invention will be specifically described below with reference to examples and experimental examples. The present invention is not limited in any way by the following examples, etc., and can be practiced with appropriate modifications within the scope of the spirit described above, and all such modifications are included in the technical scope of the present invention.

[0063] [Design of CBD-CNP Fusion Protein] 1. Regarding the linkage position between CBD and CNP, when fusing CBD to a protein or peptide, it is necessary to design the fusion protein so that it can simultaneously bind to collagen and the receptor that exerts its physiological activity. CBD has a beta-sandwich structure, with the C-terminal carboxyl group located in a sheet different from the sheet that constitutes the collagen-binding site and exposed to solution. Furthermore, the region of CNP containing the disulfide bond between two cysteine ​​residues near the C-terminus is essential for binding to the NPR-B receptor and exerting its physiological activity. Taking these factors into consideration, we decided to fuse the N-terminus of CNP to the C-terminus of CBD.

[0064] 2. Spacer: In the CBD-CNP fusion protein, a spacer was inserted between the CBD and CNP moieties to prevent them from interfering with each other's activity. The results suggest that the C-terminal 22 amino acid residues of CNP (CNP-22) are active, meaning that the N-terminal 15 amino acid residues of CNP-37 are not necessarily required for physiological activity. Therefore, a short (5 amino acid residues) spacer was inserted to improve expression efficiency. Furthermore, the spacer sequence was designed to allow for the independence of the two moieties and provide for fluctuation and free rotation. Two small glycine residues were located near the CBD, a serine residue for hydration, and a proline residue for flexibility and another small glycine residue were located a short distance from the CBD. Taking these factors into consideration, the spacer sequence was chosen as Gly-Gly-Ser-Pro-Gly.

[0065] 3. Expression System Selection and Codons: Because CNP-37 (amino acid sequence from positions 122 to 158 of SEQ ID NO: 1) fused to CBD (amino acid sequence from positions 1 to 116 of SEQ ID NO: 1) is a short 37 amino acid residues, we anticipated that it could be produced using the E. coli expression system previously used for CBD production. To improve the expression efficiency of the fusion protein in the E. coli expression system, the codons of CNP-37 were optimized for E. coli strain B, and synthetic primers containing this sequence were ordered. The protein produced in this example, in which CBD and CNP-37 are fused via a spacer (Gly Gly Ser Pro Gly), is shown in SEQ ID NO: 1. Furthermore, the protein in which CBD and CNP-53 are fused via a spacer (Gly Gly Ser Pro Gly) is shown in SEQ ID NO: 2, and the protein in which CBD and CNP-22 are fused via a spacer (Gly Gly Ser Pro Gly) is shown in SEQ ID NO: 3.

[0066] [Method for constructing a CBD-CNP fusion protein expression plasmid] A DNA fragment (SEQ ID NO: 4) encoding the CBD-CNP-37 fusion protein was obtained by PCR amplification using the CBD gene on the plasmid pCHG115 (Matsushita et al., JBC) as a template, with a forward primer encoding the N-terminus of the CBD and a reverse primer encoding the C-terminus of the CBD, a spacer, and a sequence encoding CNP-37. This DNA fragment was inserted into the TA cloning vector pMD19. The nucleotide sequence of the resulting plasmid was determined to confirm the construction of the DNA fragment encoding the fusion protein. The plasmid was digested with restriction enzymes EcoRI and XhoI to excise the desired DNA fragment, which was then inserted into the same restriction enzyme sites of the GST fusion protein expression vector pGEX-4T-2 and designated the expression plasmid pEPU005.

[0067] The polynucleotide sequence encoding the protein produced in this example in which CBD and CNP-37 are fused via a spacer (Gly Gly Ser Pro Gly) is shown in SEQ ID NO: 4. The polynucleotide sequence encoding the protein in which CBD and CNP-53 are fused via a spacer (Gly Gly Ser Pro Gly) is shown in SEQ ID NO: 5, and the polynucleotide sequence encoding the protein in which CBD and CNP-22 are fused via a spacer (Gly Gly Ser Pro Gly) is shown in SEQ ID NO: 6.

[0068] CBD-CNP Purification Method Using E. coli: Escherichia coli BL21 CodonPlus RIL (Agilent, Santa Clara, CA) was transformed with the CBD-CNP-37 fusion protein expression plasmid (pEPU005) and cultured in 4,000 mL of 2YT-G medium supplemented with 50 mg / mL ampicillin and 30 mg / mL chloramphenicol at 37°C with shaking at 150 rpm. Expression of the GST fusion protein was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside, and the E. coli cell wall was disrupted twice under French pressure at 10,000 psi. The lysate, free of cell debris, was mixed with glutathione-Sepharose beads to bind the fusion protein, followed by elution with a glutathione-containing solution. GST was cleaved with thrombin protease and dialyzed three times at 4°C against 1,000 mL of 50 mM Tris-HCl (pH 7.5), 1 mM CaCl2. The resulting fraction was mixed with glutathione-Sepharose beads, and CBD-CNP was eluted with 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM CaCl2. Molecular mass measurement using a time-of-flight mass spectrometer (TOF-MS) confirmed that the molecular weight of CBD-CNP was 18,074 Da, as predicted (Figure 3). In the following examples, the CBD-CNP-37 fusion protein is referred to as CBD-CNP.

[0069] CBD-CNP stably retains its collagen-binding ability even after heat treatment. 10 mg of collagen powder (Sigma, type I C-9879) was placed in the filter cup of a Millipore Ultrafree MC GV filter, and 100 μL of a solution containing 200 pmol of either CBD-CNP preheated at 56°C for 30 minutes or CBD-CNP without preheating was added. After incubation at room temperature for 30 minutes, the mixture was centrifuged at 15,000 rpm for 5 minutes, and the filtrate was collected and subjected to SDS-PAGE (Figure 4).

[0070] When CBD-CNP was reacted with collagen for 30 minutes without preheating, the CBD-CNP bound to the collagen and did not pass through the filter. The results were similar when CBD-CNP was preheated. From the above, it was demonstrated that CBD-CNP has collagen-binding ability, and that even when preheated at 56°C for 30 minutes, the protein did not denature and collagen-binding activity was maintained.

[0071] Binding of CBD-CNP to NPR-B Receptor: To evaluate whether CBD-CNP binds to NPR-B, the receptor for CNP-22, an in vitro protein binding assay was performed. Purified CBD-CNP (18 kDa) and His-NPR-B (DIMA BIOTECH; 49 kDa) proteins were incubated overnight at 4°C to allow binding. The protein complex was then co-precipitated with equilibrated Ni-NTA agarose beads (QIAGEN) by end-over-end mixing at 4°C for 1 hour. Nonspecific binding was then removed by washing four times with wash buffer, and the protein bound to the agarose beads was eluted with SDS sample buffer. After heating at 98°C for 5 minutes to dissociate the CBD-CNP and His-NPR-B binding, the protein was detected using anti-His-tag antibodies (MBL) and anti-CNP antibodies (Abbexa). CBD-CNP was detected only when both CBD-CNP and His-NPR-B were added, indicating the binding of CBD-CNP to the NPR-B receptor (Fig. 5A).Furthermore, the addition of FGF2 to the chondrocyte cell line RCS induced phosphorylation of ERK1 / 2, but the addition of CNP-22 or CBD-CNP suppressed this phosphorylation (Fig. 5B).

[0072] Bone formation promoting effect of CBD-CNP. Femoral fracture models were created using 9-week-old male C57BL / 6J mice as follows. Under sterile conditions, a 4 mm left medial parapatellar incision was made, and a 0.5 mm hole was drilled in the intracondylar notch. A 0.2 mm tungsten guidewire was inserted retrogradely into the medullary cavity, and a portion of the femur was resected laterally. After the guidewire was removed, a 0.5 mm diameter stainless steel screw was inserted into the medullary cavity to stabilize the fracture. Immediately after fracture model creation, 800 μg / kg of CNP-22 (Peptide Institute, Inc.) and 6576 μg / kg of CBD-CNP were administered locally to the fracture site, along with 5 mg of porcine dermis-derived type I collagen powder (Nippi). The PBS group received only PBS, the solvent for CNP-22 and CBD-CNP. The dose of CBD-CNP was set to be the same as that of CNP-22 on a molar basis, since the molecular weight of CNP-22 is 2,197.6 Da and that of CBD-CNP is 18,074 Da. Administration was performed to eight rats in each group, and micro-CT scans were performed four weeks later. Significant increases in bone mineral content and bone mass were observed in the CBD-CNP group compared to the PBS and CNP-22 groups (Figure 6).

[0073] As a specific clinical example based on the above, during osteosynthesis surgery for fractures or high tibial osteotomy for knee osteoarthritis, CBD-CNP can be administered together with collagen powder to the fracture or osteotomy site, allowing for long-term local action, thereby promoting bone formation and enabling early healing.

[0074] CBD-CNP promotes chondrocyte activity. RCS cells, a chondrocyte cell line, were seeded at 100,000 cells per well in a 6-well plate. The following day, FGF2 was added with 0.2 μM CNP-22 or 0.2 μM CBD-CNP. Cell counts were performed using the Cell Counting Kit-8 (DOJINDO) 72 hours after drug addition. Cell activity was reduced in the group treated with FGF2 alone, whereas the addition of FGF2 and CBD-CNP significantly restored cell activity. Furthermore, Alcian blue staining revealed that RCS cells produce abundant proteoglycans, which are stained with Alcian blue. However, the addition of FGF2 suppressed proteoglycan production, resulting in little Alcian blue staining. Alcian blue staining was restored in RCS cells treated with both FGF2 and CBD-CNP (Figure 7).

[0075] Long-term effects of CBD-CNP on articular cartilage and bone elongation in ex vivo culture. Tibiae were isolated from both hindlimbs of FVB / NJcl mice at embryonic day 16.5 and cultured ex vivo. Addition of 100 ng / mL FGF2 (R&D Systems) resulted in tibial shortening after 4 days, similar to that observed in achondroplasia. Next, two tibiae from the same animal were treated with 100 ng / mL FGF2 on both sides and 0.2 μM CNP-22 or CBD-CNP on the other side. The following day, the medium was changed every 24 hours, but only 100 ng / mL FGF2 was added to both sides. After 4 days, DAB staining and tibial length measurements were performed. While CNP-22 did not show any significant staining effects in DAB staining, CBD-CNP did show significant staining effects in articular cartilage (Figure 8). Furthermore, CNP-22 did not show any bone elongation effect, but CBD-CNP showed a significant bone elongation effect (Figure 9).

[0076] Long-term retention of CBD-CNP in articular cartilage and growth plate following intra-articular administration in mice. Ten-week-old male FVB / NJcl mice were intra-articularly administered CNP-22 (0.5 μg / 10 μL) or CBD-CNP (4.1 μg / 10 μL). The femurs and tibias were removed at 1 minute, 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours, and the intra-articular retention of each protein was assessed by DAB staining. CNP-22 and CBD-CNP were administered at equivalent molar doses. While CNP-22 did not show any significant staining effects beyond 1 minute post-administration, CBD-CNP stained the articular cartilage of the femur and tibia from 1 minute to 6 hours post-administration, demonstrating a longer retention in the articular cartilage than CNP-22 (Figure 10).

[0077] We generated osteoarthritis model mice by performing medial meniscal ligament resection on the right hind limb of 10-week-old male FVB / NJcl mice. Starting 2 weeks after model creation, CNP-22 or CBD-CNP was intra-articularly administered three times a week for a total of 8 weeks. Three days after the final administration at 8 weeks, hind limbs were harvested and histopathological images were analyzed by DAB staining. While CNP-22 did not significantly stain the articular cartilage of the femur and tibia, CBD-CNP stained the cartilage in the growth plate and in the articular cartilage of the femur and tibia (Figure 11A). Micro-CT analysis of the knee joint revealed thickening of the subchondral bone in the osteoarthritis model, and intra-articular administration of CBD-CNP inhibited this thickening (Figure 11B).

[0078] The present invention provides a synthetic protein (CBD-CNP) in which collagen-binding ability has been added to CNP, which has functions of promoting osteogenesis, cartilage growth, and angiogenesis.

[0079] The synthetic protein of the present invention can overcome the drawbacks of CNP, such as early deactivation and short half-life.

[0080] The collagen-binding ability of the synthetic protein enables efficient local drug delivery, which is expected to improve local efficacy, reduce the number of doses and total dose, and reduce systemic side effects compared to systemic administration.

[0081] The synthetic proteins of the present invention can be administered to the fracture site along with collagen powder to promote bone healing.

[0082] The synthetic protein of the present invention is also effective in local articular cartilage regeneration and angiogenesis, and can be applied to a wide range of diseases.

[0083] Because CNP has angiogenesis-promoting properties (Non-Patent Document 4), the angiogenesis-promoting effects of local administration of CBD-CNP could make it a potential therapeutic agent for ischemic heart disease and peripheral arterial disease. It is anticipated that intramuscular or subcutaneous injection of CBD-CNP into ischemic myocardium or peripheral tissues would promote angiogenesis.

[0084] Furthermore, artificial blood vessels, especially small-diameter ones, frequently become clogged after replacement surgery. By combining collagen with CBD-CNP to create artificial blood vessels and using them in vascular bypass surgery, aneurysms, and artificial blood vessel replacement surgery for stenotic lesions, it is expected that they will be used to prevent blockages by promoting the proliferation of vascular endothelial cells in the artificial blood vessel lumen.

[0085]

[0086]

Claims

1. A synthetic protein containing an N-terminal collagen-binding domain (CBD) and a C-terminal C-type natriuretic peptide domain (CNP).

2. The synthetic protein of claim 1, further comprising a spacer connecting the CBD and the CNP.

3. A synthetic protein according to claim 1, any one of the following (i) to (iii): (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3; (ii) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3 in which one or more amino acids have been deleted, substituted or added, and which has at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity; (iii) a protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3, and which has at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity.

4. A complex formed by binding the synthetic protein of claim 1 with collagen.

5. A composition comprising the synthetic protein of claim 1 and collagen.

6. A polynucleotide selected from any of the following (a) to (e): (a) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3; (b) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3 in which one or more amino acids have been deleted, substituted, added, or inserted, and having at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity; (c) a polynucleotide encoding a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, and having at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity; (d) a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 4, 5, or 6; (e) a polynucleotide encoding a protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence set forth in SEQ ID NO: 4, 5, or 6, and having at least one activity selected from the group consisting of osteogenesis-promoting activity, cartilage growth-promoting activity, and angiogenesis-promoting activity.

7. An expression vector comprising the polynucleotide of claim 6.

8. A transformant into which the polynucleotide according to claim 6 or the expression vector according to claim 7 has been introduced.

9. A bone formation promoter comprising the synthetic protein according to any one of claims 1 to 3, the complex according to claim 4, or the composition according to claim 5 as an active ingredient.

10. A cartilage growth promoter comprising the synthetic protein according to any one of claims 1 to 3, the complex according to claim 4, or the composition according to claim 5 as an active ingredient.

11. An angiogenesis promoter comprising the synthetic protein according to any one of claims 1 to 3, the complex according to claim 4, or the composition according to claim 5 as an active ingredient.

12. A pharmaceutical composition comprising the synthetic protein according to any one of claims 1 to 3, the complex according to claim 4, or the composition according to claim 5 as an active ingredient.

13. The pharmaceutical composition according to claim 12, which is for promoting bone formation, cartilage growth or angiogenesis.

14. A method for preventing or treating knee osteoarthritis, achondroplasia, dwarfism, congenital leg length inequality, ischemic heart disease, or peripheral arterial disease, comprising administering to a patient the synthetic protein of any one of claims 1 to 3, the complex of claim 4, or the composition of claim 5.

15. Use of the synthetic protein according to any one of claims 1 to 3, the complex according to claim 4, or the composition according to claim 5 for producing an agent for promoting bone formation, cartilage growth, or angiogenesis.

16. The synthetic protein of any one of claims 1 to 3, the complex of claim 4, or the composition of claim 5 for preventing or treating knee osteoarthritis, achondroplasia, dwarfism, congenital leg length inequality, ischemic heart disease, or peripheral arterial disease.

17. Use of the synthetic protein of any one of claims 1 to 3, the complex of claim 4, or the composition of claim 5 for the prevention or treatment of knee osteoarthritis, achondroplasia, dwarfism, congenital leg length inequality, ischemic heart disease, or peripheral arterial disease.

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