Long-acting parathyroid hormone receptor agonist fusion protein and use thereof

Abstract

A long-acting parathyroid hormone receptor agonist fusion protein and the use thereof. The fusion protein can inhibit the activity of RANKL and promote PTHR-mediated bone anabolism, and contains a first arm and a second arm. The first arm contains a functional region targeting RANKL and an Fc region, and the second arm contains a functional region capable of activating PTHR and an Fc region. The long-acting parathyroid hormone receptor agonist fusion protein has multiple advantages, e.g., better drug efficacy, longer lasting effect, better safety, higher structural stability and higher expression level, and the overall performance thereof is superior to that of an existing or potential treatment means, e.g., teriparatide, PEG-PTH, anti-RANKL monoclonal antibody, romosozumab, or the control sample synPTH-αRANKL-1.

Description

Long-acting parathyroid hormone receptor agonist fusion protein and its application

[0001] This application claims priority to Chinese Patent Application No. 2023111664518 filed on September 11, 2023, and Chinese Patent Application No. 2024112526007 filed on September 6, 2024. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to a long-acting parathyroid hormone receptor agonist fusion protein and applications thereof. Background Art

[0003] Osteoporosis is a common degenerative bone disease characterized by bone loss and bone structural degeneration. It is caused by an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts (Dirckx N, Van Hul M, Maes C. Osteoblast recruitment to sites of bone formation in skeletal development, homeostasis, and regeneration. Birth Defects Res C Embryo Today. 2013; 99(3): 170-91.). When the rate of bone resorption exceeds that of bone formation, bone density decreases and the risk of fracture increases, thereby inducing osteoporosis (Egermann M, Schneider E, Evans CH, et al. The potential of gene therapy for fracture healing in osteoporosis[J]. Osteoporos Int, 2005, 16(Suppl 2): ​​S120-S128.).

[0004] The World Health Organization has classified osteoporosis as the second most detrimental disease to human health, second only to cardiovascular disease. Because the disease lacks warning signs, its first symptom is a fracture. Among people over 50, the incidence of osteoporotic fractures is as high as 19.2%. Within one year of a hip fracture, 20% of patients die from various complications, and approximately 50% of survivors become disabled. Osteoporotic fractures (also known as fragility fractures) are the most serious consequence of osteoporosis, not only impacting patients' quality of life but also being a leading cause of disability and mortality in elderly patients, placing a heavy burden on families and society.

[0005] Currently, the commonly used drugs for the treatment of osteoporosis in clinical practice include calcitonin, bisphosphonates, estrogen drugs and monoclonal antibodies against receptor activator of nuclear factor kappa-b ligand (RANKL). Although they have achieved certain therapeutic effects, these drugs can only inhibit bone resorption but cannot promote bone formation. Therefore, they can only delay the progression of the disease but cannot cure it.

[0006] Most elderly patients with osteoporosis have very low bone metabolism and turnover rates, and many osteoporosis medications inhibit osteoclasts, which actually has an inhibitory effect on bone metabolism or turnover. In terms of bone reconstruction and regeneration, women over 65 with osteoporotic vertebral fractures and a BMD below -2.5SD, postmenopausal patients with multiple vertebral or hip fractures, patients who continue to experience osteoporotic fractures despite taking medications that inhibit bone resorption, and patients with multiple osteoporotic fractures should use more medications that promote bone formation. This has two purposes: first, to effectively increase bone strength and prevent the next fracture; second, to improve bone quality, as some patients require surgery, and poor bone quality can affect the effectiveness of surgical treatment.

[0007] In recent years, bone formation promoters have garnered significant attention. Parathyroid hormone (PTH) is an 84-amino acid peptide that plays a crucial role in bone remodeling. Binding of PTH to the parathyroid hormone receptor (PTHR) directly induces bone formation by activating cAMP and the canonical Wnt signaling pathway. Teriparatide (rhPTH(1-34)), a commercially available therapeutic PTH peptide, is a fragment of the N-terminal 34 amino acids of mature human PTH. It has been shown to increase bone formation activity in patients with osteoporosis and is FDA-approved for the treatment of primary osteoporosis, hypogonadal osteoporosis, and postmenopausal osteoporosis. Intermittent low-dose teriparatide can promote bone formation and increase bone mass, but daily subcutaneous injections are inconvenient for patients and can lead to poor patient compliance. To address the issue of poor compliance with teriparatide, some research institutions have constructed PEG-PTH 1-34 in an attempt to prolong the half-life of teriparatide. Although in vitro studies have shown that the activity of PEG-PTH 1-34 is comparable to that of teriparatide, in vivo efficacy data have shown that the effect of PEG-PTH 1-34 in promoting bone formation is significantly worse (Valderrama P, Jung RE, Thoma DS, et al. Evaluation of parathyroid hormone bound to a synthetic matrix for guided bone regeneration around dental implants: a histomorphometric study in dogs [J]. Journal of Periodontology, 2010, 81(5): 737-747. DOI: 10.1902 / jop.2010.090562.). In addition, if teriparatide is used continuously at high doses for treatment, although it can promote bone formation, it can also cause bone loss. Therefore, it is not suitable for continuous blood drug exposure treatment.On the one hand, parathyroid hormone promotes the differentiation of bone marrow stem cells into osteoblasts, promotes bone formation, and increases bone mass, thereby improving the biomechanical properties of bones (Friedl G, Turner RT, Evans GL, et al. Intermittent parathyroid hormone (PTH) treatment and age-dependent effects on rat cancellous bone and mineral metabolism[J]. J Orthop Res, 2007, 25: 1454-1464). On the other hand, it plays an important role in maintaining the body's calcium and phosphorus metabolic balance by enhancing the activity of osteoclasts, promoting bone resorption, and increasing blood calcium; acting on the kidneys to increase blood calcium and lower blood phosphorus; activating the kidney's 1α-hydroxylase, indirectly promoting intestinal calcium and phosphorus reabsorption, and increasing blood calcium and phosphorus. Studies have found that the use of PTH1-34 drugs (such as teriparatide) may cause side effects such as dizziness, nausea, back pain, and lower limb cramps (Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med 2001; 344: 1434-41; The effect of teriparatide [human parathyroid hormone (1-34)] therapy on bone density in men with osteoporosis. J Bone Miner Res 2003; 18: 9-17.). The occurrence of these side effects is related to blood calcium and phosphorus levels.

[0008] Currently, the only marketed drug that can inhibit bone resorption and promote bone formation is romosozumab, a humanized anti-sclerostin monoclonal antibody. However, this product carries an FDA black box warning, which prohibits its use in patients with a history of myocardial infarction or stroke within the past year, and limits its maximum use to one year. Furthermore, no combination therapy that can inhibit bone resorption and promote bone formation has been approved for marketing. Although neutralizing antibodies and therapeutic PTH peptides targeting RANKL are known, and the combined use of anti-RANKL monoclonal antibodies and therapeutic PTH peptides may be useful for patients with low bone mass conditions such as osteopenia, the problem of poor patient compliance remains unavoidable. Furthermore, while co-formulation of multiple agents may offer convenience, finding suitable formulation conditions for a single administration is often challenging or difficult to obtain, and both co-administration and co-formulation involve additive development, manufacturing, and regulatory costs associated with each agent. Amgen once attempted to construct a fusion compound (synPTH-αRANKL-1, which is rhPTH1-34 fused to the N-terminus of the denosumab heavy chain). When the inventors used conventional methods to prepare synPTH-αRANKL-1, they unexpectedly discovered that the prepared product of synPTH-αRANKL-1 contained a large number of broken fragments of the PTH functional region. This shows that the yield and stability of this molecule during the preparation process are low, resulting in high preparation costs.

[0009] Other bone disorders that require treatment by regulating bone formation and / or bone resorption in lesion-related areas include osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, bone injury, degenerative lumbar spondylolisthesis, or degenerative disc disease. Bone injuries include fractures (especially osteoporotic fractures, nonunion of fractures, and delayed union of fractures), bone cracks, cartilage damage, osteonecrosis, and bone damage caused by arthritis (such as rheumatoid arthritis, osteoarthritis, infectious arthritis, etc., caused by degeneration, trauma, strain, infection, and autoimmune diseases) or tumors (such as bone tumors and tumor bone metastases). Currently, common treatments have poor compliance, poor therapeutic effects, or many side effects, and there are no effective therapeutic drugs in clinical practice.

[0010] Therefore, there is an urgent need to develop a new product that can inhibit bone resorption and promote bone formation.

[0011] Summary of the Invention

[0012] Technical problems existing in existing technologies include poor structural stability of the Anti-RANKL-PTH fusion protein, poor compliance with teriparatide, the risk of hypercalcemia and / or osteoclast advantage (catabolic effects) caused by long-acting PTH (such as PEG-PTH1-34 or palopeggteriparatide), the anti-sclerostin antibody romumab, which can inhibit bone resorption and promote bone formation, has a maximum usage period of one year and is contraindicated for patients with a history of myocardial infarction or stroke within one year, the poor effect of existing marketed drugs in rapidly increasing bone density in the short term, and the side effects of anti-RANKL monoclonal antibodies such as mandibular osteonecrosis. In response to the above technical problems, the fusion protein developed by the present invention (Long-Acting PTHR Agonist Fusion protein, LAPAF for short) can inhibit bone resorption and promote bone formation through a brand-new design, which can quickly increase bone density and reach a higher level within a short period of time (such as 2 months); the fusion protein has long-term effectiveness but high safety, no risk of hypercalcemia, no osteoclast advantage, low risk of mandibular osteonecrosis, and is expected to be used for a long time; at the same time, the fusion protein has a stable structure, high preparation yield, and low preparation cost.

[0013] The present invention has attempted a variety of fusion structures. Compared to the reported fusion structures of anti-RANKL antibodies and PTH (such as US 8,992,925B2 and US11,492,386B2), the present invention has selected a completely different fusion protein structure (i.e., Structure 1 of the present invention). A single molecule of this structure contains only one RANKL binding site and one PTHR binding site, and its molecular weight is lower than that of traditional monoclonal antibodies, which is more conducive to drug distribution in the body. At the same time, when LAPAF is prepared using conventional methods, the fragmentation rate of the PTH functional region is less than 25%, and the fragmentation rate of the preferred molecule can even be less than 20%, which is much lower than the control sample synPTH-αRANKL-1 (fragmentation rate of 50%). At the same time, by adjusting the anti-RANKL functional region of LAPAF, or adjusting the linker, or adjusting the length (from a minimum of 11 amino acids to a maximum of 84 amino acids) or type (PTH or PTHrP) of the PTH functional region, or even the Fc region, LAPAF can maintain a high structural stability and have a high expression level.

[0014] In vitro activity data showed that LAPAF's inhibitory activity on the proliferation of Raw264.7 cells induced by RANKL was comparable to that of anti-RANKL monoclonal antibody and control sample synPTH-αRANKL-1, suggesting that LAPAF has a good activity in inhibiting bone resorption. At the same time, LAPAF has a good inhibitory effect on cells expressing RANKL and PTHR (RANKL) while simulating osteoblasts. + / PTHR + cells) showed a stronger in vitro activity against PTHR-induced cAMP release than teriparatide, whereas the control sample synPTH-αRANKL-1 had a stronger effect on RANKL + / PTHR + The in vitro activity of cell PTHR-induced cAMP release is only comparable to that of teriparatide. At the same time, adjusting the anti-RANKL functional region of LAPAF, or the linker, or the length or type of the PTH functional region (PTH or PTHrP), or the Fc region, the effect of LAPAF on RANKL + / PTHR + LAPAF can enhance the effects of drugs on osteoblasts.

[0015] In vivo efficacy studies showed that in an ovariectomized transgenic mouse model of osteoporosis induced by humanized RANKL, LAPAF significantly increased bone mineral density compared to the control sample, synPTH-αRANKL-1, at day 56 after a single dose. LAPAF still significantly downregulated TRAP5b levels at day 56 after a single dose, suggesting that it can sustainably inhibit osteoclast activity, whereas the control sample, synPTH-αRANKL-1, had little effect. Furthermore, LAPAF significantly upregulated osteocalcin at day 56 after a single dose, suggesting that it can sustainably activate osteoblast activity, whereas the control sample, synPTH-αRANKL-1, had little effect.

[0016] Literature reports that continuous infusion of human PTH (1-34) or abaloparatide 80 μg / kg BW / day for 2 weeks in mice can maintain strong bone formation activity, but at the same time, the activity of osteoclasts is also significantly increased, and ultimately leads to a significant decrease in bone density in mice, that is, the phenomenon of osteoclast advantage (Catabolic Effects) (Le Henaff C, Finnie B, Pacheco M, He Z, Johnson J, Partridge NC. Abaloparatide Has the Same Catabolic Effects on Bones of Mice When Infused as PTH (1-34). JBMR Plus. 2023 Jan 5; 7(2): e10710. doi: 10.1002 / jbm4.10710.). Similarly, preclinical studies of TransCon PTH (Palopegteriparatide) have also reported the phenomenon of osteoclast dominance. In the TPTx (parathyroidectomy) rat model, TransCon PTH was administered daily at a low dose of 1.2 nmol / kg and a high dose of 2.4 nmol / kg. After 4 weeks, the bone density was significantly decreased compared with the model group (Holten-Andersen L, Pihl S, Rasmussen CE, et al. Design and Preclinical Development of TransCon PTH, an investigational Sustained-Release PTH Replacement Therapy for Hypoparathyroidism J Bone Miner Res. 2019 Nov; 34(11): 2075-2086. DOI: 10.1002 / jbmr.3824). Although LAPAF was observed to have sustained and highly active blood drug exposure in a single-dose experiment in the RANKL humanized transgenic mouse osteoporosis model induced by ovariectomy (at D56, the blood sample of the 10mpk dose group diluted 10 times still had the same effect on RANKL as LYN101-2 at a concentration of 1nM), + / PTHR + However, the bone density still showed a good improvement effect, and the bone resorption markers were still in a down-regulated state. There was no phenomenon of decreased bone density and osteoclast dominance, which was completely different from the above reports.

[0017] Furthermore, in the same RANKL humanized transgenic mouse osteoporosis model caused by ovariectomy, the present invention increased the dosing frequency, adopted a weekly dosing interval, and continuously administered for four weeks to further increase the blood exposure of LAPAF. At the same time, a control group of anti-RANKL monoclonal antibody and teriparatide was set up. The results showed that LAPAF could still significantly downregulate CTX-1, indicating that it can continue to significantly inhibit the activity of osteoclasts, and can significantly upregulate osteocalcin, indicating that it can continue to significantly activate the activity of osteoblasts, and ultimately achieve a significant increase in bone density. No osteoclast advantage was observed, and the bone density improvement effect was even better than the anti-RANKL monoclonal antibody (weekly administration) and teriparatide (daily administration) combination group.

[0018] The above two experimental studies show that the present invention has uniquely and effectively overcome the problems of osteoblastic advantage and bone loss caused by continuous exposure to PTH activity, solved the problem of compliance with PTH treatment, and unexpectedly showed sustained effectiveness for up to 56 days or even longer.

[0019] In order to avoid species differences, the present invention further investigated the in vivo efficacy on non-human primates that are closer to humans. The results of the in vivo efficacy experiment of a single dose in a spontaneous elderly osteoporosis rhesus monkey model showed that LAPAF can still overcome the problem of bone loss caused by continuous exposure to PTH activity. It is not obvious that LAPAF takes effect very quickly and increases bone density very strongly. It can quickly increase bone density in a short period of time (such as 2 months), and the bone density increase ratio (>30%) is significantly better than other osteoporosis treatment drugs on the market reported in the literature. For example, after 6 months of continuous administration (once a day), L2-L4 (L is Lumbar The bone density of the lumbar spine (hereinafter referred to as L) only increased by about 5% (accessdata.fda.gov / drugsatfda_docs / nda / 2002 / 21-318_FORTEO_Pharmr_P1.pdf). The anti-sclerostin monoclonal antibody Romosozumab only increased the bone density of L1-L4 by about 8% after four months of continuous administration (once a month) (accessdata.fda.gov / drugsatfda_docs / nda / 2019 / 761062Orig1s000MultidisciplineR.pdf). The anti-RANKL monoclonal antibody Denosumab only increased the bone density of L1-L4 by about 2% after three months of single administration. There is no literature reporting that these drugs can rapidly increase bone density in the short term (such as two months). This further verifies the long-term, rapid-acting, and potent efficacy of LAPAF.

[0020] Due to the excellent characteristics demonstrated in the experiment, the present invention has the feasibility of meeting unmet clinical needs such as fracture healing, bone nonunion (such as nonunion or delayed union of fractures), and bone damage caused by osteoarthritis, which require rapid or / and continuous increase in bone mass through the promotion of osteogenesis. The in vivo efficacy results of the osteoporotic femoral fracture model (osteoporotic fracture model mice) of RANKL humanized transgenic mice with femoral fractures caused by ovarian removal and surgical modeling also showed that LAPAF can significantly accelerate the fracture healing of osteoporotic fracture model mice after continuous administration for four weeks (once a week) (for example, LAPAF can make the number of animals with closed fracture lines in osteoporotic fracture model mice more, and the bone mineral deposition rate and bone tissue formation rate at the fracture site are significantly increased). The bone density of both the fracture site (femur) and the non-fracture site (tibia) is significantly increased. In addition, the bone mineral deposition rate (Mineral Apposition Rate, MAR) and bone tissue formation rate (Bone Formation Rate) obtained by non-decalcified bone tissue sectioning technology are significantly increased. Data from the 2019 LAPAF trial showed that LAPAF significantly increased the MAR and BFR in mice with osteoporotic fracture models, with some mice even able to increase their levels to near-normal levels. The efficacy of LAPAF in fracture healing was significantly better than that of the teriparatide (daily administration) group and the combination group (anti-RANKL monoclonal antibody (weekly administration) and teriparatide (daily administration)). Furthermore, significant therapeutic effects were observed in in vivo efficacy experiments in a mouse osteoarthritis model induced by medial meniscal transection (MMT). For example, after eight weeks of continuous administration (once per week), LAPAF significantly improved the weight-bearing capacity of the modeled foot and significantly alleviated joint pain caused by MMT.

[0021] Furthermore, the present invention conducted a toxicity and toxicokinetics study on rhesus monkeys with five subcutaneous injections of LAPAF every three weeks for twelve consecutive weeks. The results showed that no obvious PTH-related toxicity such as hypercalcemia was observed in LAPAF at a dose of 0.1mpk and 0.4mpk once every three weeks for five consecutive subcutaneous injections. The hypercalcemia caused by PTH is mainly attributed to the effect of PTH on the kidney PTHR, which increases calcium reabsorption. The biggest difference between kidney tissue and bone tissue is the difference in RANKL expression. Osteoblasts are double-positive cells (PTHR) that express both RANKL and PTHR. + / RANKL + ), while the kidney tissue is a single positive cell that does not express RANKL but expresses PTHR (PTHR + / RANKL - ), for this reason, the present invention further investigated the effect of LAPAF on PTHR single positive cells (PTHR) simulating kidney cells.+ / RANKL - ) in vitro activity, and the results showed that LAPAF had an effect on PTHR + / RANKL - The in vitro activity of PTHR-induced cAMP release in cells was hundreds of times weaker than that of teriparatide, while the control sample synPTH-αRANKL-1 had similar activity to teriparatide. This shows that LAPAF exhibited unexpectedly strong RANKL selectivity compared with the control sample and teriparatide. Adjusting the anti-RANKL functional region of LAPAF, or adjusting the linker, or adjusting the length or type of the PTH functional region (PTH or PTHrP), or adjusting the Fc region still maintained significant RANKL selectivity.

[0022] In summary, the long-acting parathyroid hormone receptor agonist fusion protein (LAPAF) of the present invention has multiple advantages, including better efficacy, longer-lasting action, improved safety, greater structural stability, and higher expression levels. Its overall performance is superior to existing or potential treatments such as teriparatide, PEG-PTH 1-34, anti-RANKL monoclonal antibodies, or the control sample synPTH-αRANKL-1.

[0023] The present invention solves the above technical problems through the following technical solutions.

[0024] The first aspect of the present invention provides a long-acting parathyroid hormone receptor agonist fusion protein, which comprises a first arm and a second arm; the first arm comprises a functional region targeting RANKL and an Fc region, and the functional region targeting RANKL is Fv, ScFv, SDA, Fab or ScFab (preferably Fab or ScFab), and the functional region targeting RANKL is located at the N-terminus of the Fc region; the second arm comprises a functional region that can activate PTHR (parathyroid hormone receptor 1) and an Fc region, and the C-terminus of the functional region that can activate PTHR of the second arm is directly or through a linker (Linker) connected to the N-terminus of the Fc region, or the functional region that can activate PTHR of the second arm is connected to the amino acid residue before the CPPCP sequence of the Fc region through site-directed coupling technology.

[0025] In some embodiments of the present invention, the long-acting parathyroid hormone receptor agonist fusion protein is a dual-targeting fusion protein of the present invention.

[0026] The second aspect of the present invention provides a long-acting parathyroid hormone receptor agonist fusion protein, which comprises a first arm and a second arm; the first arm consists of a functional region targeting RANKL and an Fc region, and the functional region targeting RANKL is in the form of Fv, ScFv, SDA, Fab or ScFab (preferably Fab or ScFab), and the functional region targeting RANKL is located at the N-terminus of the Fc region; the second arm consists of a functional region that can activate PTHR (parathyroid hormone receptor 1) and an Fc region, and the C-terminus of the functional region that can activate PTHR of the second arm is directly or through a linker (Linker) connected to the N-terminus of the Fc region, or the functional region that can activate PTHR of the second arm is connected to the amino acid residue before the CPPCP sequence of the Fc region through site-directed coupling technology.

[0027] In some embodiments of the present invention, the Fc region is composed of FcA and FcB, wherein FcA and FcB are respectively the tandem heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of human IgG, including the hinge region. FcA and FcB are identical or have a Knob(s) mutation and a Hole(s) mutation, respectively. Preferably, FcA and FcB have a Knob(s) mutation and a Hole(s) mutation, respectively.

[0028] In some preferred embodiments of the present invention, the human IgG is selected from wild-type or mutant types of human IgG1, IgG2, IgG3, and IgG4.

[0029] In some more preferred embodiments of the present invention, the IgG1 is a mutant type that silences the Fc immune effector function (such as a combination of one or more of the LALA mutation, PGLALA mutation, N297A mutation and other IgG1 Fc immune effector function silent mutations), the IgG2 includes a mutant type with normal Fc immune effector function or a mutant type with silent Fc immune effector function, the IgG3 is a mutant type with silent Fc immune effector function, and the IgG4 includes a mutant type with silent Fc immune effector function (such as one or more of E233P / F234V / L235A / D265A / R409K mutations) and / or an S228P mutation that improves molecular stability. The numbering of the Fc region residues adopts the numbering of the EU index in Kabat.

[0030] In some embodiments of the present invention, when the FcA comprises the amino acid sequence shown in SEQ ID NO: 6, the FcB comprises the amino acid sequence shown in SEQ ID NO: 6.

[0031] In some embodiments of the present invention, when the FcA comprises the amino acid sequence shown in SEQ ID NO: 7, the FcB comprises the amino acid sequence shown in SEQ ID NO: 8; when the FcA comprises the amino acid sequence shown in SEQ ID NO: 8, the FcB comprises the amino acid sequence shown in SEQ ID NO: 7; when the FcA comprises the amino acid sequence shown in SEQ ID NO: 39, the FcB comprises the amino acid sequence shown in SEQ ID NO: 40; when the FcA comprises the amino acid sequence shown in SEQ ID NO: 40, the FcB comprises the amino acid sequence shown in SEQ ID NO: 39.

[0032] In some embodiments of the present invention, the functional region that can activate PTHR comprises an agonistic antigen-binding variable region targeting PTHR or comprises an agonistic antigen-binding fragment or PTH functional region targeting PTHR, the agonistic antigen-binding variable region targeting PTHR is Fv, ScFv or SDA (preferably ScFv or SDA); the agonistic antigen-binding fragment targeting PTHR is Fab or ScFab; the PTH functional region is selected from mature human parathyroid hormone (PTH), PTH truncation, PTH mutant, mutant of PTH truncation, mature human parathyroid hormone-related protein (PTHrP) truncation, mutant of PTHrP truncation and analog of PTHrP truncation; the PTH truncation is preferably a truncation of mature human PTH with no less than 11 amino acid residues truncated from the N-terminus; more preferably, the PTH truncation is a truncation of mature human parathyroid hormone with no less than 34 amino acid residues truncated from the N-terminus.

[0033] In some preferred embodiments of the present invention, the PTH functional region is a PTH (1-11) mutant, a PTH (1-14) mutant, a PTH (1-20), a PTH (1-34), a PTH (1-37), a PTH (1-38), a PTH (1-41), a PTH (1-84), a PTHrP (1-34), a PTHrP (1-36), a PTHrP (7-34) mutant or abaloparatide.

[0034] In some embodiments of the present invention, the abalotriptyline can only be linked to the Fc region via site-directed coupling technology; preferably, the site-directed coupling region is the amino acid residue before the CPPCP sequence in the Fc region.

[0035] In some more preferred embodiments of the present invention, the PTH functional region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5, 30, 31, 32, 33, 34, 35, 36, 37, 38 or sequence 69 in CN1439021A, or an amino acid sequence obtained by adding, deleting, modifying and / or conservatively substituting at least one amino acid residue, for example, 1, 2, 3, 4, 5 or 6 amino acid residues, in the amino acid sequence shown in the foregoing sequence.

[0036] In some embodiments of the present invention, the sequence of the linker is selected from (GGGGS)n, (GGGS)n, (GGS)n, (G)n, (GS)n, (EAAAK)n, or (XP)n, or a combination of the foregoing sequences, where n is a natural number such as 1-20. Preferably, the length of the linker does not exceed 20 amino acids.

[0037] In some preferred embodiments of the present invention, the sequence of the linker comprises an amino acid sequence selected from any one of SEQ ID NOs: 9-12 and 28-29.

[0038] In some specific embodiments of the present invention, the linker is selected from GGGGS (SEQ ID NO: 9), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 12), GGGS (SEQ ID NO: 28) and EAAAK (SEQ ID NO: 29).

[0039] In some specific embodiments of the present invention, the amino acid sequence of the linker is shown in SEQ ID NO:9.

[0040] In some embodiments of the present invention, the first arm comprises a first arm A chain and a first arm B chain;

[0041] The structure of the first arm A chain from N-terminus to C-terminus is VL-CL;

[0042] The structure of the first arm B chain from N-terminus to C-terminus is VH-CH1-FcA;

[0043] The VL and VH or CL and CH1 are optionally exchanged by CrossMab technology.

[0044] In some embodiments of the present invention, when the functional region of the second arm that can activate PTHR is an agonistic antigen-binding variable region targeting PTHR or an agonistic antigen-binding fragment targeting PTHR and is ScFab, ScFv or SDA, or the functional region of the second arm that can activate PTHR is a PTH functional region, the structure of the second arm from N-terminus to C-terminus is the functional region that can activate PTHR-FcB or the functional region that can activate PTHR-linker-FcB.

[0045] In some embodiments of the present invention, when the functional region of the second arm that can activate PTHR is an agonistic antigen-binding variable region targeting PTHR or an agonistic antigen-binding fragment targeting PTHR and is Fab or Fv, the second arm includes a second arm C chain and a second arm D chain; the structure of the second arm C chain from N-terminus to C-terminus is VL-CL or VL, and the structure of the second arm D chain from N-terminus to C-terminus is VH-CH1-FcB, VH-CH1-linker-FcB, VH-FcB or VH-linker-FcB; or, the structure of the second arm C chain from N-terminus to C-terminus is VH-CL or VH, and the structure of the second arm D chain from N-terminus to C-terminus is VL-CH1-FcB or VL-CH1-linker-FcB or VL-FcB or VL-linker-FcB, and the VL and VH or CL and CH1 are optionally interchanged by CrossMab technology.

[0046] In some embodiments of the present invention, the Fv or ScFv targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, and 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 16-18 or SEQ ID NOs: 22-24, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 19-21 or SEQ ID NOs: 25-27, respectively.

[0047] In some specific embodiments of the present invention, the Fv or ScFv targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, and 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 16-18, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 19-21, respectively.

[0048] In some specific embodiments of the present invention, the Fv or ScFv targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, and 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 16-18, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 25-27, respectively.

[0049] In some specific embodiments of the present invention, the Fv or ScFv targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, and 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 22-24, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 19-21, respectively.

[0050] In some specific embodiments of the present invention, the Fv or ScFv targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, and 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 22-24, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 25-27, respectively.

[0051] In some embodiments of the present invention, the Fab or ScFab targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, 3) a light chain constant region (CL) and 4) a heavy chain constant region 1 (CH1), wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 16-18 or SEQ ID NOs: 22-24, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 19-21 or SEQ ID NOs: 25-27, respectively; the Fab or ScFab can exchange the heavy chain variable region, the light chain variable region, or exchange the heavy chain constant region 1 and the light chain constant region through CrossMab technology.

[0052] In some embodiments of the present invention, the Fab or ScFab targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, 3) a light chain constant region (CL) and 4) a heavy chain constant region 1 (CH1), wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 16-18, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 19-21, respectively; the Fab or ScFab can exchange the heavy chain variable region, the light chain variable region, or exchange the heavy chain constant region 1 and the light chain constant region through CrossMab technology.

[0053] In some embodiments of the present invention, the Fab or ScFab targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, 3) a light chain constant region (CL) and 4) a heavy chain constant region 1 (CH1), wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 16-18, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 25-27, respectively; the Fab or ScFab can exchange the heavy chain variable region, the light chain variable region, or exchange the heavy chain constant region 1 and the light chain constant region through CrossMab technology.

[0054] In some embodiments of the present invention, the Fab or ScFab targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, 3) a light chain constant region (CL) and 4) a heavy chain constant region 1 (CH1), wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 22-24, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 19-21, respectively; the Fab or ScFab can exchange the heavy chain variable region, the light chain variable region, or exchange the heavy chain constant region 1 and the light chain constant region through CrossMab technology.

[0055] In some embodiments of the present invention, the Fab or ScFab targeting RANKL comprises 1) a heavy chain variable region (VH) comprising heavy chain complementary determining regions HCDR1-3, 2) a light chain variable region (VL) comprising light chain complementary determining regions LCDR1-3, 3) a light chain constant region (CL) and 4) a heavy chain constant region 1 (CH1), wherein the amino acid sequences of the HCDR1-3 are shown in SEQ ID NOs: 22-24, respectively, and the amino acid sequences of the LCDR1-3 are shown in SEQ ID NOs: 25-27, respectively; the Fab or ScFab can exchange the heavy chain variable region, the light chain variable region, or exchange the heavy chain constant region 1 and the light chain constant region through CrossMab technology.

[0056] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 1 or 3, and the amino acid sequence of the light chain variable region (VL) correspondingly comprises the amino acid sequence shown in SEQ ID NO: 2 or 4.

[0057] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region 1 (CH1) comprises the amino acid sequence shown in SEQ ID NO:14, and the amino acid sequence of the light chain constant region (CL) comprises the amino acid sequence shown in SEQ ID NO:15.

[0058] In some specific embodiments of the present invention, the long-acting parathyroid hormone receptor agonist fusion protein comprises the long-acting parathyroid hormone receptor agonist fusion protein described in the third aspect below.

[0059] The third aspect of the present invention provides a long-acting parathyroid hormone receptor agonist fusion protein, wherein the long-acting parathyroid hormone receptor agonist fusion protein consists of a first arm and a second arm, wherein the first arm includes a first arm A chain and a first arm B chain; wherein:

[0060] When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 6 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 40 linked in sequence; or

[0061] When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 6 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 40 linked in sequence; or

[0062] When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 6 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 40 linked in sequence; or

[0063] When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 6 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 40 linked in sequence; or

[0064] When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 4 and SEQ ID NO: 15 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 6 linked in sequence, or SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 40 linked in sequence; or

[0065] When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 3 and SEQ ID NO: 15 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 6 linked in sequence, or SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 40 linked in sequence;

[0066] When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 4 and SEQ ID NO: 14 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 6 linked in sequence, or SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 40 linked in sequence; or

[0067] When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 3 and SEQ ID NO: 14 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 4, SEQ ID NO: 15, SEQ ID NO: 6 linked in sequence, or SEQ ID NO: 4, SEQ ID NO: 15, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 4, SEQ ID NO: 15, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 4, SEQ ID NO: 15, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 4, SEQ ID NO: 15, SEQ ID NO: 40 linked in sequence; or

[0068] In some embodiments of the present invention:

[0069] When the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 6, the amino acid sequence of the second arm comprises a PTH functional region, a linker, and an Fc region connected in sequence, the PTH functional region is selected from SEQ ID NOs: 5 and 31-38, the linker is selected from SEQ ID NOs: 9-12, and the Fc region sequence is SEQ ID NO: 6; or

[0070] When the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 7, the amino acid sequence of the second arm comprises a PTH functional region, a linker, and an Fc region connected in sequence, the PTH functional region is selected from SEQ ID NOs: 5 and 31-38, the linker is selected from SEQ ID NOs: 9-12, and the Fc region sequence is SEQ ID NO: 8; or

[0071] When the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 8, the amino acid sequence of the second arm comprises a PTH functional region, a linker, and an Fc region connected in sequence, the PTH functional region is selected from SEQ ID NOs: 5 and 31-38, the linker is selected from SEQ ID NOs: 9-12, and the Fc region sequence is SEQ ID NO: 7; or

[0072] When the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 39, the amino acid sequence of the second arm comprises a PTH functional region, a linker, and an Fc region connected in sequence, the PTH functional region is selected from SEQ ID NOs: 5 and 31-38, the linker is selected from SEQ ID NOs: 9-12, and the Fc region sequence is SEQ ID NO: 40; or

[0073] When the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 40, the amino acid sequence of the second arm comprises a PTH functional region, a linker, and an Fc region sequentially connected, the PTH functional region is selected from SEQ ID NOs: 5 and 31-38, the linker is selected from SEQ ID NOs: 9-12, and the Fc region sequence is SEQ ID NO: 39.

[0074] In some embodiments of the present invention:

[0075] When the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 14, and SEQ ID NO: 7 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 15, and SEQ ID NO: 7 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 15, and SEQ ID NO: 7 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 14, and SEQ ID NO: 7 linked in sequence;

[0076] The amino acid sequence of the second arm comprises SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:8, or SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10, SEQ ID NO:8, or SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:8, or SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:8, or SEQ ID NO:31, SEQ ID NO:8, or SEQ ID NO:31, SEQ ID NO:9, SEQ ID NO:8, or SEQ ID NO:31, SEQ ID NO:10, SEQ ID NO:8, or SEQ ID NO:31, SEQ ID NO:11, SEQ ID NO:8, or SEQ ID NO:31, SEQ ID NO:12, SEQ ID NO:8, or SEQ ID NO:3 ... NO:8, or SEQ ID NO:32, SEQ ID NO:9, SEQ ID NO:8, or, in sequence, SEQ ID NO:32, SEQ ID NO:10, SEQ ID NO:8, or, SEQ ID NO:32, SEQ ID NO:11, SEQ ID NO:8, or, in sequence, SEQ ID NO:32, SEQ ID NO:12, SEQ ID NO:8, or, in sequence, SEQ ID NO:33, SEQ ID NO:8, or, in sequence, SEQ ID NO:33, SEQ ID NO:9, SEQ ID NO:8, or, in sequence, SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:8, or, in sequence, SEQ ID NO:33, SEQ ID NO:11, SEQ ID NO:8, or, in sequence, SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:8, or, in sequence, SEQ ID NO:34, SEQ ID NO:8, or, in sequence, SEQ ID NO:34, SEQ ID NO:8. NO:9, SEQ ID NO:8, or, SEQ ID NO:34, SEQ ID NO:10, SEQ ID NO:8 linked in sequence, or,SEQ ID NO: 34, SEQ ID NO: 11, SEQ ID NO: 8, or SEQ ID NO: 34, SEQ ID NO: 12, SEQ ID NO: 8, or SEQ ID NO: 35, SEQ ID NO: 8, or SEQ ID NO: 35, SEQ ID NO: 9, SEQ ID NO: 8, or SEQ ID NO: 35, SEQ ID NO: 10, SEQ ID NO: 8, or SEQ ID NO: 35, SEQ ID NO: 11, SEQ ID NO: 8, or SEQ ID NO: 35, SEQ ID NO: 12, SEQ ID NO: 8, or SEQ ID NO: 36, SEQ ID NO: 8, or SEQ ID NO: 36, SEQ ID NO: 9, SEQ ID NO: 8, or SEQ ID NO: 36, SEQ ID NO: 10 ...10, SEQ ID NO: 8, or SEQ ID NO: 36, SEQ ID NO: 10, SEQ ID NO: 8, or SEQ ID NO: 11, SEQ ID NO: 8, or, SEQ ID NO: 36, SEQ ID NO: 12, SEQ ID NO: 8, or, SEQ ID NO: 37, SEQ ID NO: 8, or, SEQ ID NO: 37, SEQ ID NO: 9, SEQ ID NO: 8, or, SEQ ID NO: 37, SEQ ID NO: 10, SEQ ID NO: 8, or, SEQ ID NO: 37, SEQ ID NO: 11, SEQ ID NO: 8, or, SEQ ID NO: 37, SEQ ID NO: 12, SEQ ID NO: 8, or, SEQ ID NO: 38, SEQ ID NO: 8, or, SEQ ID NO: 38, SEQ ID NO: 9, SEQ ID NO: 8, or, SEQ ID NO: 38, SEQ ID NO: 10, SEQ ID NO: 8, or, SEQ ID NO: 38, SEQ ID NO: 1 ...12, SEQ ID NO: 8, or, SEQ ID NO: 38, SEQ ID NO: 10, SEQ ID NO: 8, or, SEQ ID NO: 38, SEQ ID NO: 11, SEQ ID NO: 8, or, SEQ ID NO: 38, SEQ ID NO: 11, SEQ ID NO: 8, or, SEQ ID NO: 38, SEQ ID NO: 11, SEQ ID NO: 8, or, SEQ ID NO: 38, ID NO:38, SEQ ID NO:12, SEQ ID NO:8. ,

[0077] When the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 14, and SEQ ID NO: 8 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 15, and SEQ ID NO: 8 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 15, and SEQ ID NO: 8 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 14, and SEQ ID NO: 8 linked in sequence;

[0078] The amino acid sequence of the second arm comprises SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:7, or SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:10, SEQ ID NO:7, or SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:7, or SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO:31, SEQ ID NO:7, or SEQ ID NO:31, SEQ ID NO:9, SEQ ID NO:7, or SEQ ID NO:31, SEQ ID NO:10, SEQ ID NO:7, or SEQ ID NO:31, SEQ ID NO:11, SEQ ID NO:7, or SEQ ID NO:31, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO:32, SEQ ID NO:7, or SEQ ID NO:32, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:7, or SEQ ID NO:34, SEQ ID NO:7, or SEQ ID NO:35, SEQ ID NO:7, or SEQ ID NO:36, SEQ ID NO:7, or SEQ ID NO:37, SEQ ID NO:7, or SEQ ID NO:38, SEQ ID NO:7, or SEQ ID NO:39, SEQ ID NO:8, or SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 NO:7, or SEQ ID NO: 32, SEQ ID NO:9, SEQ ID NO:7, or, in sequence, SEQ ID NO: 32, SEQ ID NO:10, SEQ ID NO:7, or, SEQ ID NO: 32, SEQ ID NO:11, SEQ ID NO:7, or, in sequence, SEQ ID NO: 32, SEQ ID NO:12, SEQ ID NO:7, or, in sequence, SEQ ID NO: 33, SEQ ID NO:7, or, in sequence, SEQ ID NO: 33, SEQ ID NO:9, SEQ ID NO:7, or, in sequence, SEQ ID NO: 33, SEQ ID NO:10, SEQ ID NO:7, or, in sequence, SEQ ID NO: 33, SEQ ID NO:11, SEQ ID NO:7, or, in sequence, SEQ ID NO: 33, SEQ ID NO:12, SEQ ID NO:7, or, in sequence, SEQ ID NO: 34, SEQ ID NO:7, or, in sequence, SEQ ID NO: 34, SEQ ID NO:7. ID NO:9, SEQ ID NO:7, or, SEQ ID NO:34, SEQ ID NO:10, SEQ ID NO:7 linked in sequence, or,SEQ ID NO: 34, SEQ ID NO: 11, SEQ ID NO: 7, or SEQ ID NO: 34, SEQ ID NO: 12, SEQ ID NO: 7, or SEQ ID NO: 35, SEQ ID NO: 7, or SEQ ID NO: 35, SEQ ID NO: 9, SEQ ID NO: 7, or SEQ ID NO: 35, SEQ ID NO: 10, SEQ ID NO: 7, or SEQ ID NO: 35, SEQ ID NO: 11, SEQ ID NO: 7, or SEQ ID NO: 35, SEQ ID NO: 12, SEQ ID NO: 7, or SEQ ID NO: 36, SEQ ID NO: 7, or SEQ ID NO: 36, SEQ ID NO: 9, SEQ ID NO: 7, or SEQ ID NO: 36, SEQ ID NO: 10 ... SEQ ID NO: 11, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 36, SEQ ID NO: 12, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 37, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 37, SEQ ID NO: 9, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 37, SEQ ID NO: 10, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 37, SEQ ID NO: 11, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 37, SEQ ID NO: 12, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 38, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 38, SEQ ID NO: 9, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 38, SEQ ID NO: 10, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 38, SEQ ID NO: 11, SEQ ID NO: 7, or, in sequence, SEQ ID NO: 38, SEQ ID NO: 9, SEQ ID NO: 7. ID NO:38, SEQ ID NO:12, SEQ ID NO:7. ,

[0079] When the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 14, and SEQ ID NO: 39 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 15, and SEQ ID NO: 39 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 15, and SEQ ID NO: 39 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 14, and SEQ ID NO: 39 linked in sequence;

[0080] The amino acid sequence of the second arm comprises SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 40, or SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 40, or SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 40, or SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 40, or SEQ ID NO: 31, SEQ ID NO: 40, or SEQ ID NO: 31, SEQ ID NO: 9, SEQ ID NO: 40, or SEQ ID NO: 31, SEQ ID NO: 10, SEQ ID NO: 40, or SEQ ID NO: 31, SEQ ID NO: 11, SEQ ID NO: 40, or SEQ ID NO: 31, SEQ ID NO: 12 ... NO:32, SEQ ID NO:40, or, SEQ ID NO:32, SEQ ID NO:9, SEQ ID NO:40, or, SEQ ID NO:32, SEQ ID NO:10, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:32, SEQ ID NO:11, SEQ ID NO:40, or, SEQ ID NO:32, SEQ ID NO:12, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:33, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:33, SEQ ID NO:9, SEQ ID NO:40, or, SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:40, or, SEQ ID NO:33, SEQ ID NO:11, SEQ ID NO:40, or, SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:40, or, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:13, SEQ ID NO:40, or, SEQ ID NO:35, SEQ ID NO:14, SEQ ID NO:40, or, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or NO:40, or, SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:10, SEQ ID NO:40 linked in sequence, or,SEQ ID NO:34, SEQ ID NO:11, SEQ ID NO:40, or SEQ ID NO:34, SEQ ID NO:12, SEQ ID NO:40, or SEQ ID NO:35, SEQ ID NO:40, or SEQ ID NO:35, SEQ ID NO:9, SEQ ID NO:40, or SEQ ID NO:35, SEQ ID NO:10, SEQ ID NO:40, or SEQ ID NO:35, SEQ ID NO:11, SEQ ID NO:40, or SEQ ID NO:35, SEQ ID NO:12, SEQ ID NO:40, or SEQ ID NO:36, SEQ ID NO:40, or SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:40, or SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:40, or SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:40, or SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:40, or SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:40, or NO:40, or, SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:12, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:10, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:11, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:12, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:38, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:38, SEQ ID NO:9, SEQ ID NO:40, or, SEQ ID NO:38, SEQ ID NO:10, SEQ ID NO:40 linked in sequence. NO:40, or SEQ ID NO:38, SEQ ID NO:11, SEQ ID NO:40, or SEQ ID NO:38, SEQ ID NO:12, SEQ ID NO:40 linked in sequence;

[0081] When the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 14, and SEQ ID NO: 40 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 15, and SEQ ID NO: 40 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 15, and SEQ ID NO: 40 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 14, and SEQ ID NO: 40 linked in sequence;

[0082] The amino acid sequence of the second arm comprises SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:39, or SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:39, or SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:39, or SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:9, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:10, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:11, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:12 ...12, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:11, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:12, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:12, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:12, SEQ ID NO:39, or SEQ ID NO:31, SEQ ID NO:12, NO:32, SEQ ID NO:39, or SEQ ID NO:32, SEQ ID NO:9, SEQ ID NO:39, or, in sequence, SEQ ID NO:32, SEQ ID NO:10, SEQ ID NO:39, or, SEQ ID NO:32, SEQ ID NO:11, SEQ ID NO:39, or, in sequence, SEQ ID NO:32, SEQ ID NO:12, SEQ ID NO:39, or, in sequence, SEQ ID NO:33, SEQ ID NO:39, or, in sequence, SEQ ID NO:33, SEQ ID NO:9, SEQ ID NO:39, or, in sequence, SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:39, or, in sequence, SEQ ID NO:33, SEQ ID NO:11, SEQ ID NO:39, or, in sequence, SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:39, or, in sequence, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:35 NO:39, or, SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:10, SEQ ID NO:39 linked in sequence, or,SEQ ID NO:34, SEQ ID NO:11, SEQ ID NO:39, or SEQ ID NO:34, SEQ ID NO:12, SEQ ID NO:39, or SEQ ID NO:35, SEQ ID NO:39, or SEQ ID NO:35, SEQ ID NO:9, SEQ ID NO:39, or SEQ ID NO:35, SEQ ID NO:10, SEQ ID NO:39, or SEQ ID NO:35, SEQ ID NO:11, SEQ ID NO:39, or SEQ ID NO:35, SEQ ID NO:12, SEQ ID NO:39, or SEQ ID NO:36, SEQ ID NO:39, or SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:39, or SEQ ID NO:36, SEQ ID NO:10 ...11, SEQ ID NO:39, or SEQ ID NO:36, SEQ ID NO:12, SEQ ID NO:39, or SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:39, or SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:39, or SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:39, or SEQ ID NO:36 NO:39, or, SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:12, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:10, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:11, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:12, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:38, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:38, SEQ ID NO:9, SEQ ID NO:39, or, SEQ ID NO:38, SEQ ID NO:10, SEQ ID NO:39 linked in sequence. NO:39, or SEQ ID NO:38, SEQ ID NO:11, SEQ ID NO:39, or SEQ ID NO:38, SEQ ID NO:12, SEQ ID NO:39 connected in sequence.

[0083] The fourth aspect of the present invention provides an isolated nucleic acid molecule encoding the long-acting parathyroid hormone receptor agonist fusion protein as described in the first aspect, the second aspect or the third aspect.

[0084] The fifth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule as described in the fourth aspect.

[0085] The sixth aspect of the present invention provides an engineered cell comprising the recombinant expression vector as described in the fifth aspect.

[0086] In some embodiments of the present invention, the host cell of the engineered cell is a eukaryotic cell.

[0087] In some embodiments of the present invention, the eukaryotic cell is a yeast cell or a mammalian cell.

[0088] In some preferred embodiments of the present invention, the mammalian cells are CHO cells or HEK293 cells.

[0089] The seventh aspect of the present invention provides a pharmaceutical composition, comprising the long-acting parathyroid hormone receptor agonist fusion protein according to the first aspect, the second aspect, or the third aspect, the nucleic acid molecule according to the fourth aspect, the recombinant expression vector according to the fifth aspect, and the engineered cell according to the sixth aspect;

[0090] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0091] The eighth aspect of the present invention provides a use of the long-acting parathyroid hormone receptor agonist fusion protein as described in the first, second or third aspect, the nucleic acid molecule as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the engineered cell as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect in the preparation of a medicament for treating, preventing, adjuvant treating or neoadjuvant treating a bone disorder, wherein the bone disorder is a disease that requires treatment, prevention, adjuvant treating or neoadjuvant treating by regulating bone formation and / or bone resorption in lesion-related areas.

[0092] In some embodiments of the present invention, the bone disorder is osteoporosis, osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, bone injury, degenerative lumbar spondylolisthesis or degenerative disc disease; the bone injury is preferably a fracture, bone crack, cartilage injury, osteonecrosis, bone injury caused by arthritis or tumor; the fracture is preferably an osteoporotic fracture, nonunion of fracture or delayed union of fracture; the arthritis includes arthritis caused by degeneration, trauma, strain, infection, autoimmune disease or inflammation, such as rheumatoid arthritis, osteoarthritis, and infectious arthritis; the tumor includes bone tumors and tumor bone metastasis.

[0093] The ninth aspect of the present invention provides a method for preparing a long-acting parathyroid hormone receptor agonist fusion protein, the method comprising the step of culturing the engineered cells as described in the sixth aspect.

[0094] The tenth aspect of the present invention provides a method for treating, preventing, adjuvant treating or neoadjuvant treating bone disorders by regulating bone formation and / or bone resorption in lesion-related areas, the method comprising administering to a subject in need thereof an effective amount of the long-acting parathyroid hormone receptor agonist fusion protein of the first aspect, the second aspect or the third aspect, the nucleic acid molecule of the fourth aspect, the recombinant expression vector of the fifth aspect, the engineered cell of the sixth aspect or the pharmaceutical composition of the seventh aspect.

[0095] In some embodiments of the present invention, the bone disorder is osteoporosis, osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, bone injury, degenerative lumbar spondylolisthesis or degenerative disc disease; the bone injury is preferably a fracture, bone crack, cartilage injury, osteonecrosis, bone injury caused by arthritis or tumor; the fracture is preferably an osteoporotic fracture, nonunion of fracture or delayed union of fracture; the arthritis includes arthritis caused by degeneration, trauma, strain, infection, autoimmune disease or inflammation, such as rheumatoid arthritis, osteoarthritis, and infectious arthritis; the tumor includes bone tumors and tumor bone metastasis.

[0096] In some embodiments of the present invention, the method of administration is selected from oral administration (eg, administration via an oral delivery device) or injection; the injection is preferably intravenous injection, subcutaneous injection, intramuscular injection, or local injection.

[0097] The eleventh aspect of the present invention provides a long-acting parathyroid hormone receptor agonist fusion protein as described in the first, second or third aspect, the nucleic acid molecule as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the engineered cell as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect for use as a medicine.

[0098] The twelfth aspect of the present invention provides a long-acting parathyroid hormone receptor agonist fusion protein as described in the first, second or third aspect, the nucleic acid molecule as described in the fourth aspect, the recombinant expression vector as described in the fifth aspect, the engineered cell as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect, for use in the treatment, prevention, adjuvant treatment or neoadjuvant treatment of bone diseases.

[0099] In some embodiments of the present invention, the bone disorder is osteoporosis, osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, bone injury, degenerative lumbar spondylolisthesis or degenerative disc disease; the bone injury is preferably a fracture, bone crack, cartilage injury, osteonecrosis, bone injury caused by arthritis or tumor; the fracture is preferably an osteoporotic fracture, nonunion of fracture or delayed union of fracture; the arthritis includes arthritis caused by degeneration, trauma, strain, infection, autoimmune disease or inflammation, such as rheumatoid arthritis, osteoarthritis, and infectious arthritis; the tumor includes bone tumors and tumor bone metastasis.

[0100] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0101] The reagents and raw materials used in the present invention are commercially available.

[0102] The positive progress effect of the present invention is:

[0103] The present invention's long-acting parathyroid hormone receptor agonist fusion protein (LAPAF) has the effect of inhibiting bone resorption and promoting bone formation, has long-term effectiveness, no osteoclast advantage, strong bone tissue targeting, and the efficacy of multiple administrations is better than the combination group of anti-RANKL monoclonal antibody and teriparatide; It has fast-acting properties. In experiments with non-human primates, the ratio of bone density improvement of D56 after a single administration is much higher than the therapeutic effect of multiple administrations of other osteoporosis treatment drugs on the market; While ensuring efficacy, it is safer, has no side effects of persistent hypercalcemia, has an extremely low risk of mandibular osteonecrosis, has no drug-related toxicity, and is expected to be used for a long time; It significantly reduces the problem of a high ratio of PTH functional region fractures that occurs in the fusion structure of anti-RANKL antibodies and PTH in the prior art, has a high expression level, an extremely high preparation yield, low preparation cost, good serum stability, and achieves a better long-term therapeutic effect. At the same time, the present invention's long-acting parathyroid hormone receptor agonist fusion protein can accelerate fracture healing and has better effects in the treatment of diseases such as osteoarthritis caused by bone damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] FIG1 is a schematic diagram of an exemplary structure of a dual-targeting fusion protein.

[0105] FIG2 is the reduction CE spectra of LAPAF LYN101-2 of the present invention and the control sample synPTH-αRANKL-1.

[0106] FIG3 is a mass spectrometry analysis spectrum of the control sample synPTH-αRANKL-1.

[0107] FIG4 is a mass spectrometry analysis spectrum of LAPAF LYN101-1 of the present invention.

[0108] FIG5 is a mass spectrometry analysis graph of LAPAF LYN101-2 of the present invention.

[0109] FIG6 is a mass spectrometry analysis graph of LAPAF LYN101-3 of the present invention.

[0110] FIG7 shows the proliferation inhibitory activity of LAPAF LYN101-2, denosumab and the control sample synPTH-αRANKL-1 on RANKL-induced differentiation of Raw264.7 cells.

[0111] FIG8A shows the in vitro activities of LAPAF LYN101-2, teriparatide and the control sample synPTH-αRANKL-1 on PTHR-induced cAMP release in UMR106-RANKL-pool cells.

[0112] FIG8B shows the in vitro activity of LAPAFs LYN101-2, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17, LYN101-18 and teriparatide of the present invention on PTHR-induced cAMP release in UMR106-RANKL-4 monoclonal cells.

[0113] Figure 9 shows the changes in bone density of different doses of LAPAF LYN101-2 of the present invention and the control sample synPTH-αRANKL-1 in the RANKL humanized transgenic mouse osteoporosis model induced by ovariectomy. By one-way ANOVA Dunnett's multiple comparison, each drug-treated group was compared with the OVX group. n p>0.05, * p<0.05, ** p<0.01.

[0114] Figure 10 shows the changes in bone resorption markers in different doses of LAPAF LYN101-2 of the present invention and the control sample synPTH-αRANKL-1 in the RANKL humanized transgenic mouse osteoporosis model induced by ovariectomy. Two-way ANOVA Dunnett's multiple comparison was used to compare the bone resorption markers of each drug-treated group with those of the OVX group. n p>0.05, **** p<0.0001.

[0115] Figure 11 shows the changes in bone formation markers in different doses of LAPAF LYN101-2 of the present invention and the control sample synPTH-αRANKL-1 in the RANKL humanized transgenic mouse osteoporosis model caused by ovariectomy. By two-way ANOVA with Dunnett's multiple comparison, the differences in the bone formation markers in each drug-treated group were compared with the OVX group. n p>0.05, **** p<0.0001.

[0116] FIG12 shows the in vitro activity of LAPAF LYN101-2 of the present invention and mouse serum at various time points after administration on PTHR-induced cAMP release in UMR106-RANKL-pool cells.

[0117] Figure 13 shows the changes in bone density in the RANKL humanized transgenic mouse osteoporosis model induced by ovariectomy after the combination of LAPAF LYN101-2, denosumab, and teriparatide. Two-way ANOVA with Dunnett's multiple comparisons showed that the Sham group was significantly lower than the OVX group. *** p<0.001, LYN101-2 10mpk group compared with OVX group ** p<0.01, compared with the OVX group, the denosumab + teriparatide combination group * p<0.05, LYN101-2 10mpk compared with denosumab + teriparatide combination group && p<0.01.

[0118] Figure 14 shows the CTX-1 levels in the RANKL humanized transgenic mouse osteoporosis model induced by ovariectomy after the combination of LAPAF LYN101-2, denosumab, and teriparatide. Two-way ANOVA with Dunnett's multiple comparisons was used to compare the CTX-1 levels in each drug-treated group with those in the OVX group. * p<0.05, ** p<0.01, **** p<0.0001.

[0119] FIG15A shows the results of the LAPAF LYN101-2 of the present invention showing the proportion of mice with healed fracture lines in a RANKL humanized transgenic mouse model of femoral fracture induced by ovariectomy and surgical modeling.

[0120] FIG15B shows the micro-CT results of the fracture site of LAPAF LYN101-2 of the present invention in a RANKL humanized transgenic mouse model of femoral fracture induced by ovariectomy and surgical modeling.

[0121] Figure 16 shows the bone density at the fracture site of the RANKL humanized transgenic mouse model of femoral fracture induced by ovariectomy and surgical modeling after continuous administration of LAPAF LYN101-2 for 6 weeks. By one-way analysis of variance and Dunnett's multiple comparison, the bone density of each treatment group was compared with the OVX fracture model group. * p<0.05.

[0122] Figure 17 shows the bone density of the tibia (non-fracture site) after LAPAF LYN101-2 was continuously administered for 6 weeks in the RANKL humanized transgenic mouse model of femoral fracture induced by ovariectomy and surgical modeling. By one-way analysis of variance and Dunnett's multiple comparison, the bone density of each drug-treated group was compared with the OVX fracture model group. **** p<0.0001.

[0123] Figure 18 shows the bone mineral apposition rate (MAR) at the fracture site of the RANKL humanized transgenic mouse model of femoral fracture induced by ovariectomy and surgical modeling after continuous administration of LAPAF LYN101-2 for 6 weeks. By one-way ANOVA with Dunnett's multiple comparison, the LYN101 0.4 mpk group was compared with the teriparatide group. ** p<0.01; LYN101 10mpk group compared with teriparatide group **** p<0.0001; compared with the LYN10110mpk group and the denosumab + teriparatide combination group, ** p<0.01.

[0124] FIG19A shows the effects of LAPAF LYN101-2, teriparatide and the control sample synPTH-αRANKL-1 on UMR106 cells (PTHR + / RANKL - In vitro activity results of PTHR-induced cAMP release in cells.

[0125] FIG19B shows the effects of LAPAF LYN101-2, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17, LYN101-18 and teriparatide on UMR106 cells (PTHR + / RANKL - In vitro activity results of PTHR-induced cAMP release in cells. DETAILED DESCRIPTION

[0126] definition

[0127] As used herein, the term "PTH" or "parathyroid hormone" is a polypeptide secreted by the parathyroid gland, and mature human PTH contains 84 amino acids. The PTH described in the present invention refers to mature human PTH, the amino acid sequence of which is shown as amino acids 32-115 of the Uniprot number P01270, i.e., PTH (1-84). According to prior art reports, mature full-length human PTH (1-84), or PTH truncations such as PTH (1-34) (common name of rhPTH (1-34): teriparatide), PTH (1-37), PTH (1-38), PTH (1-41), PTH (1-20), or mutants of PTH truncations such as PTH (1-11) mutant and PTH (1-14) mutant all have the function of activating PTHR (parathyroid hormone receptor 1). For example, PTH (1-34) refers to a polypeptide formed by amino acids 1-34 of the mature human PTH sequence, i.e., amino acids 32-65 of the Uniprot number P01270; PTH (1-20), PTH (1-37), PTH (1-38), PTH (1-41), and PTH (1-84) refer to polypeptides formed by amino acids 1-20 of the mature human PTH sequence, amino acids 1-37 of the mature human PTH sequence, amino acids 1-38 of the mature human PTH sequence, amino acids 1-41 of the mature human PTH sequence, and amino acids 1-84 of the mature human PTH sequence, respectively. PTH (1-11) mutants and PTH (1-14) mutants refer to mutants of PTH truncations obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, such as 1-6 amino acid residues, in the mature human PTH sequence, amino acids 1-11 and amino acids 1-14, respectively, of the mature human PTH sequence, for example, SEQ ID NO: 31 or SEQ ID NO: 38 of the present invention.

[0128] As used herein, the terms "PTHR", "PTH1R", "PTHR1", "parathyroid hormone receptor 1", and "parathyroid hormone receptor" are used interchangeably herein to refer to human parathyroid hormone receptor 1 (PTHR1).

[0129] As used herein, the term "PTHrP" refers to parathyroid hormone-related protein (PTHrP), which was originally discovered in the plasma of patients with humoral hypercalcemia (HHM) caused by malignant tumors. It is a polypeptide containing 141 amino acids (Uniprot numbering is P12272, positions 37-177). Because 11 amino acids in the N-terminal fragment (Uniprot numbering is P12272, positions 37-70) consisting of the N-terminal 34 residues of mature human PTHrP (Uniprot numbering is P12272, positions 37-177) are identical to PTH (1-34), which helps to produce similar binding affinity and exert similar biological effects, PTHrP also has similar biological activity to parathyroid hormone (PTH), that is, it has the function of activating PTHR. The PTHrP truncates described herein refer to truncated forms of mature human PTHrP. For example, PTHrP(1-36) refers to a polypeptide formed from amino acids 1-36 of the mature human PTHrP sequence, i.e., amino acids 37-72 of the Uniprot number P12272. PTHrP(1-34), PTHrP(1-74), and PTHrP(1-86) refer to polypeptides formed from amino acids 1-34, 1-74, and 1-84 of the mature human PTHrP sequence, respectively. A mutant of a PTHrP truncate refers to a mutant of a PTHrP truncate obtained by adding, deleting, modifying, and / or conservatively substituting at least one amino acid residue, such as 1-3 amino acid residues, of the mature human PTHrP truncate, for example, the PTHrP(7-34) mutant, i.e., sequence 69 of CN1439021A.

[0130] As used herein, the term "abaloparatide" refers to Abaloparatide, a synthetic 34-amino acid polypeptide, an analog of PTHrP, with 41% and 76% homology to human PTH (1-34) and human PTHrP (1-34), respectively, and also has the function of activating PTHR.

[0131] As used herein, the term "functional region that can activate PTHR" refers to a functional fragment that targets PTHR and can activate PTHR, selected from an agonistic antigen-binding variable region (including a heavy chain variable region and a light chain variable region, or VHH) targeting PTHR, an agonistic antigen-binding fragment targeting PTHR, or a PTH functional region, or a combination thereof. The agonistic antigen-binding variable region targeting PTHR is selected from a heavy chain variable region (VH) and a light chain variable region (VL), or VHH, targeting PTHR, and the agonistic antigen-binding fragment of PTHR is selected from a Fab or ScFab targeting PTHR, including a heavy chain variable region, a light chain variable region, a heavy chain constant region 1 (CH1), and a light chain constant region (CL). The PTH functional region refers to mature full-length human PTH (such as PTH (1-84)), mature human PTH truncations (such as PTH (1-20), PTH (1-34), PTH (1-37), PTH (1-38), PTH (1-41)), mutants of mature human PTH truncations (such as PTH (1-11) mutant, PTH (1-14) mutant), mature human PTHrP truncations (such as PTHrP (1-34), PTHrP (1-36)), or mutants of mature human PTHrP truncations (such as PTHrP (7-34) mutant), or analogs of mature human PTHrP truncations (such as abalotriptyline).

[0132] As used herein, the term "anti-RANKL monoclonal antibody" refers to a monoclonal antibody targeting RANKL (Receptor Activator of Nuclear Factor-κB Ligand). The anti-RANKL monoclonal antibody used in the examples of the present invention is Denosumab.

[0133] As used herein, the term "anti-RANKL antibody" refers to a functional region having an antibody or half-antibody structure that targets RANKL (Receptor Activator of Nuclear Factor-κB Ligand), and is one of the components of a dual / multi-targeting antibody or dual / multi-targeting fusion protein.

[0134] As used herein, the term "Anti-RANKL functional region" or "RANKL-targeting functional region" refers to an antigen-binding fragment targeting RANKL, such as the heavy chain variable region and light chain variable region targeting RANKL (such as Fv or ScFv), VHH (SDA) targeting RANKL, and Fab region targeting RANKL (such as Fab or ScFab, including heavy chain variable region, light chain variable region, heavy chain constant region 1 (CH1) and light chain constant region (CL)).

[0135] As used herein, the term "dual-targeting fusion protein" refers to an Fc fusion protein that targets RANKL and PTHR and comprises or consists of an anti-RANKL functional region, a functional region that can activate PTHR, and an Fc region. The functional region that can activate PTHR is an agonistic antigen-binding variable region (including ScFv, SDA, or Fv) targeting PTHR, an agonistic antigen-binding fragment (including ScFab or Fab) targeting PTHR, or a PTH functional region; when the functional region that can activate PTHR is a PTH functional region, the PTH functional region can be connected to the anti-RANKL functional region or the Fc region of human IgG via a linker, or can be directly connected or connected via site-directed coupling technology. When the agonistic antigen-binding fragment comprising the anti-RANKL functional region or targeting PTHR is a Fab or ScFab, those skilled in the art can also use common dual antibody technologies such as Crossmab to optimize the structure of the Fab region, such as swapping the VH and VL in the Fab, or swapping the CH1 and CL in the Fab. The CH2 and CH3 domains of the IgG constant region and the hinge region constitute the Fc region. As used herein, the term "dual-targeting fusion protein of the present invention" refers to the dual-targeting fusion protein of Structure 1.

[0136] As used herein, the terms "long-acting parathyroid hormone receptor agonist fusion protein", "Long-Acting PTHR Agonist Fusion protein", and "LAPAF" can be used interchangeably in the present invention to refer to the dual-targeting fusion protein of the present invention, or a fusion protein comprising the dual-targeting fusion protein sequence of the present invention and targeting bone tissue cells (such as osteoblasts) expressing RANKL, such as a triple-targeting / multi-targeting fusion protein, or a dual-targeting fusion protein or triple-targeting / multi-targeting fusion protein of the present invention coupled with a polypeptide, a small molecule compound, an oligonucleotide or other molecule.

[0137] As used herein, the term "silencing Fc immune effector function" refers to reducing or eliminating the ADCP and / or ADCC and / or CDC effects of the Fc region. Common Fc immune effector function silencing Fc mutants of IgG1 include the LALA mutation (L234A / L235A), the PGLALA mutation (L234A, L235A, and P329G), and N297A, and Fc mutants of IgG4 include one or more of the E233P / F234V / L235A / D265A / R409K mutations. The numbering of Fc region residues herein adopts the EU index numbering in Kabat.

[0138] As used herein, the term "linking" refers to the connection of two peptide chains into a single peptide chain via a peptide bond or peptide chains, or the connection of two peptide chains via a site-directed coupling technique.

[0139] As used herein, the term "Fab" is composed of the variable region (VL) of the light chain, the constant region (CL) of the light chain, the variable region (VH) of the heavy chain, and the constant region 1 (CH1) domain of the heavy chain, can bind to the antigen, and comprises two peptide chains, chain a and chain b; when chain a is VH-CH1, chain b is VL-CL, and vice versa; when chain a is VH-CL, chain b is VL-CH1, and vice versa.

[0140] As used herein, the term "ScFab" refers to a single peptide chain formed by linking the two peptide chains of the Fab described above via a linker.

[0141] As used herein, the term "Fv" comprises two peptide chains, namely the variable region of the heavy chain (VH) and the variable region of the light chain (VL).

[0142] As used herein, the term "ScFv" refers to a single peptide chain with the variable region of the heavy chain (VH) and the variable region of the light chain (VL) connected by a linker.

[0143] As used herein, the term "SDA" or "VHH" refers to the antigen-binding region of a single domain antibody (Nanobody), ie the Nanobody heavy chain variable region.

[0144] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The heavy chain variable region CDRs (HCDR1, HCDR2, HCDR3) and light chain variable region CDRs (LCDR1, LCDR2, LCDR3) described in the present invention are defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined based on the heavy chain / light chain variable region sequences by other systems such as Chothia and IMGT, AbM and other numbering systems / methods. It should be noted that the boundaries of the CDRs of the same antibody variable region obtained by different numbering systems may be different. That is, the CDR sequences of the same antibody variable region defined by different numbering systems are different. Therefore, when it comes to defining an antibody with a specific CDR sequence defined by the present invention, the scope of the antibody also covers such antibodies whose variable region sequence comprises a specific CDR sequence, but due to the application of different schemes (e.g., different numbering system rules or combinations thereof) the CDR boundaries for which protection is claimed are different from the specific CDR boundaries defined by the present invention.

[0145] As used herein, the term "knob(s)-into-hole(s) technology" or "knob-into-hole" technology or "button" technology refers to the use of genetic engineering technology to introduce different mutations into the two CH3 domains of the heavy chain to promote heterodimerization of the heavy chains. A knob is made on one heavy chain and a hole is made on the other heavy chain, and then the two preferentially engage together to form an asymmetric antibody (Ridgway JB, et al. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996, 9(7): 617-621). As known to those skilled in the art, multiple knobs and / or holes can be made on one heavy chain, and correspondingly, multiple holes and / or knobs can be made on the other heavy chain.

[0146] As used herein, the term "host cell" generally includes a single cell, a cell line or a cell culture, which comprises a polynucleotide disclosed herein, a recombinant expression vector, or expresses a long-acting parathyroid hormone receptor agonist fusion protein of the present invention. Host cells can include the offspring of a single host cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original parent cell (morphologically or on the genomic total DNA complement). Host cells can include cells transfected in vitro with a vector disclosed herein. Host cells are eukaryotic cells, such as yeast cells, HEK293 cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells or myeloma cells. In some embodiments, host cells are mammalian cells. In some embodiments, the mammalian cells are CHO cells or HEK293 cells.

[0147] As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into a host cell and / or between host cells. The term can include vectors primarily used to insert DNA into cells, vectors primarily used for replication of DNA, and expression vectors for transcription and / or translation of DNA or RNA. Nucleic acid molecules encoding different chains of the long-acting parathyroid hormone receptor agonist fusion protein of the present invention can be in the same DNA molecule or in different DNA molecules. Also included are vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. The host cell herein generally comprises an expression vector capable of producing a desired expression yield.

[0148] The term "composition" refers to a composition comprising the long-acting parathyroid hormone receptor agonist fusion protein of the present invention and an optional adjuvant, excipient or pharmaceutically acceptable carrier. The composition may contain a pharmaceutically acceptable carrier. The composition may be in the form of any pharmaceutical preparation, including but not limited to an injection, powder, freeze-dried powder, etc., or a formulation that can be built into an oral delivery therapeutic device. The composition in the form of a pharmaceutical preparation, also referred to herein as a "pharmaceutical composition," can be prepared according to conventional pharmaceutical preparation techniques, including fusing the pharmaceutically active ingredient, the long-acting parathyroid hormone receptor agonist fusion protein of the present invention, with a pharmaceutical carrier, and preparing the desired dosage form according to conventional pharmaceutical preparation techniques. The composition may also include an expression vector encoding a nucleic acid molecule of the long-acting parathyroid hormone receptor agonist fusion protein and an optional pharmaceutically acceptable carrier.

[0149] The term "therapeutic" refers to the long-acting parathyroid hormone receptor agonist fusion protein or composition of the present invention.

[0150] The term "effective amount" or "therapeutically effective amount" refers to an amount of a long-acting parathyroid hormone receptor agonist fusion protein or composition (e.g., a composition comprising a long-acting parathyroid hormone receptor agonist fusion protein or a long-acting parathyroid hormone receptor agonist fusion protein as described herein) sufficient to achieve the intended application (including but not limited to disease treatment, disease prevention, disease alleviation). The therapeutically effective amount may vary depending on the intended application (e.g., in vitro or in vivo) or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. The term can also be applied to a dose that induces a specific response (e.g., target gene induction, proliferation and / or apoptosis) in the target cell. The specific dose will vary depending on the specific molecule selected, the dosing regimen followed, whether it is administered in combination with other molecules, the timing of administration, the tissue to which it is administered, and the physical delivery system in which it is administered.

[0151] The terms "treat" or "alleviate" or "improve" are used interchangeably herein and refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. As used herein, therapeutic benefit generally refers to eradication or lessening of the severity of the underlying condition being treated. In addition, therapeutic benefit is achieved by eradicating, lessening the severity, or reducing the incidence of one or more physiological symptoms associated with the underlying condition, such that an improvement is observed in the subject (although the subject may still be afflicted with the underlying condition). For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, to reduce the risk of the subject developing the particular disease, even though a diagnosis of the disease may not have yet been made. "Adjuvant therapy" refers to treatment given after surgery, and "pre-adjuvant therapy" refers to treatment given before surgery.

[0152] As used herein, the term "administer" refers to delivering a therapeutically effective amount of a therapeutic or control sample to a subject. Administration can be systemic or local. Administration can be performed using an administration device, such as a syringe or a device for oral delivery of the therapeutic. Modes of administration include, but are not limited to, oral administration and injection. Routes of administration include intravenous, subcutaneous, or intramuscular administration, local injection, and the like.

[0153] As used herein, the terms "co-administration," "administered in combination with," or "in conjunction with," and grammatical equivalents thereof, generally refer to administering two or more agents to a subject such that the agents and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0154] As used herein, the term "subject" or "individual" or "animal" or "patient" refers to a human or non-human animal, including a mammal or primate, for whom diagnosis, prognosis, alleviation, prevention and / or treatment of a disease or condition is desired. Mammalian subjects include humans, livestock animals, farm animals, and zoo, sports or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0155] As used herein, the term "in vivo" generally refers to events that occur within the body of a subject (in vivo).

[0156] As used herein, the term "in vitro" generally refers to an event that occurs outside the body of a subject (in vitro or ex vivo). For example, an in vitro assay includes any assay performed outside a subject. In vitro assays include cell-based assays in which dead or living cells are used. In vitro assays also include cell-free assays in which intact cells are not used.

[0157] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0158] Example 1: Structural design of dual-targeting fusion protein and construction of expression vector

[0159] The present invention designs dual-targeting fusion proteins of various structures, the structural form of which is shown in FIG1 . In the Knob(s)-into-Hole(s) structure in FIG1 , the Knob(s) and the Hole(s) can be interchanged in the two Fc regions.

[0160] The dual-targeting fusion protein and control sample, synPTH-αRANKL-1, were synthesized directly by GENEWIZ and Universal Biotechnology based on the protein sequence after codon optimization, ligated into the pCDNA3.4 plasmid, and confirmed by sequencing. The aforementioned expression plasmids were mixed and paired to transfect expression cells to obtain the dual-targeting fusion protein or control sample (see Table 1). Subsequent experimental materials were extracted from expression cells transfected with these plasmids.

[0161] Table 1 Exemplary molecular sequences of dual-targeting fusion proteins and control samples

[0162] Example 2: Preparation of expression plasmid, cell transfection, and expression and purification of target protein

[0163] 1. Preparation of expression plasmid

[0164] Glycerol culture containing the expression plasmid (1 mL of E. coli containing the expression plasmid was inoculated into liquid LB medium at a ratio of 1:1000 (0.5 mL of 60% sterile glycerol solution was added to 1 mL of the E. coli culture containing the expression plasmid and mixed thoroughly). Culture was shaken at 37°C, 220 rpm, and then harvested by centrifugation. The expression plasmid was extracted using the Endotoxin-Free Plasmid Extraction Kit (DP117, purchased from Tiangen Biotech (Beijing) Co., Ltd.) according to the standard protocol provided in the kit's instructions.

[0165] 2. Cell transfection and protein expression

[0166] After filtering the obtained expression plasmid with a 0.22 μm filter membrane, 50 μg of plasmid was drawn (wherein, the mass ratio of the first arm heavy chain, the first arm light chain, and the second arm chain expression plasmid of the dual-targeting fusion protein (dual-targeting fusion protein of structure 1) of the present invention was 1:2:2; the mass ratio of the dual-targeting fusion protein chain 1 and chain 2 expression plasmid of structure 2, structure 3, structure 4, and structure 5 was 2:3) and added to 2 mL of Opti PRO SFM Medium (GIBCO) and mixed evenly. 160 μL of the transfection reagent Expi Fectamine CHO Reagent was drawn and added to 2 mL of Opti PRO SFM Medium and mixed evenly. The obtained transfection reagent mixed solution was added to the mixed solution containing the plasmid and mixed evenly. The mixture of plasmid and transfection reagent was slowly and evenly added to a 50 mL volume with a cell density of 6×10 6 The cells were suspended in ExpiCHO-S (Thermo Fisher) host cells at 100 viable cells / mL and cultured in a 37°C, 8% CO2 incubator. On day 1 (18-22 hours later), 300 μL of ExpiCHO Enhancer and 8 mL of ExpiCHO Feed were added, and the culture temperature was lowered to 32°C. A second feeding was performed on day 5, with 8 mL of ExpiCHO Feed added, and the cells were harvested after 12 days of culture. The cell suspension was centrifuged at 8000 rpm for 15 minutes, and the supernatant obtained from the centrifugation, the cell culture harvest, was used for purification of the target protein.

[0167] 3. Protein purification

[0168] 1) Sample capture (Protein A affinity capture)

[0169] The cell culture medium was centrifuged at 10,000 rpm for 30 min to remove cells and debris. The cells were then loaded onto a Protein A affinity column (GE Healthcare) to elute the target protein. Protein purity was assessed by SDS-PAGE.

[0170] The purification method of Protein A affinity capture is a conventional protein purification method well known to those skilled in the art. For specific experimental methods, please refer to the GE Healthcare Protein A product instructions and the GE Antibody Purification Manual.

[0171] 2) Sample purity

[0172] a) The following method is applicable to the dual-targeting fusion protein of structure 1 (the dual-targeting fusion protein of the present invention).

[0173] Use equilibration solution (50mM Tris-HCl 150mM NaCl, pH7.2) to equilibrate the chromatography column (anti-kappa chromatography column) until the UV detection line is stable; use a sample pump to load the sample, with a retention time of 5min and a load of ≤30mg / mL; use eluent (0.1M Gly-HCl pH2.7) to elute the target protein, adjust the pH of the eluent to 5.5 with 1MTris, and detect the protein purity by SDS-PAGE.

[0174] A chromatography column (Sperdex 200 pg chromatography column (Cytiva 16 / 600)) was equilibrated with an equilibration solution (50 mM NaAC-HAC, pH 5.5) until the UV detection line stabilized. Samples were loaded using a sample pump with a retention time of 120 min, a sample volume ≤ 2 mL, and a loading capacity ≤ 30 mg. The column was flushed with an equilibration solution (50 mM NaAC-HAC, pH 5.5). Proteins of different sizes were fractionated based on UV 280, and protein purity was determined by SDS-PAGE and SEC-HPLC. The purified dual-targeting fusion protein of the present invention was used in the studies of Examples 3-12.

[0175] b) The following method is applicable to dual-targeting fusion proteins of Structure 2, Structure 3, Structure 4, and Structure 5.

[0176] A chromatography column (Sperdex 200 pg chromatography column (Cytiva 16 / 600)) was equilibrated with an equilibration solution (50 mM NaAC-HAC, pH 5.5) until the UV detection line stabilized. Samples were loaded using a sample pump with a retention time of 120 min, a sample volume ≤ 2 mL, and a loading capacity ≤ 30 mg. The column was flushed with an equilibration solution (50 mM NaAC-HAC, pH 5.5). Proteins of different sizes were fractionated based on UV 280, and protein purity was determined by SDS-PAGE and SEC-HPLC. The purified dual-targeting fusion protein was used in the study of Example 3.

[0177] c) The following method is applied to the control sample synPTH-αRANKL-1

[0178] First, treat the chromatography column with a disinfectant (0.5M NaOH), then rinse the chromatography column (AKTA AVANT / Pure (cytiva) cationic chromatography column) with a balancing solution (20mM HAc-NaAc, pH 5.0). After the UV absorption reading stabilizes, start loading the sample. After the sample is loaded, continue to rinse the chromatography column with the balancing solution. The chromatography column is then rinsed with a washing solution (50mM HAc-NaAc + 0-0.5M NaCl, pH 5.0), and then with an eluent (20mM HAc-NaAc + 1.5M NaCl, pH 5.0). After the UV absorption reading rises, collect the eluted sample until the UV reading decreases and stabilizes. The chromatography column is then rinsed with a disinfectant and a balancing solution, respectively, and finally the chromatography column is preserved with a preservative (20% ethanol). The purified sample is subsequently used in the studies of Examples 3, 4, 5, and 6.

[0179] Example 3: Protein Structural Stability Analysis of Long-Acting Parathyroid Hormone Receptor Agonist Fusion Protein

[0180] 1) Expression titer analysis

[0181] The cell culture harvested fluid in Example 2 was used to detect the expression titer (titer, expression amount) of the dual-targeting fusion protein in the supernatant by the HPLC method. The cell culture harvested fluid of the dual-targeting fusion protein and the control sample was centrifuged at 13000rpm for 10min, and the supernatant was taken for detection. The specific detection method is: the expression titer in the cell supernatant was evaluated using HPLC (Agilent 1260) and Protein A affinity chromatography column (Thermo Fisher / POROS20A), mobile phase A: 50mM PBS+150mM NaCl, mobile phase B: 12mM HCl+150mM NaCl, gradient elution, detection time of 3 minutes, flow rate of 2.0mL / min, column temperature: 25°C, injection volume of 10μL, detection wavelength of 280nm. The test results are shown in Table 2.

[0182] Table 2 Dual-targeting fusion protein expression titer (Titer)

[0183] As shown in Table 2, the test results are as follows:

[0184] When the structure of the dual-targeting fusion protein is structure 1, its expression titer (expression titer ≥ 0.66 g / L) is significantly higher than that of other structure types (expression titer 0.07 g / L-0.54 g / L).

[0185] When the Anti-RANKL functional region sequence is the same, among the dual-targeting fusion proteins with different structural types or Linker or PTH functional regions, LYN101-10 has the highest titer (expression titer of 1g / L), which is significantly higher than other dual-targeting fusion proteins, such as the expression titer of LYN101-7 of structure 5 (expression titer of 0.07g / L).

[0186] When the structure, linker, PTH functional region, and Fc region of the dual-targeting fusion protein are identical, replacing the anti-RANKL functional region has almost no effect on protein production, that is, the expression titer remains at the same level. As shown in the data in Table 2, the expression titers of LYN101-2 and LYN101-8 are both greater than 0.8 g / L, indicating a relatively high expression level.

[0187] When the structure, anti-RANKL functional region, linker, and Fc region of the dual-targeting fusion protein are the same, changing the length (11 amino acids to 84 amino acids) or type (PTH or PTHrP) of the PTH functional region has almost no effect on protein production, that is, the expression titer remains at the same level (expression titer ≥0.72g / L).

[0188] When the structure of the dual-targeting fusion protein, the Anti-RANKL functional region, the Linker, and the PTH functional region are the same, changing the Fc region sequence (human IgG2, IgG4, Fc immune function-silent IgG1) or swapping the first and second arm Fc region sequences does not affect the protein expression titer, that is, the expression titer is at the same level. As shown in Table 2, the expression titers of LYN101-17, LYN101-18, and LYN101-2 are all greater than 0.8 g / L, which is at a relatively high expression level.

[0189] In summary, among the dual-targeting fusion proteins with different structures, the dual-targeting fusion protein of structure 1 (i.e., the dual-targeting fusion protein of the present invention) has the highest protein expression amount (expression titer), suggesting that the dual-targeting fusion protein (LAPAF) of the present invention can achieve a higher expression level, higher yield, and lower production cost; when the dual-targeting fusion protein is structure 1 (i.e., the dual-targeting fusion protein of the present invention), when Linker selects Linker1 (SEQ ID NO: 9), its protein expression amount (yield) is relatively higher; changes in the Anti-RANKL functional region, changes in the PTH functional region (length or type), and changes in the Fc region in the dual-targeting fusion protein of the present invention do not affect the expression amount (yield) of the dual-targeting fusion protein (LAPAF) of the present invention.

[0190] 2) Analysis of structural stability of PTH functional regions

[0191] 2.1) CE-SDS analysis

[0192] The purified Protein A sample was further analyzed by reduced CE-SDS to determine the proportion of impurities. The procedure was as follows: the sample was diluted with water to 2.5 mg / mL, 40 μL was taken to prepare the reduced sample, and then heated at 70°C for 10 minutes. The sample was then loaded for CE-SDS separation using a SCIEX PA800Plus instrument with a UV detector at a wavelength of 220 nm. The results are shown in Table 3.

[0193] Table 3 Analysis of the breakage ratio of PTH functional regions in dual-targeting fusion proteins

[0194] Note: The PTH impurity ratio is calculated based on peak area percentage. PTH impurities refer to peaks isolated slightly earlier than the PTH chain, and the PTH impurity ratio refers to the ratio of the peak area isolated slightly earlier than the PTH chain (chain containing the PTH functional region) to the total PTH-related peak area. To ensure comparability among dual-targeting fusion proteins of varying structures, the PTH functional region of the dual-targeting fusion proteins listed in Table 3 is PTH (1-34), i.e., the sequence is shown in SEQ ID NO: 5.

[0195] As shown in Table 3:

[0196] When the PTH functional region sequences are identical, when the dual-targeting fusion protein has a structure type of structure 1, the PTH impurity ratio is the lowest (≤25%), significantly lower than other dual-targeting fusion protein structure types, such as structure 4 (the PTH impurity ratios of the control samples synPTH-αRANKL-1 and LYN101-6 are as high as 50%). This indicates that the dual-targeting fusion protein (LAPAF) of the present invention has the lowest rate of breakage in the PTH functional region during preparation, and the structural stability of the PTH functional region is the highest. Furthermore, changes in the Anti-RANKL functional region in the dual-targeting fusion protein (LAPAF) of the present invention do not affect the structural stability of the PTH functional region in the dual-targeting fusion protein (LAPAF) of the present invention. For example, the dual-targeting fusion proteins (LAPAF) LYN101-8 and LYN101-2 of the present invention also have the characteristics of a low rate of breakage in the PTH functional region and high structural stability.

[0197] Furthermore, when the PTH functional region is PTH (1-34), the PTH functional region structural stability of LYN101-2 (when Linker 1 (SEQ ID NO: 9) is selected as the linker sequence) in the dual-targeting fusion protein (LAPAF) of the present invention is optimal, with a PTH impurity ratio of only 17%, far lower than the control sample synPTH-αRANKL-1 (50%). The reduced CE spectra of LYN101-2 and the control sample synPTH-αRANKL-1 are shown in Figure 2. As shown in Figure 2, the peak area of ​​the main peak of the heavy chain (PTH chain) of the control sample synPTH-αRANKL-1 is substantially equivalent to the peak area of ​​the PTH impurity, while the peak area of ​​the impurity peak (PTH impurity) of the second arm (PTH chain) of the dual-targeting fusion protein LYN101-2 of the present invention is significantly lower than the peak area of ​​the main peak (PTH chain). This indicates that the PTH functional region in the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention is highly structurally stable and has a low rate of breakage.

[0198] At the same time, the present invention also analyzes the structural stability of the PTH functional region of the dual-targeting fusion protein (LAPAF) of the present invention with other lengths or types of PTH functional regions, such as LYN101-9, whose PTH impurity ratio is only 1%.

[0199] In addition, the present invention also analyzed the effects of changes in the Fc region in the dual-targeting fusion protein of the present invention on the structural stability of the PTH functional region in the dual-targeting fusion protein (LAPAF) of the present invention. The results showed that there was no significant difference in the breakage ratio of the PTH functional region among the dual-targeting fusion proteins (LAPAF) LYN101-17, LYN101-18 and LYN101-2 of the present invention.

[0200] In summary, among the dual-targeting fusion proteins with different structures, the dual-targeting fusion protein with structure 1 (i.e., the dual-targeting fusion protein of the present invention) has the lowest breakage rate in the PTH functional region and the most stable structure; when the dual-targeting fusion protein is structure 1 (i.e., the dual-targeting fusion protein of the present invention) and the PTH functional region is PTH (1-34), when the Linker sequence is Linker1 (SEQ ID NO: 9), the breakage is minimized and the structure is the most stable; changes in the Anti-RANKL functional region, PTH functional region, or Fc region in the dual-targeting fusion protein of the present invention have no or almost no effect on the structural stability of the (LAPAF) PTH functional region in the dual-targeting fusion protein of the present invention.

[0201] 2.2) Mass spectrometry analysis

[0202] To further confirm the molecular fragmentation pattern, the inventors performed mass spectrometry molecular weight analysis on the dual-targeted fusion protein sample of the present invention and the control sample. PNGaseF enzyme was used to remove the N-glycosylation modification of the test sample and then the sample was injected for analysis. The mass spectrometer used was a Thermo QE, and the scan mode was positive ion mode, with a scan range of 2000-4000 m / z. Mobile phase A was 0.1% FA aqueous solution, and mobile phase B was 0.1% FA acetonitrile solution. The gradient elution was at a flow rate of 0.3 mL / min. The chromatographic column was a desalting column (Waters Mass PREP™ Micro Desalting), and the column temperature was 80°C. The data analysis software was Thermo's Biopharma Finder 4.0.

[0203] As shown in Figure 3, the control sample synPTH-αRANKL-1 showed a relatively low intensity of the intact molecule target peak (153342.42 Da) after deglycosylation, and a large number of non-target molecule peaks with higher intensity were present. By matching the sequence with the measured values, it is speculated that the control sample synPTH-αRANKL-1 has severe fragmentation to varying degrees at different sites within the PTH functional region. Correspondingly, as shown in Figures 4, 5, and 6, the mass spectrometry results of the dual-targeting fusion proteins (LAPAF) LYN101-1, LYN101-2, and LYN101-3 of the present invention after deglycosylation showed that the intact molecule target peaks (102003.72, 102320.49, and 103264.58, respectively) were all the highest intensity peaks, and the low molecular weight impurity peaks had extremely low intensities.

[0204] Mass spectrometry analysis of the dual-targeting fusion protein of the present invention and control samples once again verified that the dual-targeting fusion protein of the present invention (LAPAF) has a stable protein structure, the proportion of breakage in the PTH functional region is extremely low, and the yield of the target protein is extremely high.

[0205] Based on the above-mentioned expression titer analysis and protein structure stability analysis, such as the PTH functional region structural stability analysis, it can be found that the dual-targeting fusion protein (LAPAF) of the present invention has the highest expression titer and the least occurrence of PTH functional region cleavage. Therefore, it can be expected that under the same preparation process, the dual-targeting fusion protein of the present invention will ultimately obtain the most target protein, with the highest yield, the highest production volume, and the lowest production cost. Modification of the Anti-RANKL functional region, or Linker or PTH functional region, or Fc region has little or no effect on the expression titer of the dual-targeting fusion protein (LAPAF) of the present invention and the structural stability of the PTH functional region. Among them, when the PTH functional region is PTH (1-34), LYN101-2 is the preferred molecule in the dual-targeting fusion protein (LAPAF) of the present invention with the highest expression titer and the least occurrence of PTH functional region cleavage.

[0206] Example 4: LAPAF inhibitory activity on RANKL-induced osteoclast proliferation

[0207] In this example, RANKL was used to induce the differentiation of Raw264.7 cells (mouse mononuclear macrophage leukemia cells) into osteoclasts to test the ability of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention to inhibit the binding of RANKL to cell surface RANK. The following method uses LYN101-2 as an example and is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention, the control sample synPTH-αRANKL-1, and the anti-RANKL monoclonal antibody.

[0208] Raw264.7 cells in the logarithmic growth phase (purchased from ATCC, catalog number TIB-71) were taken and washed once with 6 mL of DPBS. Then, 3 mL of preheated trypsin was added and digested in a 37°C, 5% CO2 incubator for 6-10 minutes. After the cells were completely digested, cells still attached to the wall were scraped off with a cell scraper and 10 mL of DMEM complete medium was added and gently washed. Raw264.7 cells were prepared into a cell suspension and the cell density was adjusted to 5 × 10 4Cells / mL, 100 μL / well was added to a 96-well cell culture plate and placed in a constant temperature incubator (NUNRE / NU-8700E) at 37°C and 5% CO2 for 1 hour. The sample to be tested (the dual-targeting fusion protein LYN101-2 of the present invention, denosumab, synPTH-αRANKL-1) was pre-diluted to 15nM as the starting concentration and then diluted 1.5 times downward for a total of 11 concentration gradients. An equal volume of 200ng / mL RANKL diluent (purchased from Sino Biological, catalog number: 11682-HNCH) was then added to the sample dilution well and mixed, and then placed in a constant temperature incubator at 37°C and 5% CO2 for 2 hours. The mixed sample after the above incubation was added to each well of a 96-well cell culture plate on which Raw264.7 cells had been plated at 100 μL / well, and then placed in a constant temperature incubator at 37°C and 5% CO2 for 96h±3h. After incubation, the supernatant was removed and 100 μL / well of Citrate buffer (Sigma, Catalog No. C3434-250G) was added. The cells were shaken at 450 rpm and lysed for 40 min. 80 μL / well of the supernatant was added to 80 μL / well of prepared pNPP solution (Santa, Catalog No. sc-3720), mixed thoroughly, and incubated in a 37°C, 5% CO2 incubator for 30 min. The reaction was terminated by adding 40 μL / well of 2.5 M NaOH (Sino-pharm, Catalog No. 10019718). The 96-well cell culture plate was placed in a microplate reader (Molecular Devices\Spectra Max M2) and shaken for 30 seconds. The absorbance at 405 nm was read and recorded. Data were analyzed using Graphpad Prism software to calculate IC50 values.

[0209] The results showed that the proliferation inhibitory activity of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention on Raw264.7 cells induced to differentiate into osteoclasts by RANKL was comparable to that of anti-RANKL monoclonal antibodies (such as denosumab) and the control sample synPTH-αRANKL-1, that is, the dual-targeting fusion protein (LAPAF) of the present invention can inhibit the activation of osteoclasts and inhibit bone resorption by blocking the binding of RANKL and RANK.

[0210] As shown in Figure 7, the dual-targeting fusion protein LYN101-2 of the present invention has an inhibitory activity on the proliferation of Raw264.7 cells induced by RANKL differentiation, which is comparable to that of Denosumab (trade name: Proli, an anti-RANKL monoclonal antibody) and the control sample synPTH-αRANKL-1, indicating that the long-acting parathyroid hormone receptor agonist fusion protein LYN101-2 can inhibit the binding of RANKL and RANK, thereby inhibiting the activation of osteoclasts and inhibiting bone resorption.

[0211] In addition, the present invention also simultaneously tested the dual-targeting fusion proteins LYN101-1, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention, which have different anti-RANKL functional regions, linkers, PTH functional regions, or Fc regions from LYN101-2. The results were similar to those of LYN101-2, with EC50 values ​​of approximately 1 nM. It can be seen that replacing the Anti-RANKL functional region, Linker, the length or type (PTH or PTHrP) of the PTH functional region, and the Fc region in the dual-targeting fusion protein (LAPAF) of the present invention does not affect the inhibitory activity of the dual-targeting fusion protein (LAPAF) of the present invention on RANKL-induced osteoclast proliferation, and the activity is comparable to that of the anti-RANKL monoclonal antibody and the control sample synPTH-αRANKL-1. It can well block the binding of RANKL and RANK, thereby inhibiting the activation of osteoclasts and inhibiting bone resorption. In summary, the dual-targeting fusion protein (LAPAF) of the present invention has significant proliferation inhibitory activity on RANKL-induced differentiated Raw264.7 cells, and the activity is comparable to that of denosumab. By blocking the binding of RANKL and RANK, it inhibits the activation of osteoclasts and inhibits bone resorption. Replacing the Anti-RANKL functional region in the dual-targeting fusion protein (LAPAF) of the present invention, or adjusting the length or type of the PTH functional region, or the Linker, or the Fc region has no effect on the inhibitory activity of the dual-targeting fusion protein (LAPAF) of the present invention on RANKL-induced osteoclast proliferation.

[0212] Example 5: LAPAF on PTHR + / RANKL + In vitro activity of cellular PTHR-induced cAMP release

[0213] 1) Construction of the UMR106-RANKL cell line for LAPAF in vitro activity

[0214] In this example, the following method was used to construct a UMR106-RANKL stable transfection cell line.

[0215] a) Wash UMR106 cells (purchased from ATCC, catalog number: CRL-1661) once with 6 mL of DPBS, then add 3 mL of preheated trypsin and incubate in a 37°C, 5% CO2 incubator for 5 minutes. After complete digestion, add 10 mL of DMEM complete medium and gently rinse the cells to prepare a cell suspension. Adjust the cell density to 1 × 10 6 cells / mL, 1.5 mL / well was added to a 6-well cell culture plate, and the plate was placed in a constant temperature incubator (NUNRE / NU-8700E) at 37°C and 5% CO2 for overnight culture.

[0216] b) The next day, pre-plated UMR106 cells were infected with RANKL lentivirus at an MOI of 50. 48 h later, 2 μg / mL puromycin was added for pressure selection. The cells were treated with DMEM + 10% FBS + 2 μg / mL puromycin every 2-3 days. After 2 weeks, a pool of stably transfected cells expressing RANKL (UMR106-RANKL-pool) was obtained.

[0217] c) The RANKL stably transfected cell pool was washed once with 6 mL of DPBS, and then 3 mL of preheated trypsin was added. The cells were digested in a 37°C, 5% CO2 incubator for 5 minutes. After complete digestion, 10 mL of DMEM + 10% FBS + 2 μg / mL puromycin medium was added and gently washed. The cells were prepared into a cell suspension, diluted to 1 cell / well, and plated in a 96-well plate with 100 μL per well. The plates were cultured in a 37°C, 5% CO2 incubator (NUNRE / NU-8700E) for 2 weeks. During the culture period, the supernatant in the 96-well plate was aspirated every 2-3 days, and 100 μL / well of DMEM + 10% FBS + 2 μg / mL puromycin medium was added. The wells with single clones were selected and marked. After the monoclonal cells in the 96-well plate filled up the wells, 30 clones were selected and transferred to 6-well plates for culture. After the cells in the 6-well plates filled up, they were expanded into T25 medium. At the same time, some cells were taken to detect the expression level of RANKL by flow cytometry to obtain UMR106-RANKL monoclonal cells UMR106-RANKL-4.

[0218] 2) Functional verification of LAPAF activity in vitro using the UMR106-RANKL cell line

[0219] a) Wash the UMR106-RANKL-pool or UMR106-RANKL-4 monoclonal cells once with 6 mL of DPBS, then add 3 mL of preheated trypsin and incubate in a 37°C, 5% CO2 incubator for 5 min. After complete digestion, add 10 mL of DMEM complete medium and gently rinse the cells to prepare a cell suspension. Adjust the cell density to 1 × 10 6 cells / mL, 100 μL / well was added to a 96-well U-shaped plate for later use;

[0220] b) Dilute denosumab to a starting concentration of 20 nM, then perform a 5-fold dilution series for a total of six concentrations. Centrifuge the cells treated in step a) at 1000 rpm for 5 minutes, discard the supernatant, add 100 μL / well of the above antibody, incubate at 4°C for 1 hour, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0221] c) hIgG(H+L)-Alexa After 488 fluorescent secondary antibody was diluted 500 times, 100 μL / well was added to the above cells, incubated at 4°C for 1 hour, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended with 100 μL / well buffer.

[0222] d) The 96-well plate was placed in a BD C6 flow cytometer, and the MFI of FL1-H was read and recorded. The data were analyzed using Graphpad Prism software.

[0223] Denosumab (an anti-RANKL monoclonal antibody) binds to the RANKL stably transfected cell pool (UMR106-RANKL-pool) in a gradient pattern, confirming the successful construction of the RANKL stably transfected cell pool. The binding curve of denosumab to the RANKL stably transfected monoclonal cell line UMR106-RANKL-4 is similar to that of the UMR106-RANKL-pool, confirming the successful construction of the RANKL stably transfected monoclonal cell line.

[0224] To simplify the expression, UMR106-RANKL-pool and UMR106-RANKL-4 are collectively referred to as UMR106-RANKL, referring to UMR106-RANKL-pool or UMR106-RANKL-4.

[0225] 3) LAPAF for UMR106-RANKL (PTHR + / RANKL + ) In vitro activity assay of PTHR-induced cAMP release in cells

[0226] In this example, the following method was used to determine the in vitro activity of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention on PTHR-induced cAMP release in UMR106-RANKL cells. The following method, using LYN101-2 as an example, is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention, the control sample synPTH-αRANKL-1, teriparatide, and agonistic antibodies targeting PTHR.

[0227] One day in advance, UMR106-RANKL-pool or UMR106-RANKL-4 cells were washed once with 6 mL of DPBS, and then 3 mL of preheated trypsin was added. The cells were digested in a 37°C, 5% CO2 incubator for 5 minutes. After the cells were completely digested, 10 mL of DMEM complete medium was added and gently washed. The two cells were prepared into cell suspensions and the cell density was adjusted to 3 × 10 5Cells were plated at 100 μL / well in a 96-well cell culture plate at 100 cells / mL and cultured overnight in a 37°C, 5% CO2 incubator (NUNRE / NU-8700E). The next morning, the supernatant from the 96-well plate was discarded, and DMEM / F12 medium was added and incubated in a 37°C, 5% CO2 incubator for 1 hour. The supernatant was discarded, and the test samples (LYN101-2, teriparatide, and control sample synPTH-αRANKL-1) were diluted to the starting concentration (the starting sample concentration was 100 nM). Five-fold dilutions were then performed downward, for a total of eight concentration gradients (diluent composition: DMEM / F12 + 1 mM IBMX + 0.1% BSA). 100 μL / well of the above sample was added to each well of a 96-well cell culture plate seeded with UMR106-RANKL-pool or UMR106-RANKL-4 cells and incubated in a 37°C, 5% CO2 incubator for 40 minutes. Then, 130 μL / well of 1× lysis buffer was added and incubated at room temperature for 30 minutes to obtain the test sample (cell lysate supernatant). Subsequent procedures were performed using the cAMP Parameter Assay Kit (purchased from R&D, Cat. No. SKGE002b). Briefly, 50 μL / well of the primary antibody from the kit was added to a 96-well plate pre-coated with Goat Anti-mouse. The plate was incubated at 500 rpm / min for 1 hour at room temperature and then washed. Then, 100 μL / well of the cell lysate supernatant and 50 μL / well of cAMP Conjugate were added and incubated at 500 rpm / min for 2 hours at room temperature and then washed. After adding the substrate and reacting at room temperature for 30 minutes, the stop solution was added and the absorbance at 450 / 570 nm was read and recorded using a microplate reader. The data were analyzed using Graphpad Prism software to determine the EC50 value.

[0228] The results showed that the control sample synPTH-αRANKL-1 and teriparatide were + / RANKL + The in vitro activity of the dual-targeting fusion proteins (LAPAF) LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17, and LYN101-18 of the present invention on UMR106-RANKL cells (PTHR) was comparable. + / RANKL +The results of this experiment indicate that the in vitro activity of PTHR-induced cAMP release in double-positive cells (PTHR) expressing both PTHR and RANKL is not obviously better than that of teriparatide. + / RANKL + cells), at the same molar concentration, the dual-targeting fusion protein (LAPAF) of the present invention LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17, and LYN101-18 had a stronger in vitro activity on PTHR-induced cAMP release than teriparatide or the control sample synPTH-αRANKL-1. This indicates that the dual-targeting fusion protein (LAPAF) of the present invention is effective in simulating PTHR of osteoblasts. + / RANKL + Cells have a stronger effect and can enhance the effect of drugs on osteoblasts.

[0229] As shown in Figure 8A, in UMR106-RANKL-pool cells (PTHR + / RANKL + In vitro activity of the dual-targeting fusion protein (LAPAF) LYN101-2 on PTHR-induced cAMP release was superior to that of teriparatide (EC50 LYN101-2 =1.771nM, EC50 特立帕肽 =6.532nM, EC50 LYN101-2 <EC50 特立帕肽 ), while the activity of the control sample synPTH-αRANKL-1 was comparable to that of teriparatide (EC50synPTH-αRANKL-1=8.969nM, EC50 特立帕肽 =6.532nM, EC50synPTH-αRANKL-1≈EC50 特立帕肽 ).

[0230] At the same time, in order to evaluate whether different selections of the Anti-RANKL functional region, Linker, PTH functional region, and Fc region in the dual-targeting fusion protein (LAPAF) of the present invention have an effect on the in vitro activity experimental results of PTHR-induced cAMP release in UMR106-RANKL cells, the inventors also simultaneously tested LYN101-8, which has a different Anti-RANKL functional region from LYN101-2, LYN101-1 and LYN101-3, which have different Linkers from LYN101-2, LYN101-11, LYN101-12, LYN101-13, LYN101-14, and LYN101-15, which have different PTH functional regions from LYN101-2, and LYN101-16. YN101-2 has LYN101-17 and LYN101-18 with different Fc regions. The results showed that the in vitro activity of LYN101-8, LYN101-1, LYN101-3, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17 and LYN101-18 on PTHR-induced cAMP release in UMR106-RANKL cells was similar to that of LYN101-2. It can be seen that changes in the Anti-RANKL functional region, or Linker, or PTH functional region, or Fc region do not affect the activity of the dual-targeting fusion protein (LAPAF) of the present invention on UMR106-RANKL cells (PTHR). + / RANKL + The in vitro activity of PTHR-induced cAMP release in WT cells showed superior efficacy compared with teriparatide.

[0231] The specific experimental results are as follows: The dual-targeting fusion protein (LAPAF) LYN101-8 (different from the LYN101-2 Anti-RANKL functional region) of the present invention was detected in UMR106-RANKL-pool cells (PTHR + / RANKL +The in vitro activity of LYN101-8 on PTHR-induced cAMP release in UMR106-RANKL-pool cells was similar to that of LYN101-2, that is, the in vitro activity of LYN101-8 on PTHR-induced cAMP release in UMR106-RANKL-pool cells was stronger than that of teriparatide and the control sample synPTH-αRANKL-1. The in vitro activity of LYN101-8 on PTHR-induced cAMP release in UMR106-RANKL-pool cells was comparable to that of LYN101-2. For example, the EC50 of the activation activity of LYN101-8 and LYN101-2 on PTHR-induced cAMP activation in UMR106-RANKL-pool cells was 1.7 nM (LYN101-2) and 1.4 nM (LYN101-8), respectively. It can be seen that changing the Anti-RANKL functional region does not affect the effect of the dual-targeting fusion protein (LAPAF) of the present invention on PTHR. + / RANKL + The in vitro activity of PTHR-induced cAMP release in cells is superior to that of teriparatide.

[0232] The dual-targeting fusion proteins (LAPAF) LYN101-1 and LYN101-3 (different from LYN101-2 Linker selection) of the present invention were expressed in UMR106-RANKL-pool cells (PTHR + / RANKL + The in vitro activity of LYN101-1 and LYN101-3 on PTHR-induced cAMP release in UMR106-RANKL-pool cells was similar to that of LYN101-2, that is, the in vitro activity of LYN101-1 and LYN101-3 on PTHR-induced cAMP release in UMR106-RANKL-pool cells was stronger than that of teriparatide and the control sample synPTH-αRANKL-1. It can be seen that changing the linker does not affect the targeting fusion protein (LAPAF) of the present invention on PTHR. + / RANKL + The in vitro activity of PTHR-induced cAMP release in cells is superior to that of teriparatide.

[0233] In addition, in order to evaluate the effect of the length or type of the PTH functional region and the Fc region of the dual-targeting fusion protein (LAPAF) of the present invention on UMR106-RANKL cells (PTHR + / RANKL +To investigate whether the in vitro activity of PTHR-induced cAMP release in UMR106-RANKL-4 cells (PTHR) is affected, the inventors also simultaneously tested the effects of the dual-targeting fusion proteins (LAPAF) of the present invention, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17, and LYN101-18, which have different PTH functional region lengths or types (PTH or PTHrP) or different Fc regions from LYN101-2, on the in vitro activity of PTHR-induced cAMP release in UMR106-RANKL-4 cells (PTHR). + / RANKL + The results showed that, as shown in Figure 8B, the release of cAMP induced by PTHR in UMR106-RANKL-4 cells (PTHR) + / RANKL + The in vitro activity of cAMP release induced by PTHR in cells, the dual-targeting fusion proteins (LAPAF) LYN101-2, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17, LYN101-18 of the present invention are all stronger than teriparatide, for example, EC50 特立帕肽 =39.7nM, EC50 LYN101- 12 =1.4nM, EC50 LYN101-14 =3.6nM, which is similar to the result of LYN101-2. It can be seen that changing the length or type of the PTH functional region and changing the Fc region do not affect the targeting fusion protein (LAPAF) of the present invention on PTHR. + / RANKL + The in vitro activity of PTHR-induced cAMP release in cells is superior to that of teriparatide.

[0234] Example 6: Efficacy and pharmacokinetic study of a single dose of a test substance in an ovariectomized RANKL humanized transgenic mouse osteoporosis model

[0235] In this example, the following method was used to evaluate the efficacy of a single administration of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention in an ovariectomized RANKL humanized transgenic mouse osteoporosis model. The following method uses LYN101-2 as an example and is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention.

[0236] Thirty humanized RANKL transgenic mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., B-hRANKL mice, catalog number: 111072) were randomly divided into 6 groups: G1 Sham group (n=3), G2 OVX group (model group) (n=6), G3 control sample synPTH-αRANKL-1 10mpk group (n=6), G4 LYN101-2 2mpk group (n=6), G5 LYN101-2 10mpk group (n=6), G6 LYN101-2 10mpk group (n=3); the Sham group underwent sham surgery, and the other groups underwent ovariectomy to establish osteoporosis mouse models. Drug administration began 4 weeks after surgery, and the day of administration was counted as Day 0. The G1 and G2 groups were subcutaneously administered with an equal volume of buffer (normal saline solution containing 0.005% Tween 80), and the G3 to G6 groups were subcutaneously administered once. Serum was collected from groups G1 and G2 on D (Day, hereinafter abbreviated as D)-28 (before modeling), D-21, D-14, and D-7; serum was collected from groups G1 to G5 on D0 (before administration), 6h, 24h, D2, D3, D5, D7, D14, D21, D28, D42, and D56 after administration; serum was collected from group G6 at 312h, 360h, 1200h, and 1368h after administration. Serum from groups G1 to G5 was used for PD marker (osteocalcin, TRAP-5b) detection, and serum from group G6 was used for PTH functional activity detection; at the end of the experiment, tibia, femur, and vertebrae samples from groups G1 to G5 were collected for tibial bone density detection. The algorithm for the percentage change of bone density is as follows: percentage change of BMD in the sham group = (BMD of the sham group - BMD of the OVX group) / BMD of the sham group * 100, percentage change of BMD in the synPTH-αRANKL-1 group = (BMD of the synPTH-αRANKL-1 group - BMD of the OVX group) / BMD of the OVX group, percentage change of BMD in the LYN101-2,2mpk group = (BMD of the LYN101-2,2mpk group - BMD of the OVX group) / BMD of the OVX group, percentage change of BMD in the LYN101-2,10mpk group = (BMD of the LYN101-2,10mpk group - BMD of the OVX group) / BMD of the OVX group.

[0237] This example uses LYN101-2 as a representative dual-targeting fusion protein (LAPAF) of the present invention and synPTH-αRANKL-1 as a representative control sample to demonstrate the efficacy of LAPAF and the control sample in a transgenic mouse osteoporosis model induced by ovariectomy and humanized RANKL.

[0238] As shown in Figure 9, the bone density of the proximal tibia of mice decreased significantly after modeling (see OVX group, model group). At D56 (week 8), the control sample synPTH-αRANKL-1 had an extremely weak effect on improving the bone density of the proximal tibia of mice (p>0.05), while the dual-targeting fusion protein (LAPAF) LYN101-2 2mpk and 10mpk groups of the present invention had a very significant effect on improving the bone density of the proximal tibia of mice (p<0.05, p<0.05), and there was a dose-effect trend. It can be seen that 8 weeks after a single administration (D56), the dual-targeting fusion protein (LAPAF) of the present invention was significantly better than the control sample synPTH-αRANKL-1 in improving the bone density of mice in the osteoporosis model caused by ovariectomy. This suggests that a single administration of the dual-targeted fusion protein (LAPAF) of the present invention has a better and long-lasting effect on improving bone density, and no osteoclastogenic advantage was observed with continuous infusion of teriparatide or abalotriptyline, or TransCon PTH.

[0239] As shown in Figure 10, the level of tartrate-resistant acid phosphatase 5b (TRAP5b), a bone resorption marker, in the serum of mice was significantly increased after modeling (data not shown). At D56 (week 8), the levels of TRAP5b in the serum of the control sample synPTH-αRANKL-1 and the OVX group were basically equivalent, while the levels of TRAP5b in the dual-targeting fusion protein (LAPAF) LYN101-2 2mpk and 10mpk groups of the present invention were significantly downregulated compared with the OVX group (p<0.0001, p<0.0001). It can be seen that 8 weeks after a single administration, the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention can effectively inhibit the activity of osteoclasts, and its efficacy is significantly better than that of the control sample synPTH-αRANKL-1. This suggests that a single administration of the dual-targeted fusion protein (LAPAF) of the present invention has superior and long-lasting effects on the levels of bone resorption markers, and further verifies that the dual-targeted fusion protein (LAPAF) of the present invention did not show osteoclastogenic advantages over continuous infusion of teriparatide or abalotriptyline, or TransCon PTH in the model of this example.

[0240] As shown in Figure 11, the level of osteocalcin, a bone formation marker, decreased significantly after modeling (data not shown). At D56 (week 8), the levels of osteocalcin in the serum of the control sample synPTH-αRANKL-1 and the OVX group were basically equivalent, while the levels of osteocalcin in the serum of the dual-targeting fusion protein (LAPAF) LYN101-2 2mpk and 10mpk groups of the present invention were significantly increased compared with the OVX group (p < 0.0001, p < 0.0001). It can be seen that 8 weeks after a single administration, the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention can still maintain a high efficacy in promoting bone formation, and the efficacy is significantly better than that of the control sample synPTH-αRANKL-1. This suggests that a single administration of the dual-targeting fusion protein (LAPAF) of the present invention has superior and long-lasting effects on the levels of bone formation markers.

[0241] At the same time, the inventors also investigated the blood drug exposure of the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention in mice and the activity of the corresponding PTH functional area. The inventors mixed the serum of 3 mice at each blood collection time point in the LYN101-2 10mpk group (G6 group) and diluted it 10 times to detect its in vitro activity on PTHR-induced cAMP release in UMR106-RANKL-pool cells (PTH functional activity experiment, for specific detection methods, see Example 5). The results are shown in Figure 12. The serum at each time point still maintains a strong in vitro activity on PTHR-induced cAMP release in UMR106-RANKL-pool cells. After a single dose of D56 (week 8, 1368h), the serum diluted 10 times still has a strong PTH functional activity, and the activity is equivalent to the LYN101-2 1nM level.

[0242] In summary, the present invention unexpectedly discovered that LAPAF (the dual-targeting fusion protein of the present invention) was exposed to long-term high blood concentrations in mice, but the osteoclast advantage phenomenon reported in the literature did not occur. It can be seen that LAPAF (the dual-targeting fusion protein of the present invention) solves the osteoclast advantage that occurs with long-acting PTH (such as TransCon PTH) or continuous infusion of teriparatide or abaloparatide.

[0243] Example 7: In vivo efficacy study of multiple doses of the test substance in an ovariectomized RANKL humanized transgenic mouse osteoporosis model

[0244] In this example, the following method was used to evaluate the in vivo efficacy of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention after multiple administration in an ovariectomized RANKL humanized transgenic mouse osteoporosis model. The following method uses LYN101-2 as an example and is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention.

[0245] Twenty-four humanized RANKL transgenic mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., B-hRANKL mice, catalog number: 111072) were randomly divided into four groups, with six mice in each group: G1 sham group (subcutaneously administered with an equal volume of buffer (saline solution containing 0.005% Tween 80)), G2 OVX group (model group, subcutaneously administered with an equal volume of buffer (saline solution containing 0.005% Tween 80)), G3 denosumab (10 mpk, subcutaneously administered once a week) + teriparatide (trade name: Zhen Gu) (0.1 mpk, subcutaneously administered once a day) group (combination group), and G4 LYN101-2 (10 mpk, subcutaneously administered once a week) group (LYN101-2 group). The sham group underwent sham surgery, while the other groups underwent ovariectomy to establish an osteoporosis mouse model (ovariectomy was performed using procedures known in the art). Drug administration began four weeks after surgery and continued for four consecutive weeks. Blood samples were collected before, for three weeks, and four weeks after drug administration to measure serum CTX-1, calcium, and phosphorus levels. Four weeks after drug administration, mice were sacrificed, and the tibia, femur, and fourth lumbar vertebra were harvested for analysis. Micro-CT analysis of the left tibia was performed. The percentage change in bone density was calculated as follows: percentage change in BMD in the sham group = (BMD in the sham group minus BMD in the OVX group) / BMD in the sham group; percentage change in BMD in the denosumab + teriparatide group = (BMD in the denosumab + teriparatide group minus BMD in the OVX group) / BMD in the OVX group; and percentage change in BMD in the LYN101-2 group = (BMD in the LYN101-2 group minus BMD in the OVX group) / BMD in the OVX group.

[0246] This example uses LYN101-2 as a representative of the dual-targeting fusion protein (LAPAF) of the present invention and a combination of denosumab (an anti-RANKL monoclonal antibody) and teriparatide (rhPTH1-34) as a representative control sample to demonstrate the efficacy of multiple doses of LAPAF in an ovariectomized RANKL humanized transgenic mouse osteoporosis model.

[0247] As shown in Figure 13, after oophorectomy, the bone density of the proximal tibia in the OVX group (G2 group) decreased significantly (p < 0.001). After 4 weeks of treatment, the bone density of the combination group (G3 group) and the LYN101-2 group (G4 group) increased. However, the percentage change of the improvement of the bone density of the proximal tibia in the dual-targeting fusion protein (LAPAF) LYN101-2 group (G4 group) of the present invention was significantly better than that in the combination group (G3 group) of denosumab (anti-RANKL monoclonal antibody) and teriparatide.

[0248] As shown in Figure 14, after oophorectomy, the level of CTX-1 in the serum of the OVX group (G2 group) increased significantly. After 4 weeks of treatment, compared with the OVX group (G2 group), the levels of CTX-1 in the serum of the combination group of denosumab (anti-RANKL monoclonal antibody) and teriparatide (G3 group) and the dual-targeting fusion protein (LAPAF) LYN101-2 group of the present invention (G4 group) decreased significantly (G3, p < 0.05; G4, p < 0.01), and the LYN101-2 group (G4) can reduce the serum CTX-1 to a greater extent than the combination group (G3), thereby slowing down the rate of bone loss.

[0249] Example 8: Preliminary study on the efficacy of a single dose of LAPAF in a spontaneous osteoporosis model of elderly rhesus monkeys

[0250] In this example, the following method was used to evaluate the in vivo efficacy of a single dose of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 in a spontaneous elderly osteoporosis rhesus monkey model. The following method uses LYN101-2 as an example and is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention.

[0251] Two elderly female rhesus monkeys with spontaneous osteoporosis were divided into two groups: LYN101-2 0.1 mpk and LYN101-2 0.5 mpk. Dosing began on D0, and serum calcium and phosphorus levels were measured on D1, D3, D7, D10, D14, D18, D21, D28, D35, D42, D49, and D56. Levels of the bone formation markers PINP and TRAP5b were measured before the first dose and on D56. Lumbar vertebrae L1-L4 bone density scans were performed using a GE Prodigy dual-energy X-ray absorptiometry before the first dose and on D56.

[0252] This example uses LYN101-2 as a representative of LAPAF to demonstrate the results of a preliminary in vivo efficacy study in a spontaneous elderly osteoporosis rhesus monkey model.

[0253] The results showed that after a single dose of LYN101-2 0.1mpk on D56, the bone formation marker PINP in spontaneously aged osteoporotic rhesus monkeys decreased by 8.5% compared with the baseline before administration, and the bone resorption marker TRAP5b increased by 8.8% compared with the baseline before administration, indicating that the activity of osteoblasts on D56 was slightly decreased while the activity of osteoclasts was slightly increased compared with the baseline before administration. This data suggests that the bone density of the model animals should theoretically decrease on D56, but the inventors unexpectedly observed the opposite result. Compared with the baseline before administration, the L1-L4 (lumbar vertebrae) of spontaneously aged osteoporotic rhesus monkeys on D56 decreased by 8.5%. The bone density of the vertebrae increased by 33.1%, which is significantly better than other osteoporosis treatment drugs on the market. For example, after 6 months of continuous administration of teriparatide (once a day), the bone density of L2-L4 only increased by about 5% (accessdata.fda.gov / drugsatfda_docs / nda / 2002 / 21-318_FORTEO_Pharmr_P1.pdf), and the anti-sclerostin monoclonal antibody Romosozumab increased by only about 5% after continuous administration. After 4 months of treatment with the drug (administered once a month), the bone density of L1-L4 only increased by about 8% (accessdata.fda.gov / drugsatfda_docs / nda / 2019 / 761062Orig1s000MultidisciplineR.pdf). The anti-RANKL monoclonal antibody Denosumab only increased the bone density of L1-L4 by about 2% 3 months after a single dose. There is no literature report that the above drugs can quickly increase bone density in the short term (such as within 2 months). It can be seen that the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention has fast-acting and long-lasting effects. After a single low-dose (0.1 mpk) administration, D56 can significantly increase the bone density of spontaneously aged osteoporotic rhesus monkeys, and the bone density increase rate in the short term (such as 2 months) is as high as 33.1%, which is significantly better than the bone density improvement effect (2%-8%) of existing osteoporosis treatment drugs administered once or continuously for 3-6 months.

[0254] In addition, after a single dose, serum calcium and phosphorus levels in spontaneously osteoporotic elderly female rhesus monkeys were observed. The results showed that regardless of whether LYN101-2 was administered at a dose of 0.1 mpk or 0.5 mpk, serum calcium levels at all observed time points remained within the normal range (9.2-11.6 mg / dL) (normal range reference: Cui Shufang, Experimental Animal Science, Shanghai, Second Military Medical University Press, 2013). No hypercalcemia was observed as a result of other long-acting PTH drugs, such as TransCon PTH (generic name: palopegteriparatide, trade name: Yorvipath).

[0255] Example 9: In vivo efficacy study of multiple doses of the test substance in an ovariectomized RANKL humanized transgenic mouse model of osteoporosis and surgically prepared femoral fracture

[0256] In this example, the following method was used to evaluate the in vivo efficacy of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention after multiple administration in an ovariectomized RANKL humanized transgenic mouse model of osteoporosis and surgically induced femoral fracture. The following method uses LYN101-2 as an example and is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention.

[0257] Thirty-nine RANKL humanized transgenic mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., B-hRANKL mice, catalog number: 111072) were divided into 7 groups: Sham group, Sham + fracture group (non-OVX fracture model group), OVX (ovariectomy) + fracture group (OVX fracture model group), teriparatide group (4ug / kg), teriparatide (4ug / kg) + denosumab (10mpk) (combination group), LYN101-2 0.4mpk group, and LYN101-2 2mpk group. There were 3 mice in the Sham group and 6 mice in the other groups. Ovariectomy (OVX) surgery was performed on day -31 to establish an osteoporosis model. A left femoral fracture model (open fracture) was established on day -3. LYN101-2 0.4 mpk and LYN101-2 2 mpk groups were subcutaneously injected 3 days later (Day 0) for 6 consecutive weeks. Except for teriparatide, which was administered daily from Monday to Friday, all other drugs were administered weekly. The sham and model groups were injected with an equal volume of buffer (saline solution containing 0.005% Tween 80) weekly. Serum calcium and phosphorus levels were measured on day -31 (before OVX), day -3 (before fracture), day 7, day 28, and day 42. Serum ALP, CTX-1, osteocalcin, PINP, and TRAP5b levels were also measured. The left femur was harvested for fracture healing analysis, the right tibia was harvested for bone mineral density analysis, the right tibia and L4 lumbar vertebra were harvested for future use, and the right femur was harvested for future use. Micro-CT analysis of the right proximal tibia and left femoral fracture sites was performed. Calcein was injected intraperitoneally on the 10th and 3rd days before collecting the mouse fracture samples, and alizarin red was injected intraperitoneally on the 7th day before collecting the mouse fracture samples to fluorescently label the areas with vigorous bone metabolism in the mice. At the end of the animal experiment, the mice were humanely sacrificed and their femoral fracture specimens were obtained. The fluorescent double labeling method was used to visually study the mineralization process of the mouse femoral fracture specimens. Ultrathin sections were obtained using the non-decalcified bone tissue sectioning technique, and then photographed under a laser confocal microscope to observe the spacing of the fluorescent labeled substances to calculate the bone mineral apposition rate (MAR, in μm / day) and the bone tissue formation rate (BFR, in μm 3 / μm 2 The specific calculation method is as follows: Bone mineral apposition rate (MAR) refers to the thickness of newly deposited mineralized bone on the active bone formation surface per day. It is calculated as the average width of the double-labeled band divided by the number of days between double labeling. Bone formation rate (BFR) = (MS / TP) * (1 / MAR), where MS represents the bone formation area and TP represents trabecular thickness. Results are expressed as mean ± standard deviation.

[0258] This example uses LYN101-2 as a representative of LAPAF to demonstrate the efficacy of multiple administrations in a model of osteoporosis and surgically prepared femoral fracture in ovariectomized RANKL humanized transgenic mice.

[0259] As shown in Figures 15A and 15B: After the RANKL humanized transgenic mice were ovariectomized and surgically fractured, a very obvious fracture line appeared at the fracture site. The drug was administered once a week. After 6 weeks of treatment, only 1 mouse in the OVX fracture model group showed fracture line closure, and 5 mice still had relatively obvious fracture lines; correspondingly, in the dual-targeting fusion protein (LAPAF) LYN101-2 0.4mpk group of the present invention, fracture lines were observed to be closed in 4 mice, the fracture line of 1 mouse was partially closed, and only 1 mouse still had a relatively obvious fracture line; in the dual-targeting fusion protein (LAPAF) LYN101-2 2mpk group of the present invention, the fracture lines of 5 mice were closed, and only 1 mouse had a relatively obvious fracture line.

[0260] As shown in Figure 16, after RANKL humanized transgenic mice underwent ovariectomy and femoral fracture, the bone density at the femur (fracture site) decreased significantly. After 6 weeks of treatment (once a week), compared with the OVX fracture model group, the bone density at the fracture site of mice in the LYN101-2 0.4mpk group of the dual-targeting fusion protein (LAPAF) of the present invention increased, and the bone density at the fracture site of mice in the LYN101-2 2mpk group was significantly increased.

[0261] As shown in Figure 17, after RANKL humanized transgenic mice underwent ovariectomy and femoral fracture, the bone density of the tibia (non-fracture site) decreased significantly. After 6 weeks of treatment (once a week), the bone density of the proximal tibia of mice in the dual-targeting fusion protein (LAPAF) LYN101-2 0.4mpk and 2mpk groups of the present invention was significantly improved, and the bone density of some mice was even close to normal levels.

[0262] As shown in Figure 18, after ovariectomy and femoral fracture, the MAR of RANKL humanized transgenic mice was significantly reduced, indicating that the model was successfully established. After 6 weeks of administration, the MAR of mice in the 0.4 mpk and 2 mpk groups of the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention was significantly increased, and a dose-dependent increase trend was observed. Compared with the teriparatide group, the MAR of the 0.4 mpk group of the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention was significantly increased. Compared with the teriparatide group and the combination group of teriparatide and denosumab, the MAR of the LYN101-2 2 mpk group was also significantly increased, indicating that 0.4 mpk of LYN101-2 is significantly better than teriparatide in the treatment of fracture healing in mice with osteoporotic fractures, and 2 mpk of LYN101-2 is significantly better than teriparatide in the treatment of fracture healing in mice with osteoporotic fractures. Furthermore, the MAR of mice treated with the dual-targeting fusion protein (LAPAF) LYN101-2 (2 mpk) showed no statistically significant difference between the sham group and the non-OVX fracture model group, suggesting that the dual-targeting fusion protein (LAPAF) LYN101-2 can restore the MAR of mice with osteoporotic fractures to near-normal levels. The BFR trend was consistent with the MAR.

[0263] Example 10: Evaluation of the efficacy of LAPAF in a mouse osteoarthritis model induced by medial meniscus transection (MMT)

[0264] In this example, the following method was used to evaluate the efficacy of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention in a mouse osteoarthritis model induced by medial meniscus transection (MMT). The following method uses LYN101-2 as an example and is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention.

[0265] This example mainly studies 15 11-week-old mice using Bio-Book, and is randomly divided into 3 groups according to animal weight: Sham group, MMT model group, MMT model group + LYN101-2 0.4mpk group (LYN101-2 0.4mpk group). The surgical modeling process is as follows: the medial side of the femoral-tibial joint will be incised, the medial meniscus will be exposed by blunt dissection, and a full-thickness incision will be made at the narrowest part of the meniscus. After completion, the joint will be repositioned, the knee joint muscles, ligaments and skin will be sutured, and the surgical site will be disinfected. Subcutaneous injections will be started two weeks after surgery. This experiment will be administered once a week, with subcutaneous administration for the first four weeks and intra-articular injection for the last four weeks, for a total of 8 doses. The Sham group and the MMT model group will be injected with equal amounts of Buffer (a physiological saline solution containing 0.005% Tween 80) subcutaneously or intra-articularly. During the administration period, the main indicators observed were body weight, clinical signs, and foot balance test. After the end of the experiment, the right knee joints of all animals were fixed in 10% NBF for 48 hours and then transferred to 70% ethanol. The samples were decalcified with formic acid, embedded in paraffin, sectioned coronally, and stained with H&E and Safranin-O. Professional pathologists scored the sections for degeneration (main tissue properties, surface regularity, structural integrity, chondrocyte clustering, cartilage degeneration, inflammatory response in the subchondral bone area, neovascularization, and bone spurs). The scoring criteria were based on the Osteoarthritis Research Society International (OARSI) scoring system put forth in 2010 (20).

[0266] This example uses LYN101-2 as a representative of LAPAF to demonstrate the efficacy of LAPAF on a mouse osteoarthritis model induced by medial meniscus transection (MMT).

[0267] The experimental results showed that the foot balance test results showed that the dual-targeting fusion protein (LAPAF) LYN101-2 group of the present invention observed significant therapeutic effects. For example, the weight-bearing capacity of the modeling side foot of the mice in the LYN101-2 0.4mpk group was significantly improved compared with the MMT model group (p<0.01), indicating that the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention can significantly relieve joint pain caused by MMT.

[0268] Example 11: Evaluation of the toxicity of repeated administration of LAPAF in rhesus monkeys after five consecutive subcutaneous injections every three weeks followed by a six-week recovery period

[0269] In this example, the following method was used to evaluate the repeated administration toxicology study of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 in rhesus monkeys. The toxicity and toxicokinetics of the dual-targeting fusion proteins of the present invention were studied in rhesus monkeys after different subcutaneous injection regimens. The recovery of toxicity (if any) or possible delayed toxicity was observed during the 6-week recovery period. The following method uses LYN101-2 as an example and is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention.

[0270] Thirty rhesus monkeys, half male and half female, were randomly divided into three groups based on body weight and sex: a control group (vehicle group), a LYN101-2 0.1 mpk group, and a LYN101-2 0.4 mpk group. In this study, the drug was administered once every three weeks for a total of five doses. The low dose of 0.1 mpk was more than twice the effective dose, and the high dose of 0.4 mpk was approximately 10 times the effective dose. A vehicle control group was also included. The drug was administered subcutaneously once on Days 1, 22, 43, 64, and 85. During the administration period, the following indicators were mainly tested: cage-side observation, detailed clinical observation, administration site irritation evaluation, body weight, food consumption, ophthalmological examination, clinical pathological examination (including hematology, coagulation, serum biochemistry, immunoglobulin, complement, urinalysis), lymphocyte phenotype, and safety pharmacology (electrocardiogram, blood pressure, body temperature, respiratory system, central nervous system) studies, biomarker detection, immunogenicity and toxicokinetics studies were carried out simultaneously.

[0271] This example uses LYN101-2 as a representative of LAPAF to demonstrate the toxicity and toxicokinetics of five consecutive doses of LAPAF administered subcutaneously once every three weeks in rhesus monkeys.

[0272] Specific results are as follows: During the trial, both male and female monkeys administered LYN101-2 (0.1 mpk) and 0.4 mpk, a dual-targeting fusion protein (LAPAF) of the present invention, were in good general condition, exhibited normal locomotor activity, and had clean fur. All animals survived until the scheduled dissection date. No erythema or edema was observed at the injection site. At all time points during the trial, no significant changes in body weight, food intake, or ophthalmological examinations were observed in both male and female monkeys administered LYN101-2 (0.1 mpk) and 0.4 mpk. Compared with the control group and before the first administration, the LYN101-2 0.1mpk and 0.4mpk administration groups had significant changes in hematology (WBC and cell counts and percentages of each type, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, %RET, RET), blood biochemistry (ALP, TBIL, DBIL, AST, ALT, GGT, CK, UREA, CREA, TP, ALB, GLO, A / G, GLU, TCHO, TG, Ca, P, K) at each time point after administration. + 、Cl － 、Na + ), coagulation function (APTT, PT, Fbg, TT), complement (C3, C4), immunoglobulins (IgM, IgG, IgA), urine tests (pH, protein, glucose, bilirubin, ketone bodies, occult blood, white blood cells, etc.), and lymphocyte phenotypic analysis showed no obvious test article-related changes. The results of the safety pharmacology assessment of the cardiovascular system, respiratory system, and central nervous system showed no abnormal changes related to the test article. Changes in biomarkers such as PINP and TRAP-5b were consistent with expectations. Toxicokinetic results showed that there was no difference in drug exposure between male and female animals; the increase in exposure was linearly related to the increase in dose; and there was no obvious accumulation after the last dose compared to the first dose.

[0273] Under the conditions of this study, rhesus monkeys were administered the dual-targeting fusion protein (LAPAF) LYN101-2 0.1 mpk and LYN101-2 0.4 mpk of the present invention once every three weeks for five consecutive subcutaneous injections, and no toxicity related to the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention was observed.

[0274] Example 12: LAPAF on PTHR + / RANKL - In vitro activity of cellular PTHR-induced cAMP release

[0275] In this example, the following method was used to determine the effects of the dual-targeting fusion proteins LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 on PTHR. + / RANKL - In vitro activity of PTHR-induced cAMP release in cells. The following method uses LYN101-2 as an example and is applicable to all long-acting parathyroid hormone receptor agonist fusion proteins (LAPAF) of the present invention, the control sample synPTH-αRANKL-1, teriparatide, and agonistic antibodies targeting PTHR.

[0276] One day in advance, UMR106 cells were washed once with 6 mL of DPBS, and then 3 mL of preheated trypsin was added. The cells were digested in a 37°C, 5% CO2 incubator for 5 minutes. After the cells were completely digested, 10 mL of DMEM complete medium was added and gently washed. The two cell suspensions were prepared separately and the cell density was adjusted to 3 × 10 5Cells were plated at 100 μL / well of the 96-well cell culture plate and incubated overnight in a 37°C, 5% CO2 incubator (NUNRE / NU-8700E). The next morning, the supernatant from the 96-well plate was discarded, and DMEM medium was added and incubated in a 37°C, 5% CO2 incubator for 1 hour. The supernatant was discarded, and the test samples (LYN101-2, teriparatide, and the control sample, synPTH-αRANKL-1) were diluted to the starting concentration (200 nM), followed by a 5-fold dilution series for a total of eight concentrations. The samples were then added to each well of a 96-well cell culture plate containing UMR106 cells at 100 μL / well and incubated in a 37°C, 5% CO2 incubator for 40 minutes. After adding 130 μL / well of 1× lysis buffer and incubating at room temperature for 30 minutes, the test sample (cell lysate supernatant) was obtained. Subsequent procedures were then performed according to the cAMP Parameter Assay Kit (purchased from R&D, Cat. No. SKGE002b). Briefly, 50 μL / well of the primary antibody from the kit was added to a 96-well plate pre-coated with Goat Anti-mouse. The plate was incubated at 500 rpm / min for 1 hour at room temperature, and the plate was washed. Then, 100 μL / well of the cell lysate supernatant and 50 μL / well of cAMP Conjugate were added and incubated at 500 rpm / min for 2 hours at room temperature, and the plate was washed. Substrate was added and reacted at room temperature for 30 minutes, followed by the addition of stop solution. The absorbance at 450 / 570 nm was read and recorded using a microplate reader. Data were analyzed using Graphpad Prism software to determine the EC50 value.

[0277] The results showed that both the control samples synPTH-αRANKL-1 and teriparatide had the effect of UMR106 cells (PTHR single positive cells, i.e. cells expressing PTHR but not expressing RANKL, PTHR + / RANKL - The in vitro activity of the dual-targeting fusion proteins (LAPAF) LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, and LYN101-18 of the present invention on UMR106 cells (PTHR) was comparable. + / RANKL -The in vitro activity of PTHR-induced cAMP release in cells (PTHR) was significantly weaker than that of teriparatide. + / RANKL - cells), at the same molar concentration, the dual-targeting fusion protein (LAPAF) of the present invention LYN101-1, LYN101-2, LYN101-3, LYN101-8, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17, LYN101-18 to PTHR + / RANKL - The in vitro activity of cAMP release induced by PTHR in cells was significantly weaker than that of teriparatide or the control sample synPTH-αRANKL-1, indicating that the dual-targeting fusion protein (LAPAF) of the present invention is effective for PTHR single positive cells (PTHR) simulating renal cells. + / RANKL - ) in vitro activity was significantly weaker than that of teriparatide and the control sample synPTH-αRANKL-1, reducing the toxic side effects caused by binding to non-target tissues such as kidney cells that only express PTHR.

[0278] As shown in Figure 19A, in UMR106 cells (PTHR + / RANKL - In vitro activity of the dual-targeting fusion protein (LAPAF) LYN101-2 on PTHR-induced cAMP release was significantly weaker than that of teriparatide (EC50 LYN101-2 =~515279nM, EC50 特立帕肽 =0.8267nM, EC50 LYN101-2 >>EC50 特立帕肽 ), while the activity of the control sample synPTH-αRANKL-1 was comparable to that of teriparatide (EC50synPTH-αRANKL-1=0.5863nM, EC50 特立帕肽 =0.8267nM, EC50synPTH-αRANKL-1≈EC50 特立帕肽 ). It can be seen that at the same molar concentration, the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention has a significant effect on PTHR. + / RANKL -The activity of cells was significantly weaker than that of teriparatide and the control sample synPTH-αRANKL-1. At concentrations of 100 nM and below, the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention hardly activated UMR106 cells (PTHR + / RANKL - The in vitro activity of cAMP release induced by PTHR in cells, that is, at a concentration of 100 nM and below, the dual-targeting fusion protein (LAPAF) LYN101-2 of the present invention hardly binds to PTHR + / RANKL - cells (such as kidney cells).

[0279] At the same time, in order to evaluate the effects of different selections of Anti-RANKL functional region, Linker, PTH functional region and Fc region in the dual-targeting fusion protein (LAPAF) of the present invention on UMR106 cells (PTHR + / RANKL - To investigate whether the results of in vitro activity experiments on cAMP release induced by PTHR in cells were affected, the inventors also simultaneously detected LYN101-8, which has a different Anti-RANKL functional region from LYN101-2, LYN101-1 and LYN101-3, which have different Linkers from LYN101-2, and LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, and LYN101-2, which have different PTH functional regions from LYN101-2. 01-15, and LYN101-16, as well as LYN101-17 and LYN101-18 with different Fc regions from LYN101-2, the results showed that LYN101-8, LYN101-1, LYN101-3, LYN101-9, LYN101-10, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-16, LYN101-17 and LYN101-18 were effective against UMR106 cells (PTHR + / RANKL - The results of in vitro activity of PTHR-induced cAMP release in cells were similar to those of LYN101-2. It can be seen that changes in the Anti-RANKL functional region, or Linker, or PTH functional region, or Fc region have an important effect on the effect of the dual-targeting fusion protein (LAPAF) of the present invention on PTHR. + / RANKL - The in vitro activity of cellular PTHR-induced cAMP release was essentially unaffected.

[0280] The specific experimental results are as follows:

[0281] The dual-targeting fusion protein (LAPAF) LYN101-8 (different from the LYN101-2 Anti-RANKL functional region) of the present invention is located in UMR106 (PTHR + / RANKL - The in vitro activity of LYN101-8 on PTHR-induced cAMP release in UMR106 cells was similar to that of LYN101-2, that is, the in vitro activity of LYN101-8 on PTHR-induced cAMP release in UMR106 cells was significantly weaker than that of teriparatide and the control sample synPTH-αRANKL-1.

[0282] The dual-targeting fusion protein (LAPAF) LYN101-1 and LYN101-3 (different from LYN101-2 Linker selection) of the present invention are in UMR106 (PTHR + / RANKL - The in vitro activity of LYN101-1 and LYN101-3 on PTHR-induced cAMP release in UMR106 cells (PTHR) was similar to that of LYN101-2. + / RANKL - The in vitro activity of PTHR in inducing cAMP release in cells was weaker than that of teriparatide (more than 40-fold) and the control sample synPTH-αRANKL-1.

[0283] In addition, in order to evaluate the length or type of the PTH functional region in the dual-targeting fusion protein (LAPAF) of the present invention, the Fc region in the dual-targeting fusion protein of the present invention and the PTH functional region in the PTHR + / RANKL - In order to investigate whether the in vitro activity of cAMP release induced by PTHR of cells was affected, the inventors also simultaneously detected the effects of the dual-targeting fusion proteins (LAPAF) of the present invention LYN101-2, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17, and LYN101-18 with different PTH functional region lengths or types (PTH or PTHrP) or different Fc regions on UMR106 cells (PTHR). + / RANKL - In vitro activity of PTHR-induced cAMP release in cells.

[0284] As shown in Figure 19B, in UMR106 cells (PTHR + / RANKL -The in vitro activity of the dual-targeting fusion proteins (LAPAF) LYN101-2, LYN101-11, LYN101-12, LYN101-13, LYN101-14, LYN101-15, LYN101-17, and LYN101-18 on PTHR-induced cAMP release in UMR106 cells was significantly weaker than that of teriparatide, and the results were similar to those of LYN101-2. For example, the EC50 特立帕肽 =1.4nM, EC50 LYN101-12 =162.3nM, EC50 LYN101- 14 =115nM. It can be seen that changing the length or type of the PTH functional region and changing the Fc region also do not affect the targeting fusion protein (LAPAF) of the present invention in UMR106 cells (PTHR + / RANKL - In vitro activity of PTHR-induced cAMP release in cells.

[0285] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0286] All publications and patent applications cited in this specification are incorporated herein by reference, as if each independent publication or patent application is specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance understanding of the present invention, and is not intended to limit the present invention to such theory, mechanism, proof or discovery in any way. Although the present invention has been shown and described in detail in the accompanying drawings and the preceding description, the present invention should be considered to be illustrative rather than restrictive.

[0287] Partial sequences used in the present invention:

[0288] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A long-acting parathyroid hormone receptor agonist fusion protein, characterized in that: The long-acting parathyroid hormone receptor agonist fusion protein comprises a first arm and a second arm; the first arm comprises a functional region targeting RANKL and an Fc region, the functional region targeting RANKL is Fab or ScFab, and the functional region targeting RANKL is located at the N-terminus of the Fc region; the second arm comprises a functional region capable of activating PTHR and an Fc region, and the C-terminus of the functional region capable of activating PTHR of the second arm is directly or through a linker connected to the N-terminus of the Fc region.

2. The long-acting parathyroid hormone receptor agonist fusion protein according to claim 1, characterized in that: The Fc region is composed of FcA and FcB, wherein FcA and FcB are respectively CH2 and CH3 in series containing the hinge region of human IgG, and FcA and FcB are respectively Knob(s) mutation and Hole(s) mutation; Preferably, the human IgG is selected from IgG1, IgG2, and IgG4, wherein the IgG1 is a mutant type in which the Fc immune effector function is silent, the IgG2 includes a mutant type in which the Fc immune effector function is normal or the Fc immune effector function is silent, and the IgG4 includes a mutant type in which the Fc immune effector function is silent and / or an S228P mutation that improves molecular stability.

3. The long-acting parathyroid hormone receptor agonist fusion protein according to claim 2, characterized in that: When the FcA comprises the amino acid sequence shown in SEQ ID NO:7, the FcB comprises the amino acid sequence shown in SEQ ID NO:8; when the FcA comprises the amino acid sequence shown in SEQ ID NO:8, the FcB comprises the amino acid sequence shown in SEQ ID NO:7; when the FcA comprises the amino acid sequence shown in SEQ ID NO:39, the FcB comprises the amino acid sequence shown in SEQ ID NO:40; when the FcA comprises the amino acid sequence shown in SEQ ID NO:40, the FcB comprises the amino acid sequence shown in SEQ ID NO:

39.

4. The long-acting parathyroid hormone receptor agonist fusion protein according to any one of claims 1 to 3, characterized in that: The functional region capable of activating PTHR comprises an agonistic antigen-binding variable region targeting PTHR or a PTH functional region, wherein the agonistic antigen-binding variable region targeting PTHR is ScFv or SDA; the PTH functional region is selected from mature human parathyroid hormone (PTH), PTH truncations, PTH mutants, mutants of PTH truncations, mature human parathyroid hormone-related protein (PTHrP) truncations and mutants of PTHrP truncations; the PTH truncations are preferably derived from mature human P The PTH truncation is preferably a truncation of not less than 11 amino acid residues from the N-terminus of mature human PTH; the PTH truncation is more preferably a truncation of not less than 34 amino acid residues from the N-terminus of mature human PTH; further preferably, the PTH functional region is PTH (1-34), PTH (1-37), PTH (1-38), PTH (1-41), PTH (1-84), PTHrP (1-34), PTHrP (1-36); further preferably, the PTH functional region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5, 33-37, or an amino acid sequence obtained by adding, deleting, modifying and / or conservatively replacing at least one amino acid residue, such as 1, 2, 3, 4, 5 or 6 amino acid residues in the amino acid sequence shown in the aforementioned sequence; and / or, The sequence of the linker is selected from (GGGGS)n, (GGGS)n, (GGS)n, (G)n, (GS)n, (EAAAK)n, or (XP)n, or a combination of the foregoing sequences, n is a natural number, and the length of the linker does not exceed 20 amino acids; preferably, the sequence of the linker comprises an amino acid sequence selected from any one of SEQ ID NOs:9-12; more preferably, the sequence of the linker comprises an amino acid sequence as shown in SEQ ID NO:

9.

5. The long-acting parathyroid hormone receptor agonist fusion protein according to any one of claims 1 to 4, characterized in that: The first arm includes a first arm A chain and a first arm B chain; The structure of the first arm A chain from N-terminus to C-terminus is VL-CL; The structure of the first arm B chain from N-terminus to C-terminus is VH-CH1-FcA; Optionally, the VL and VH or CL and CH1 are exchanged by CrossMab technology. and / or, The structure of the second arm from the N-terminus to the C-terminus is a functional region that can activate PTHR-FcB or a functional region that can activate PTHR-linker-FcB; 6. The long-acting parathyroid hormone receptor agonist fusion protein according to any one of claims 1 to 5, characterized in that: The Fab or ScFab targeting RANKL includes 1) VH comprising heavy chain complementary determining regions HCDR1-3, 2) VL comprising light chain complementary determining regions LCDR1-3, 3) CL and 4) CH1, the amino acid sequences of HCDR1-3 are shown in SEQ ID NO:16-18 or SEQ ID NO:22-24, respectively, and the amino acid sequences of LCDR1-3 are shown in SEQ ID NO:19-21 or SEQ ID NO:25-27, respectively; optionally, the Fab or ScFab interchanges VH and VL or interchanges CH1 and CL by CrossMab technology.

7. The long-acting parathyroid hormone receptor agonist fusion protein according to claim 5 or 6, characterized in that: The amino acid sequence of the VH comprises the amino acid sequence shown in SEQ ID NO: 1 or 3, and the amino acid sequence of the VL comprises the amino acid sequence shown in SEQ ID NO: 2 or 4; And / or, the amino acid sequence of CH1 comprises the amino acid sequence shown in SEQ ID NO:14, and the amino acid sequence of CL comprises the amino acid sequence shown in SEQ ID NO:

15.

8. A long-acting parathyroid hormone receptor agonist fusion protein, characterized in that: The long-acting parathyroid hormone receptor agonist fusion protein comprises a first arm and a second arm, wherein the first arm comprises a first arm A chain and a first arm B chain; wherein: When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 40 linked in sequence; or, When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 40 linked in sequence; or, When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 7 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 8 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 39 linked in sequence, or SEQ ID NO: 1, SEQ ID NO: 15, SEQ ID NO: 40 linked in sequence; or, When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 7 linked in sequence or SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 8 linked in sequence or SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 39 linked in sequence or SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 40 linked in sequence; or, When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO:4 and SEQ ID NO:15 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:7 linked in sequence, or SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:8 linked in sequence, or SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:39 linked in sequence, or SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:40 linked in sequence; or, When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO:3 and SEQ ID NO:15 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:7 linked in sequence, or SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:8 linked in sequence, or SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:39 linked in sequence, or SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:40 linked in sequence; or, When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO:4 and SEQ ID NO:14 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:7 linked in sequence, or SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:8 linked in sequence, or SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:39 linked in sequence, or SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:40 linked in sequence; or, When the amino acid sequence of the A chain of the first arm comprises SEQ ID NO:3 and SEQ ID NO:14 linked in sequence, the amino acid sequence of the B chain of the first arm comprises SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:7 linked in sequence or SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:8 linked in sequence or SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:39 linked in sequence or SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:40 linked in sequence; Preferably, When the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 7, the amino acid sequence of the second arm comprises a PTH functional region, a linker and an Fc region connected in sequence, the PTH functional region is selected from SEQ ID NO: 5, 33-37, the linker is selected from SEQ ID NO: 9-12, and the Fc region sequence is SEQ ID NO: 8; or, When the amino acid sequence of the B chain of the first arm comprises SEQ ID NO: 8, the amino acid sequence of the second arm comprises a PTH functional region, a linker and an Fc region connected in sequence, the PTH functional region is selected from SEQ ID NO: 5, 33-37, the linker is selected from SEQ ID NO: 9-12, and the Fc region sequence is SEQ ID NO: 7; or, When the amino acid sequence of the B chain of the first arm comprises SEQ ID NO:39, the amino acid sequence of the second arm comprises a PTH functional region, a linker and an Fc region connected in sequence, the PTH functional region is selected from SEQ ID NOs:5, 33-37, the linker is selected from SEQ ID NOs:9-12, and the Fc region sequence is SEQ ID NO:40; or, When the B chain amino acid sequence of the first arm comprises SEQ ID NO:40, the amino acid sequence of the second arm comprises a PTH functional region, a linker and an Fc region connected in sequence, the PTH functional region is selected from SEQ ID NO:5, 33-37, the linker is selected from SEQ ID NO:9-12, and the Fc region sequence is SEQ ID NO:

39.

9. The long-acting parathyroid hormone receptor agonist fusion protein according to claim 8, characterized in that: When the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 14 and SEQ ID NO: 7 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 7 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 15 and SEQ ID NO: 7 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 14 and SEQ ID NO: 7 linked in sequence; The amino acid sequence of the second arm comprises SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:8 linked in sequence, or SEQ ID NO:5, SEQ ID NO:8 linked in sequence, or SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:8 linked in sequence, or SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:8 linked in sequence, or SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:9, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:11, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:9, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:11, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:8 linked in sequence, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:8 linked in sequence. NO:34, SEQ ID NO:8, or, SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:8 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:10, SEQ ID NO:8 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:11, SEQ ID NO:8 linked in sequence. NO:8, or, SEQ ID NO:34, SEQ ID NO:12, SEQ ID NO:8, or, SEQ ID NO:35, SEQ ID NO:8, or, SEQ ID NO:35, SEQ ID NO:9, SEQ ID NO:8, or, SEQ ID NO:35, SEQ ID NO:10, SEQ ID NO:8, or, SEQ ID NO:35, SEQ ID NO:11, SEQ ID NO:8, or, SEQ ID NO:35, SEQ ID NO:12, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:12, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:8, or, SEQ ID NO:36, SEQ ID NO:12, SEQ ID NO:8 NO:36, SEQ ID NO:12, SEQ ID NO:8, or, SEQ ID NO:37, SEQ ID NO:8 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:8 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:10, SEQ ID NO:8 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:11, SEQ ID NO:8 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:12, SEQ ID NO:8 linked in sequence; When the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 14 and SEQ ID NO: 8 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 8 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 15 and SEQ ID NO: 8 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 14 and SEQ ID NO: 8 linked in sequence; The amino acid sequence of the second arm comprises SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:7, or SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:10, SEQ ID NO:7, or SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:7, or SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:9, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:11, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:12 ...12, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO: NO:34, SEQ ID NO:7, or, SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:7, or, SEQ ID NO:34, SEQ ID NO:10, SEQ ID NO:7, or, SEQ ID NO:34, SEQ ID NO:11, SEQ ID NO:7, or, SEQ ID NO:34, SEQ ID NO:12, SEQ ID NO:7, or, SEQ ID NO:35, SEQ ID NO:7, or, SEQ ID NO:35, SEQ ID NO:9, SEQ ID NO:7, or, SEQ ID NO:35, SEQ ID NO:10, SEQ ID NO:7, or, SEQ ID NO:35, SEQ ID NO:11, SEQ ID NO:7, or, SEQ ID NO:35, SEQ ID NO:12, SEQ ID NO:7, or, SEQ ID NO:36, SEQ ID NO:7, or, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 NO:7, or, SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:7 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:7 linked in sequence, or,SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:7, or SEQ ID NO:36, SEQ ID NO:12, SEQ ID NO:7, or SEQ ID NO:37, SEQ ID NO:7, or SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:7, or SEQ ID NO:37, SEQ ID NO:10, SEQ ID NO:7, or SEQ ID NO:37, SEQ ID NO:11, SEQ ID NO:7, or SEQ ID NO:37, SEQ ID NO:12, SEQ ID NO:7, connected in sequence; When the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 14 and SEQ ID NO: 39 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 39 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 15 and SEQ ID NO: 39 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 14 and SEQ ID NO: 39 linked in sequence; The amino acid sequence of the second arm comprises SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:40 linked in sequence, or SEQ ID NO:5, SEQ ID NO:40 linked in sequence, or SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:40 linked in sequence, or SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:40 linked in sequence, or SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:40 linked in sequence, or SEQ ID NO:33, SEQ ID NO:40 linked in sequence, or SEQ ID NO:33, SEQ ID NO:9, SEQ ID NO:40 linked in sequence, or SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:40 linked in sequence, or SEQ ID NO:33, SEQ ID NO:11, SEQ ID NO:40 linked in sequence, or SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:40 linked in sequence. NO:40, or, SEQ ID NO:34, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:10, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:11, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:12, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:9, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:10, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:11, SEQ ID NO:40 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:12, SEQ ID NO:40 linked in sequence. NO:40, or, SEQ ID NO:36 and SEQ ID NO:40 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:9 and SEQ ID NO:40 linked in sequence, or,SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:40, or SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:40, or SEQ ID NO:36, SEQ ID NO:12, SEQ ID NO:40, or SEQ ID NO:37, SEQ ID NO:40, or SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:40, or SEQ ID NO:37, SEQ ID NO:10, SEQ ID NO:40, or SEQ ID NO:37, SEQ ID NO:11, SEQ ID NO:40, or SEQ ID NO:37, SEQ ID NO:12, SEQ ID NO:40, connected in sequence; When the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 14 and SEQ ID NO: 40 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 2 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 40 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 14 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 15 and SEQ ID NO: 40 linked in sequence; or, the amino acid sequence of the first arm A chain comprises SEQ ID NO: 1 and SEQ ID NO: 15 linked in sequence, and the amino acid sequence of the first arm B chain comprises SEQ ID NO: 2, SEQ ID NO: 14 and SEQ ID NO: 40 linked in sequence; The amino acid sequence of the second arm comprises SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 39, or SEQ ID NO: 5, SEQ ID NO: 39, or SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 39, or SEQ ID NO: 5, SEQ ID NO: 1 ...0, SEQ ID NO: 39, or SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 39, or SEQ ID NO: 5, SEQ ID NO: SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:39, or, SEQ ID NO:33, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:33, SEQ ID NO:9, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:33, SEQ ID NO:10, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:33, SEQ ID NO:11, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:33, SEQ ID NO:12, SEQ ID NO:39, or, SEQ ID NO:34, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:39, or, SEQ ID NO:34, SEQ ID NO:10, SEQ ID NO:39, or, SEQ ID NO:34, SEQ ID NO:11, SEQ ID NO:39, or, SEQ ID NO:34, SEQ ID NO:12, SEQ ID NO:39, or, SEQ ID NO:34, SEQ ID NO:39 linked in sequence. NO:34, SEQ ID NO:12, SEQ ID NO:39, or, SEQ ID NO:35, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:9, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:10, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:11, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:12, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:10, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:35, SEQ ID NO:12, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:36, SEQ ID NO:9, SEQ ID NO:39 linked in sequence. NO:36, SEQ ID NO:12, SEQ ID NO:39, or, SEQ ID NO:37, SEQ ID NO:39 linked in sequence, or, SEQ ID NO:37, SEQ ID NO:9, SEQ ID NO:39 linked in sequence, or,SEQ ID NO:37, SEQ ID NO:10, SEQ ID NO:39 are linked in sequence, or SEQ ID NO:37, SEQ ID NO:11, SEQ ID NO:39 are linked in sequence, or SEQ ID NO:37, SEQ ID NO:12, SEQ ID NO:39 are linked in sequence.

10. An isolated nucleic acid molecule encoding the long-acting parathyroid hormone receptor agonist fusion protein according to any one of claims 1 to 9.

11. A recombinant expression vector comprising the nucleic acid molecule of claim 10.

12. An engineered cell comprising the recombinant expression vector of claim 11; preferably, the host cell of the engineered cell is a eukaryotic cell; more preferably, the eukaryotic cell is a yeast cell or a mammalian cell; further more preferably, the mammalian cell is a CHO cell or a HEK293 cell.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the long-acting parathyroid hormone receptor agonist fusion protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the recombinant expression vector according to claim 11, and / or the engineered cell according to claim 12; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

14. Use of the long-acting parathyroid hormone receptor agonist fusion protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the recombinant expression vector according to claim 11, the engineered cell according to claim 12, or the pharmaceutical composition according to claim 13 in the preparation of a drug for treating, preventing, adjuvanting or neoadjuvanting a bone disorder, wherein the bone disorder is a disease that needs to be treated, prevented, adjuvanted or neoadjuvanted by regulating bone formation and / or bone resorption at a lesion-related site; Preferably, the bone disorder is osteoporosis, osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, bone injury, degenerative spondylolisthesis or degenerative disc disease; the bone injury is preferably a fracture, a bone crack, a cartilage injury, osteonecrosis, a bone injury caused by arthritis or a tumor; the fracture is preferably an osteoporotic fracture, a nonunion of a fracture or a delayed union of a fracture; the arthritis includes arthritis caused by degeneration, trauma, strain, infection, autoimmune disease, such as rheumatoid arthritis, osteoarthritis, infectious arthritis; the tumor includes bone tumors and tumor bone metastases.

15. A method for preparing a long-acting parathyroid hormone receptor agonist fusion protein, characterized in that: The method comprises the step of culturing the engineered cell of claim 12.

16. A method for treating, preventing, adjuvant therapy or neoadjuvant therapy of bone disorders by regulating bone formation and / or bone resorption at lesion-related sites, characterized in that: The method comprises administering to a subject in need thereof an effective amount of the long-acting parathyroid hormone receptor agonist fusion protein of any one of claims 1 to 9, the nucleic acid molecule of claim 10, the recombinant expression vector of claim 11, the engineered cell of claim 12, or the pharmaceutical composition of claim 13; Preferably, the bone disorder is osteoporosis, osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, bone injury, degenerative spondylolisthesis or degenerative disc disease; the bone injury is preferably fracture, bone crack, cartilage injury, osteonecrosis, bone injury caused by arthritis or tumor; the fracture is preferably osteoporotic fracture, nonunion of fracture or delayed union of fracture; the arthritis includes arthritis caused by degeneration, trauma, strain, infection, autoimmune disease or inflammation, such as rheumatoid arthritis, osteoarthritis, infectious arthritis; the tumor includes bone tumor and tumor bone metastasis; And / or, the method of administration is selected from oral administration (eg, administration via an oral delivery device) or injection; the injection is preferably intravenous injection, subcutaneous injection, intramuscular injection, or local injection.

17. Use of the long-acting parathyroid hormone receptor agonist fusion protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the recombinant expression vector according to claim 11, the engineered cell according to claim 12 or the pharmaceutical composition according to claim 13 as a medicine.

18. A long-acting parathyroid hormone receptor agonist fusion protein according to any one of claims 1 to 9, a nucleic acid molecule according to claim 10, a recombinant expression vector according to claim 11, an engineered cell according to claim 12, or a pharmaceutical composition according to claim 13, for use in the treatment, prevention, adjuvant therapy, or neoadjuvant therapy of a bone disorder; Preferably, the bone disorder is osteoporosis, osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, bone injury, degenerative spondylolisthesis or degenerative disc disease; the bone injury is preferably a fracture, a bone crack, a cartilage injury, osteonecrosis, a bone injury caused by arthritis or a tumor; the fracture is preferably an osteoporotic fracture, a nonunion of a fracture or a delayed union of a fracture; the arthritis includes arthritis caused by degeneration, trauma, strain, infection, autoimmune disease or inflammation, such as rheumatoid arthritis, osteoarthritis, infectious arthritis; the tumor includes bone tumors and tumor bone metastases.

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