Bifunctional fusion protein and use thereof

Abstract

The present invention relates to the field of biomedicine. More specifically, the present invention relates to a polypeptide construct comprising an antigen binding domain capable of specifically binding to RANKL, and a PTH peptide, and a related use thereof for disease treatment.

Description

Bifunctional fusion proteins and their applications

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411613381.0, filed on November 12, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedicine, and more specifically, to polypeptide constructs comprising an antigen-binding domain capable of specifically binding RANKL and a PTH peptide, and their related uses in the treatment of diseases. Background Technology

[0004] Osteoporosis is a systemic bone disease characterized by low bone mass, damage to bone microstructure, increased bone fragility, and a predisposition to fractures. In 2001, the National Institutes of Health (NIH) defined it as a skeletal disease characterized by decreased bone strength and increased fracture risk. Osteoporosis can occur at any age, but is most common in postmenopausal women and older men. Based on etiology, osteoporosis is divided into two main categories: primary and secondary. Primary osteoporosis includes postmenopausal osteoporosis (Type I), geriatric osteoporosis (Type II), and idiopathic osteoporosis (juvenile type). Postmenopausal osteoporosis generally occurs within 5–10 years after menopause in women; geriatric osteoporosis generally refers to osteoporosis that occurs after age 70; idiopathic osteoporosis mainly occurs in adolescents, and its cause is still unknown. Secondary osteoporosis refers to osteoporosis caused by diseases, medications, or other identifiable causes that affect bone metabolism.

[0005] With the increasing aging of my country's population, the prevalence of osteoporosis is rising rapidly, becoming a significant public health issue. The Seventh National Population Census showed that my country has 264 million people aged 60 and above (approximately 18.7% of the total population), and over 190 million people aged 65 and above (approximately 13.5% of the total population), making it the country with the largest elderly population in the world. A national epidemiological survey of osteoporosis showed that the prevalence of osteoporosis in people aged 50 and above is 19.2%, with 32.1% in women and 6.9% in men; the prevalence in people aged 65 and above is 32.0%, with 51.6% in women and 10.7% in men. Based on these epidemiological data, it is estimated that there are currently approximately 90 million people with osteoporosis in my country, of whom approximately 70 million are women.

[0006] Osteoporotic fractures (or fragility fractures) are fractures that occur from minor trauma (equivalent to a fall from standing height or lower) and are a serious consequence of osteoporosis. Common sites for osteoporotic fractures include the vertebrae, distal forearm, hip, proximal humerus, and pelvis, with vertebral fractures being the most common. The prevalence of vertebral fractures in women over 50 years of age is approximately 15.0%, and the prevalence increases with age, reaching as high as 36.6% in women over 80 years of age. A recent screening study of vertebral fractures in Shanghai communities showed that the prevalence of vertebral fractures in people over 60 years of age was roughly equal in men and women, with 17.0% in men and 17.3% in women. A nationwide random sampling study showed that the prevalence of vertebral fractures in people over 40 years of age in my country was 10.5% in men and 9.5% in women. Hip fractures are the most serious type of osteoporotic fracture, and the incidence of hip fractures in my country has shown a significant upward trend in recent years. Recent big data analysis from urban employee and resident medical insurance systems shows that in 2016, the incidence of hip fractures in people over 55 years old in my country was 99 per 100,000 for men and 177 per 100,000 for women. The total number of hip fractures increased threefold from 16,587 in 2012 to 66,575 in 2016. Overall, with the increasing aging of my country's population, the incidence of osteoporotic fractures is still growing rapidly. Osteoporotic fractures are extremely harmful, being one of the leading causes of disability and death among elderly patients. Within one year of a hip fracture, 20% of patients may die from various complications; approximately 50% of patients become disabled, resulting in a significant decline in their quality of life. It is estimated that by 2035, medical expenses in my country for major osteoporotic fractures (wrist, vertebral, and hip) will reach 132 billion yuan; and by 2050, this medical expenditure will climb to 163 billion yuan.

[0007] Osteoporosis treatment drugs are mainly classified into four categories based on their biological mechanisms: basal modulators, bone resorption inhibitors, bone formation promoters, and bone metabolism modulators. Among them, denosumab (DMAB) and teriparatide have been used clinically for many years as the main anti-osteoporosis biological drugs, with clear advantages and disadvantages.

[0008] Denosumab is a RANKL (receptor activator of NF-κB ligand) neutralizing antibody that inhibits bone resorption. In clinical use, it can significantly reduce the risk of vertebral, non-vertebral, and hip fractures in postmenopausal women. However, due to its biological mechanisms, denosumab is less effective in patients with severe osteoporosis, and discontinuation of denosumab leads to increased bone resorption, causing BMD (Bone Mineral Density) to return to pre-treatment levels. Furthermore, it increases the risk of multiple vertebral fractures.

[0009] Teriparatide is recombinant human PTH 1-34, primarily acting on PTH1R (Parathyroid hormone receptor 1) to regulate bone metabolism and promote bone remodeling. PTH can promote osteoblast differentiation and maturation, thereby promoting bone formation. Simultaneously, PTH can upregulate RANKL expression on osteoblasts, further promoting osteoclast differentiation and bone resorption. In clinical use, teriparatide has significantly reduced the risk of vertebral and non-vertebral fractures in postmenopausal women, but its effect on reducing the risk of hip fractures has not been confirmed. Furthermore, due to its short half-life, teriparatide requires daily administration and is expensive, leading to poor patient compliance. Additionally, because PTH1R has a broad expression spectrum, with high expression in tissues such as the kidneys and intestines, its targeting to bone tissue is poor. Teriparatide's high activation activity also poses a risk of hypercalcemia during use, potentially leading to nephrotoxicity.

[0010] From a biological mechanism perspective, denosumab and teriparatide have complementary biological mechanisms. Some retrospective studies have found that the combination of denosumab and teriparatide can significantly increase bone mineral density in the vertebrae, femoral neck, and hip bones, with a greater increase than teriparatide or denosumab alone. However, combination therapy still cannot solve the problems of teriparatide's short half-life, high dosing frequency, and poor bone tissue targeting. Summary of the Invention

[0011] This invention provides a polypeptide construct (also referred to herein as a bifunctional fusion protein) comprising an antigen-binding domain specifically binding to RANKL and a PTH peptide. The polypeptide construct exhibits significantly superior stability, a significantly increased half-life at the PTH terminus, and enhanced pharmacological activity in regulating bone metabolism. Furthermore, the polypeptide construct of this invention can reduce the potential toxic side effects of PTH1R by decreasing the activation activity of the PTH peptide terminus on PTH1R in cells expressing only PTH1R. Simultaneously, by utilizing the targeting ability of RANKL, it achieves conditional activation of PTH1R in cells expressing both PTH1R and RANKL, thereby achieving targeted activation of the PTH peptide and enhancing the role of the polypeptide construct in regulating bone metabolism and its bone tissue-targeted activation activity.

[0012] Therefore, the aforementioned polypeptide construct is further provided as a medicine and method for preventing and / or treating bone metabolism-related diseases (such as osteoporosis) or diseases caused by parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism) (such as hypoparathyroidism, hyperparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria).

[0013] peptide constructs

[0014] In one aspect, the present invention provides a polypeptide construct comprising an antigen-binding domain capable of specifically binding to RANKL, a PTH peptide, and an Fc domain; said Fc domain comprising a first Fc domain monomer and a second Fc domain monomer.

[0015] The antigen-binding domain and the PTH peptide are each linked to one of the first and second Fc domain monomers.

[0016] In some embodiments, the PTH peptide is selected from wild-type PTH peptide or variants thereof.

[0017] In some embodiments, the wild-type PTH peptide is an active fragment of the wild-type PTH protein.

[0018] In some embodiments, the wild-type PTH peptide possesses the biological activity of the wild-type PTH protein from which it is derived (e.g., PTH1R binding activity and / or PTH1R activation activity).

[0019] In some embodiments, the wild-type PTH peptide comprises amino acid residues in the wild-type PTH protein at positions 1-16 corresponding to SEQ ID NO:31.

[0020] As used herein, the expression "amino acid residues in the wild-type PTH protein that correspond to positions 1-16 of SEQ ID NO:31" refers to the amino acid residues in the wild-type PTH protein sequence that are located at the same positions as amino acid residues 1-16 of SEQ ID NO:31 when the wild-type PTH protein sequence is optimally aligned with SEQ ID NO:31, that is, when the wild-type PTH protein sequence is aligned with SEQ ID NO:31 to obtain the highest percentage identity, respectively.

[0021] Unless otherwise specified or clearly contradicted by the context, the meaning of the rest of the similar expressions in this document shall be defined in a manner similar to that described above.

[0022] In some embodiments, the wild-type PTH peptide comprises, or is composed of, amino acid residues in the wild-type PTH protein at positions corresponding to positions 3-16 (or 3-17, or 3-27, or 3-33, or 3-34, or 3-35, or 3-40, or 3-50, or 3-60, or 3-70, or 3-80, or 3-84, or 1-16, or 1-17, or 1-27, or 1-33, or 1-34, or 1-35, or 1-40, or 1-50, or 1-60, or 1-70, or 1-80, or 1-84) of SEQ ID NO:31.

[0023] In some embodiments, the wild-type PTH peptide comprises, or is composed of, amino acid residues in the wild-type PTH protein at positions corresponding to positions 1-16 (or 1-17, or 1-27, or 1-33, or 1-34, or 1-35, or 1-40, or 1-50, or 1-60, or 1-70, or 1-80, or 1-84) of SEQ ID NO:31.

[0024] In some embodiments, the wild-type PTH peptide comprises, or is composed of, amino acid residues in the wild-type PTH protein at positions 1-33, 1-34, or 1-40 of SEQ ID NO:31.

[0025] In some embodiments, the wild-type PTH peptide is selected from PTH. 3-16 PTH 3-17 PTH 3-27 PTH 3- 33 PTH 3-34 PTH 3-35 PTH 3-40 PTH 3-50 PTH 3-60 PTH 3-70 PTH 3-80 PTH 3-84 PTH 1- 16 PTH 1-17 PTH 1-27 PTH 1-33 PTH 1-34 PTH 1-35 PTH 1-40 PTH 1-50 PTH 1-60 PTH 1- 70PTH 1-80 and PTH 1-84 .

[0026] In some embodiments, the wild-type PTH peptide is selected from PTH. 1-33 PTH 1-34 and PTH 1-40 .

[0027] In some embodiments, the wild-type PTH protein is the human PTH protein.

[0028] In some embodiments, the wild-type PTH protein has: (a) an amino acid sequence as shown in SEQ ID NO:31; (b) an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence shown in SEQ ID NO:31; or (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO:31.

[0029] In some embodiments, the PTH peptide variant, compared to its derived wild-type PTH peptide, possesses one or more of the following characteristics:

[0030] (i) In the PTH peptide variant, compared to the wild-type PTH peptide, the amino acid residue (e.g., asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by an amino acid residue other than the asparagine residue, preferably replaced by a glutamic acid residue, a glycine residue or a serine residue.

[0031] (ii) In the PTH peptide variant, compared to the wild-type PTH peptide, the amino acid residue (e.g., serine residue) at the position corresponding to the 17th position of SEQ ID NO:31 is replaced by an amino acid residue other than serine residue, glycine residue and proline residue, preferably replaced by glutamic acid residue;

[0032] (iii) Compared to the wild-type PTH peptide, the PTH peptide variant has an increased number of glycosylation sites (e.g., N-glycosylation sites); preferably, the PTH peptide variant comprises the characteristic sequence NXS (or T), wherein N represents asparagine, X represents any amino acid other than proline, S represents serine, and T represents threonine; in some embodiments, the amino acid residue (e.g., asparagine residue) of the PTH peptide variant at the position corresponding to position 33 of SEQ ID NO:31 is capable of N-glycosylation modification; in some embodiments, compared to the wild-type PTH peptide, the amino acid residue (e.g., valine residue) of the PTH peptide variant at the position corresponding to position 35 of SEQ ID NO:31 is replaced by a serine residue or a threonine residue; in some embodiments, compared to the wild-type PTH peptide, the amino acid residue (e.g., valine residue) of the PTH peptide variant at the position corresponding to position 35 of SEQ ID NO:31 is replaced by a serine residue or a threonine residue; The amino acid residue at position 34 of NO:31 (e.g., phenylalanine residue) is replaced by an alanine residue.

[0033] As used herein, the expression "the amino acid residue at the position corresponding to the 16th amino acid residue of SEQ ID NO:31 in the PTH peptide variant" means, when the sequence of the PTH peptide variant is optimally aligned with SEQ ID NO:31, that is, when the sequence of the PTH peptide variant is aligned with SEQ ID NO:31 to obtain the highest percentage identity, the amino acid residue in the sequence of the compared PTH peptide variant that is located at the same position as the 16th amino acid residue of SEQ ID NO:31.

[0034] Unless otherwise specified or clearly contradicted by the context, the meaning of the rest of the similar expressions in this document shall be defined in a manner similar to that described above.

[0035] In some embodiments, the wild-type PTH peptide comprises, or is composed of, amino acid residues at positions 1-33 or 1-34 of the wild-type PTH protein corresponding to SEQ ID NO:31; and, compared to the wild-type PTH peptide, in the PTH peptide variant, the amino acid residue (e.g., asparagine residue) at position 16 of SEQ ID NO:31 is replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, the amino acid residue (e.g., serine residue) at position 17 of SEQ ID NO:31 is replaced by a glutamic acid residue.

[0036] In some embodiments, the wild-type PTH peptide comprises, or is composed of, amino acid residues at positions 1-40 of the wild-type PTH protein corresponding to SEQ ID NO:31; and, compared to the wild-type PTH peptide, (a) the PTH peptide variant has an increased number of glycosylation sites (e.g., N-glycosylation sites), for example, the amino acid residue (e.g., asparagine residue) at position 33 of SEQ ID NO:31 in the PTH peptide variant is capable of N-glycosylation modification; and / or, (b) in the PTH peptide variant, the amino acid residue (e.g., asparagine residue) at position 16 of SEQ ID NO:31 is replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) the amino acid residue (e.g., serine residue) at position 17 of SEQ ID NO:31 is replaced by a glutamic acid residue.

[0037] In some embodiments, the wild-type PTH peptide comprises, or is composed of, amino acid residues in the wild-type PTH protein corresponding to positions 1-40 of SEQ ID NO:31; and, compared to the wild-type PTH peptide, (a) the PTH peptide variant has an amino acid residue (e.g., a valine residue) at position 35 of SEQ ID NO:31 replaced by a serine or threonine residue; and / or, (b) in the PTH peptide variant, an amino acid residue (e.g., an asparagine residue) at position 16 of SEQ ID NO:31 replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) the amino acid residue (e.g., a serine residue) at position 17 of SEQ ID NO:31 replaced by a glutamic acid residue.

[0038] In some embodiments, the wild-type PTH peptide comprises, or is composed of, amino acid residues at positions 1-40 of the wild-type PTH protein corresponding to SEQ ID NO:31; and, compared to the wild-type PTH peptide, (a) the PTH peptide variant has an amino acid residue (e.g., valine residue) at position 35 of SEQ ID NO:31 replaced by a serine or threonine residue, and an amino acid residue (e.g., phenylalanine residue) at position 34 of SEQ ID NO:31 replaced by an alanine residue; and / or, (b) in the PTH peptide variant, an amino acid residue (e.g., asparagine residue) at position 16 of SEQ ID NO:31 replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) an amino acid residue (e.g., serine residue) at position 17 of SEQ ID NO:31 replaced by a glutamic acid residue.

[0039] In some embodiments, the PTH polypeptide (e.g., the wild-type PTH peptide or the PTH polypeptide variant) has an amino acid sequence as shown in any one of SEQ ID NO: 7-9, 11-16, 31.

[0040] In some embodiments, the antigen-binding domain is linked to the first Fc domain monomer, and the PTH peptide is linked to the second Fc domain monomer; or, the antigen-binding domain is linked to the second Fc domain monomer, and the PTH peptide is linked to the first Fc domain monomer.

[0041] In some embodiments, the first and second Fc domain monomers each independently contain modifications of one or more amino acids that promote heterodimerization of the first and second Fc domain monomers.

[0042] In some embodiments, the first Fc domain monomer contains an amino acid modification capable of forming a hole structure, and the second Fc domain monomer contains an amino acid modification capable of forming a knob structure, wherein the hole structure can pair with the knob structure to form a heterodimeric Fc domain.

[0043] Those skilled in the art will readily understand that the hole structure can pair with the knot structure to form a "knob in hole" structure, which can be used to reduce heavy chain mismatches in peptide constructs. A "knob" and "hole" structure can be introduced by mutating the corresponding positions in the CH3 domains of the two Fc domain monomers, thereby forming a specific interaction interface between the two Fc domain monomers (see Ridgway et al., Protein Eng., 9: 617-621 (1996); WO 2006 / 028936; the entire text of which is incorporated herein by reference). Amino acid substitutions that can be used to form the "knob" structure include, but are not limited to, S354C and / or T366W (e.g., T366W), where the numbering follows the Eu numbering scheme. Amino acid substitutions that can be used to form the "hole" structure include, but are not limited to, Y349C, T366S, L368A and / or Y407V (e.g., T366S, L368A and Y407V), where the numbering follows the Eu numbering scheme. Due to the mutual attraction between the "knob" and "hole" structures, as well as the mutual repulsion between the "knob" and "knob" structures, the "knob in hole" structure can effectively reduce heavy chain mismatches in peptide constructs.

[0044] In some embodiments, the Fc domain monomer is derived from the Fc domain of human immunoglobulin and includes modifications capable of forming a knock or hole structure. In some embodiments, the Fc domain monomer is derived from human immunoglobulin IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain monomer (e.g., the second Fc domain monomer) includes one or more modifications forming a knock structure, wherein the one or more modifications include a T366W amino acid substitution, wherein the numbering follows the Eu numbering scheme. In some embodiments, the Fc domain monomer (e.g., the second Fc domain monomer) includes a T366W amino acid substitution, wherein the numbering follows the Eu numbering scheme. In some embodiments, the Fc domain monomer (e.g., the first Fc domain monomer) includes one or more modifications forming a hole structure, wherein the one or more modifications include T366S, L368A, and / or Y407V amino acid substitutions, wherein the numbering follows the Eu numbering scheme. In some embodiments, the Fc domain monomer (e.g., the first Fc domain monomer) includes T366S, L368A, and Y407V amino acid substitutions, wherein the numbering follows the Eu numbering scheme.

[0045] In some embodiments, the first Fc domain monomer and / or the second Fc domain monomer are derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4).

[0046] In some embodiments, the CH2 domain (residues 231-340 of human IgG1, numbered according to the Eu numbering system), CH3 domain (residues 341-447 of human IgG1, numbered according to the Eu numbering system), and / or hinge region (residues 216-230, numbered according to the Eu numbering system) of the first Fc domain monomer and / or the second Fc domain monomer are introduced with one, two, or more mutations (e.g., amino acid substitutions) to alter one or more functional properties of the polypeptide construct, such as serum half-life, complement binding, Fc receptor binding, ADCC activity, CDC activity, and / or ADCP activity.

[0047] In some embodiments, the CH2 domain, CH3 domain and / or hinge region of the first Fc domain monomer and / or the second Fc domain monomer are introduced with one, two or more mutations (e.g., amino acid substitutions) to reduce or ablate the effector function of the Fc region.

[0048] In some embodiments, compared to the wild-type Fc domain monomer from which they are derived, the first Fc domain monomer and / or the second Fc domain monomer each independently have amino acid modifications capable of forming hole or knob structures, altered (e.g., enhanced, reduced, or ablated) effector functions (e.g., ADCC, CDC, ADCP activities), prolonged half-life (e.g., enhanced FcRn binding activity), and / or altered (e.g., enhanced, reduced, or eliminated) protein A binding activity.

[0049] In some embodiments, compared to the wild-type Fc domain monomer from which it is derived, the first Fc domain monomer has amino acid modifications capable of forming a hole structure, altered (e.g., enhanced or reduced) effector function, and / or prolonged half-life (e.g., enhanced FcRn binding activity); the second Fc domain monomer has amino acid modifications capable of forming a knob structure, altered (e.g., enhanced or reduced) effector function, prolonged half-life (e.g., enhanced FcRn binding activity), and / or altered (e.g., enhanced, reduced, or eliminated) protein A binding activity.

[0050] In some embodiments, compared to the wild-type Fc domain monomer from which it is derived, the first Fc domain monomer has amino acid modifications capable of forming a hole structure, altered (e.g., enhanced or reduced) effector function, prolonged half-life (e.g., enhanced FcRn binding activity), and / or altered (e.g., enhanced, reduced, or eliminated) protein A binding activity; the second Fc domain monomer has amino acid modifications capable of forming a knob structure, altered (e.g., enhanced or reduced) effector function, and / or prolonged half-life (e.g., enhanced FcRn binding activity).

[0051] In some embodiments, the first Fc domain monomer and / or the second Fc domain monomer are derived from human immunoglobulin IgG4, and the first Fc domain monomer contains substitution mutations T366S, L368A and / or Y407V; and / or the second Fc domain monomer contains substitution mutation T366W.

[0052] In some embodiments, the first Fc domain monomer and / or the second Fc domain monomer are derived from human immunoglobulin IgG4, and the first Fc domain monomer contains substitution mutations: (i) F234A, L235A, and / or, (ii) T366S, L368A, Y407V; and / or, the second Fc domain monomer contains substitution mutations: (i) F234A, L235A, and / or, (ii) T366W.

[0053] In some embodiments, one of the first Fc domain monomer and the second Fc domain monomer further comprises substitution mutations H435R and / or Y436F.

[0054] In some embodiments, the first Fc domain monomer contains an amino acid sequence as shown in SEQ ID NO:20, and the second Fc domain monomer contains an amino acid sequence as shown in SEQ ID NO:32.

[0055] In some embodiments, the antigen-binding domain and the PTH peptide are each connected to the Fc domain monomer (e.g., the first Fc domain monomer or the second Fc domain monomer) via a hinge region.

[0056] In some embodiments, the hinge region is derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4).

[0057] In some embodiments, the hinge region is selected from the wild-type human immunoglobulin hinge region and its variants.

[0058] In some embodiments, the hinge region is derived from human immunoglobulin IgG4 and contains the substitution mutation S228P.

[0059] In some embodiments, the hinge region comprises an amino acid sequence as shown in SEQ ID NO:10.

[0060] In some embodiments, the antigen-binding domain is selected from Fab, Fab', F(ab')2, scFab, VHH, Fv, disulfide-linked Fv and scFv.

[0061] In some implementations, the antigen-binding domain is Fab.

[0062] In some embodiments, the antigen-binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL), which together form an antigen-binding domain that specifically binds to RANKL.

[0063] In some embodiments, the VH includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:1; the VL includes LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:3.

[0064] In some implementations, the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0065] In some embodiments, the VH includes HCDR1 as shown in SEQ ID NO:34, HCDR2 as shown in SEQ ID NO:35, and HCDR3 as shown in SEQ ID NO:36; and / or, the VL includes LCDR1 as shown in SEQ ID NO:37, LCDR2 as shown in SEQ ID NO:38, and LCDR3 as shown in SEQ ID NO:39.

[0066] The CDRs are defined by the Kabat numbering system.

[0067] In some embodiments, the VH comprises an amino acid sequence as shown in SEQ ID NO:1, and / or the VL comprises an amino acid sequence as shown in SEQ ID NO:3.

[0068] In some embodiments, the polypeptide construct comprises peptide chain IA, peptide chain IB, and peptide chain IC; wherein, peptide chain IA comprises the VL and a light chain constant region, peptide chain IB comprises: the VH, a heavy chain CH1 region, a hinge region, and a first Fc domain monomer (or a second Fc domain monomer), and peptide chain IC comprises: the PTH peptide, a hinge region, and a second Fc domain monomer (or a first Fc domain monomer).

[0069] In some embodiments, the peptide chain IA includes the VL and a light chain constant region from the N-terminus to the C-terminus, the peptide chain IB includes the VH, a heavy chain CH1 region, a hinge region, and a first Fc domain monomer (or a second Fc domain monomer) from the N-terminus to the C-terminus, and / or the peptide chain IC includes the PTH peptide, a hinge region, and a second Fc domain monomer (or a first Fc domain monomer) from the N-terminus to the C-terminus.

[0070] In some embodiments, adjacent domains of peptide chain IA are optionally connected by or without a connector, adjacent domains of peptide chain IB are optionally connected by or without a connector, and / or adjacent domains of peptide chain IC are optionally connected by or without a connector.

[0071] In some embodiments, the peptide connectors are each independently the same or different peptide connectors (e.g., rigid peptide connectors or flexible peptide connectors). In some embodiments, the peptide connectors are each independently selected from peptide connectors containing one or more glycine (G) and / or serine (S), for example, possessing (G) m S) nThe peptide linker shown has the following structure, wherein m and n are each independently an integer not less than 0, for example, each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the peptide linker each independently comprises an amino acid sequence as shown in any one of SEQ ID NO: 17-19.

[0072] In some embodiments, the peptide chain IC comprises, from the N-terminus to the C-terminus: the PTH peptide, a peptide linker, a hinge region, and a second Fc domain monomer (or a first Fc domain monomer), wherein the peptide linker comprises an amino acid sequence as shown in any one of SEQ ID NO:17-19. In some embodiments, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO:18.

[0073] In some embodiments, the light chain constant region is the constant region of the human immunoglobulin κ or λ light chain.

[0074] In some embodiments, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:4.

[0075] In some embodiments, the heavy chain CH1 region is the heavy chain CH1 region of human immunoglobulins (such as IgG1, IgG2, IgG3, or IgG4).

[0076] In some embodiments, the heavy chain CH1 region contains an amino acid sequence as shown in SEQ ID NO:2.

[0077] In some embodiments, the polypeptide construct comprises:

[0078] The peptide chain IA contains the amino acid sequence shown in SEQ ID NO:6, the peptide chain IB contains the amino acid sequence shown in SEQ ID NO:5, and the peptide chain IC contains the amino acid sequence shown in any one of SEQ ID NOs:21-30.

[0079] Modified PTH protein

[0080] On the other hand, this application provides a modified PTH protein that, compared to its derived wild-type PTH protein, possesses one or more of the following characteristics:

[0081] (i) In the modified PTH protein, compared to the wild-type PTH protein, the amino acid residue (e.g., asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by an amino acid residue other than the asparagine residue, preferably replaced by a glutamic acid residue, a glycine residue or a serine residue.

[0082] (ii) In the modified PTH protein, compared to the wild-type PTH protein, the amino acid residue (e.g., serine residue) at the position corresponding to the 17th position of SEQ ID NO:31 is replaced by an amino acid residue other than serine residue, glycine residue and proline residue, preferably replaced by glutamic acid residue.

[0083] In some embodiments, the modified PTH protein has an increased number of glycosylation sites (e.g., N-glycosylation sites) compared to the wild-type PTH protein; preferably, the modified PTH protein comprises the characteristic sequence NXS (or T), wherein N represents asparagine, X represents any amino acid other than proline, S represents serine, and T represents threonine; in some embodiments, the amino acid residue (e.g., asparagine residue) at the position corresponding to position 33 of SEQ ID NO:31 in the modified PTH protein is capable of N-glycosylation modification; in some embodiments, compared to the wild-type PTH protein, the amino acid residue (e.g., valine residue) at the position corresponding to position 35 of SEQ ID NO:31 in the modified PTH protein is replaced by a serine residue or a threonine residue; in some embodiments, compared to the wild-type PTH protein, the amino acid residue (e.g., valine residue) at the position corresponding to position 35 of SEQ ID NO:31 in the modified PTH protein is replaced by a serine residue, and ... The amino acid residue at position 34 of NO:31 (e.g., phenylalanine residue) is replaced by an alanine residue.

[0084] In some embodiments, the wild-type PTH protein is the human PTH protein.

[0085] In some embodiments, the wild-type PTH protein has: (a) an amino acid sequence as shown in SEQ ID NO:31; (b) an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence shown in SEQ ID NO:31; or (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO:31.

[0086] In some embodiments, compared to the wild-type PTH protein, the modified PTH protein has an amino acid residue (e.g., asparagine residue) at position 16 of SEQ ID NO:31 replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, an amino acid residue (e.g., serine residue) at position 17 of SEQ ID NO:31 replaced by a glutamic acid residue.

[0087] In some embodiments, compared to the wild-type PTH protein, (a) the modified PTH protein has increased glycosylation sites (e.g., N-glycosylation sites), for example, the modified PTH protein is capable of N-glycosylation modification of the amino acid residue (e.g., asparagine residue) at position 33 of SEQ ID NO:31; and / or, (b) in the modified PTH protein, the amino acid residue (e.g., asparagine residue) at position 16 of SEQ ID NO:31 is replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) the amino acid residue (e.g., serine residue) at position 17 of SEQ ID NO:31 is replaced by a glutamic acid residue.

[0088] In some embodiments, compared to the wild-type PTH protein, (a) the modified PTH protein has an amino acid residue (e.g., a valine residue) at position 35 of SEQ ID NO:31 replaced with a serine residue or a threonine residue; and / or, (b) the modified PTH protein has an amino acid residue (e.g., an asparagine residue) at position 16 of SEQ ID NO:31 replaced with a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) the amino acid residue (e.g., a serine residue) at position 17 of SEQ ID NO:31 is replaced with a glutamic acid residue.

[0089] In some embodiments, compared to the wild-type PTH protein, (a) the modified PTH protein has an amino acid residue (e.g., valine residue) at position 35 of SEQ ID NO:31 replaced with a serine or threonine residue, and an amino acid residue (e.g., phenylalanine residue) at position 34 of SEQ ID NO:31 replaced with an alanine residue; and / or, (b) in the modified PTH protein, an amino acid residue (e.g., asparagine residue) at position 16 of SEQ ID NO:31 replaced with a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) an amino acid residue (e.g., serine residue) at position 17 of SEQ ID NO:31 replaced with a glutamic acid residue.

[0090] Modified PTH peptide

[0091] On the other hand, this application provides a modified PTH peptide, which is an active fragment of the modified PTH protein as described above.

[0092] In some embodiments, the modified PTH peptide has an amino acid sequence that differs from that of the wild-type PTH peptide.

[0093] In some embodiments, the modified PTH peptide possesses the biological activity of the modified PTH protein from which it is derived (e.g., PTH1R binding activity and / or PTH1R activation activity).

[0094] In some embodiments, the modified PTH peptide comprises amino acid residues in the modified PTH protein at positions 3-16 of SEQ ID NO:31.

[0095] In some embodiments, the modified PTH peptide comprises, or is composed of, amino acid residues in the modified PTH protein at positions corresponding to positions 3-16 (or 3-17, or 3-27, or 3-33, or 3-34, or 3-35, or 3-40, or 3-50, or 3-60, or 3-70, or 3-80, or 3-84, or 1-16, or 1-17, or 1-27, or 1-33, or 1-34, or 1-35, or 1-40, or 1-50, or 1-60, or 1-70, or 1-80, or 1-84) of SEQ ID NO:31.

[0096] In some embodiments, the modified PTH peptide comprises, or is composed of, amino acid residues in the modified PTH protein at positions 1-33, 1-34, or 1-40 of SEQ ID NO:31.

[0097] In some embodiments, compared to the wild-type PTH protein, the modified PTH protein has an amino acid residue (e.g., an asparagine residue) at position 16 of SEQ ID NO:31 replaced by a glutamic acid residue, a glycine residue, or a serine residue, and / or an amino acid residue (e.g., a serine residue) at position 17 of SEQ ID NO:31 replaced by a glutamic acid residue; and the modified PTH peptide comprises, or is composed of, the amino acid residues at positions 1-33 or 1-34 of SEQ ID NO:31 in the modified PTH protein.

[0098] In some embodiments, compared to the wild-type PTH protein, (a) the modified PTH protein has increased glycosylation sites (e.g., N-glycosylation sites), for example, the modified PTH protein is capable of N-glycosylation modification of the amino acid residue (e.g., asparagine residue) at position 33 of SEQ ID NO:31; and / or, (b) in the modified PTH protein, the amino acid residue (e.g., asparagine residue) at position 16 of SEQ ID NO:31 is replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) the amino acid residue (e.g., serine residue) at position 17 of SEQ ID NO:31 is replaced by a glutamic acid residue; and the modified PTH peptide comprises, or is composed of, the amino acid residues at positions 1-40 of SEQ ID NO:31 in the modified PTH protein.

[0099] In some embodiments, compared to the wild-type PTH protein, (a) the modified PTH protein has an amino acid residue (e.g., a valine residue) at position 35 of SEQ ID NO:31 replaced by a serine residue or a threonine residue; and / or, (b) the modified PTH protein has an amino acid residue (e.g., an asparagine residue) at position 16 of SEQ ID NO:31 replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) the amino acid residue (e.g., a serine residue) at position 17 of SEQ ID NO:31 replaced by a glutamic acid residue; and the modified PTH peptide comprises, or is composed of, the amino acid residues at positions 1-40 of SEQ ID NO:31 in the modified PTH protein.

[0100] In some embodiments, compared to the wild-type PTH protein, (a) the modified PTH protein has an amino acid residue (e.g., valine residue) at position 35 of SEQ ID NO:31 replaced by a serine or threonine residue, and an amino acid residue (e.g., phenylalanine residue) at position 34 of SEQ ID NO:31 replaced by an alanine residue; and / or, (b) the modified PTH protein has an amino acid residue (e.g., asparagine residue) at position 16 of SEQ ID NO:31 replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or, (c) the amino acid residue (e.g., serine residue) at position 17 of SEQ ID NO:31 replaced by a glutamic acid residue; and the modified PTH peptide comprises, or is composed of, the amino acid residues at positions 1-40 of SEQ ID NO:31 in the modified PTH protein.

[0101] In some embodiments, the modified PTH peptide has an amino acid sequence as shown in any one of SEQ ID NO:9, 11-16.

[0102] peptide constructs

[0103] On the other hand, this application provides a polypeptide construct comprising the modified PTH peptide and the Fc domain as described above.

[0104] In some embodiments, the polypeptide construct further includes an antigen-binding domain capable of specifically binding to RANKL.

[0105] In some implementations, the Fc domain comprises a first Fc domain monomer and a second Fc domain monomer.

[0106] In some embodiments, the PTH peptide is covalently linked to the Fc domain and optionally the antigen-binding domain (e.g., through a covalent link comprising peptide bonds, isopeptide bonds and / or disulfide bonds).

[0107] In some embodiments, the first Fc domain monomer and / or the second Fc domain monomer are derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4).

[0108] In some embodiments, the first Fc domain monomer and / or the second Fc domain monomer are selected from the Fc domain monomers of wild-type human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4).

[0109] In some embodiments, the CH2 domain (residues 231-340 of human IgG1, numbered according to the Eu numbering system), CH3 domain (residues 341-447 of human IgG1, numbered according to the Eu numbering system), and / or hinge region (residues 216-230, numbered according to the Eu numbering system) of the first Fc domain monomer and / or the second Fc domain monomer are introduced with one, two, or more mutations (e.g., amino acid substitutions) to alter one or more functional properties of the polypeptide construct, such as serum half-life, complement binding, Fc receptor binding, ADCC activity, CDC activity, and / or ADCP activity.

[0110] In some embodiments, the CH2 domain, CH3 domain and / or hinge region of the first Fc domain monomer and / or the second Fc domain monomer are introduced with one, two or more mutations (e.g., amino acid substitutions) to reduce or ablate the effector function of the Fc region.

[0111] In some embodiments, the hinge region is derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4).

[0112] In some embodiments, the hinge region is selected from the wild-type human immunoglobulin hinge region and its variants.

[0113] In some embodiments, the hinge region is derived from human immunoglobulin IgG4 and contains the substitution mutation S228P.

[0114] In some embodiments, the hinge region comprises an amino acid sequence as shown in SEQ ID NO:10.

[0115] In some embodiments, the antigen-binding domain is selected from Fab, Fab', F(ab')2, scFab, VHH, Fv, disulfide-linked Fv and scFv.

[0116] In some implementations, the antigen-binding domain is Fab.

[0117] In some embodiments, the antigen-binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL), which together form an antigen-binding domain that specifically binds to RANKL.

[0118] In some embodiments, the VH includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:1; the VL includes LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:3.

[0119] In some implementations, the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0120] In some embodiments, the VH includes HCDR1 as shown in SEQ ID NO:34, HCDR2 as shown in SEQ ID NO:35, and HCDR3 as shown in SEQ ID NO:36; and / or, the VL includes LCDR1 as shown in SEQ ID NO:37, LCDR2 as shown in SEQ ID NO:38, and LCDR3 as shown in SEQ ID NO:39.

[0121] The CDRs are defined by the Kabat numbering system.

[0122] In some embodiments, the VH comprises an amino acid sequence as shown in SEQ ID NO:1, and / or the VL comprises an amino acid sequence as shown in SEQ ID NO:3.

[0123] preparation

[0124] The polypeptide constructs, modified PTH proteins, and modified PTH peptides of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, nucleic acid molecules encoding the polypeptide constructs, modified PTH proteins, or modified PTH peptides of the present invention can be obtained by chemical synthesis or PCR amplification, the obtained nucleic acid molecules can be inserted into an expression vector, and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the polypeptide constructs, modified PTH proteins, and modified PTH peptides of the present invention.

[0125] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the polypeptide construct, modified PTH protein, or modified PTH peptide of the present invention. Based on codon degeneracy in the art, in some embodiments, the nucleotide sequence in the isolated nucleic acid molecule encoding the polypeptide construct, modified PTH protein, or modified PTH peptide of the present invention can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0126] In some embodiments, the isolated nucleic acid molecule encodes the polypeptide construct of the present invention.

[0127] It is readily understood that the polypeptide construct of the present invention may consist of one or more polypeptide chains, and there is no limitation on the number of nucleic acid molecules in which the isolated nucleic acid molecules encoding the polypeptide construct of the present invention are located.

[0128] In some embodiments, the polypeptide construct of the present invention comprises peptide chain IA, peptide chain IB and peptide chain IC as described above, wherein the isolated nucleic acid molecule comprises a first nucleotide sequence encoding peptide chain IA of the polypeptide construct of the present invention, a second nucleotide sequence encoding peptide chain IB thereof and a third nucleotide sequence encoding peptide chain IC thereof, wherein the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence are present on the same or different nucleic acid molecules.

[0129] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) that contains the nucleic acid molecule of the present invention.

[0130] In some embodiments, the vector of the present invention is, for example, a plasmid, a granulosome, a bacteriophage, a lentivirus, etc. In some embodiments, the vector is capable of expressing the polypeptide construct, modified PTH protein, or modified PTH peptide of the present invention in a subject (e.g., a mammal, such as a human).

[0131] It is readily understood that the isolated nucleic acid molecules described above can exist in the vector in any form. For example, when the isolated nucleic acid molecule contains multiple nucleotide sequences encoding different peptide chains, these multiple nucleotide sequences can be located in the same vector or in different vectors. There are no restrictions on the orientation, relative position, or connection method of the multiple nucleotide coding sequences on the vector.

[0132] In some embodiments, the polypeptide construct of the present invention comprises peptide chain IA, peptide chain IB and peptide chain IC as described above, and the vector comprises a first nucleotide sequence encoding peptide chain IA of the polypeptide construct of the present invention, a second nucleotide sequence encoding peptide chain IB thereof and a third nucleotide sequence encoding peptide chain IC thereof, wherein the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence are present on the same or different vector molecules.

[0133] In another aspect, the present invention provides a host cell comprising the nucleic acid molecules or vectors described above. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-EBNA, CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0134] In another aspect, the present invention provides a method for preparing the polypeptide construct or modified PTH protein or modified PTH peptide of the present invention, comprising the following steps:

[0135] Under conditions that allow protein expression, host cells are cultured, and the polypeptide construct or modified PTH protein or modified PTH peptide is recovered from the cultured host cell culture; wherein the host cells contain a nucleotide sequence encoding the polypeptide construct or modified PTH protein or modified PTH peptide of the present invention.

[0136] Conjugate

[0137] The polypeptide constructs, modified PTH proteins, and modified PTH peptides of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization of the polypeptide constructs, modified PTH proteins, and modified PTH peptides does not adversely affect PTH1R binding. Therefore, the polypeptide constructs, modified PTH proteins, and modified PTH peptides of the present invention are also intended to include such derivatized forms. For example, the polypeptide constructs, modified PTH proteins, or modified PTH peptides of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as detection reagents, pharmaceutical reagents, and / or proteins or polypeptides (e.g., avidin or multihistidine tags) capable of mediating the binding of the polypeptide constructs, modified PTH proteins, or modified PTH peptides to another molecule.

[0138] As one of the antibody derivatives, the present invention provides conjugates comprising the polypeptide construct of the present invention or a modified PTH protein or a modified PTH peptide, and a coupling moiety.

[0139] In some embodiments, the coupling portion is selected from purified tags and therapeutic agents.

[0140] In some embodiments, the polypeptide construct of the present invention is optionally conjugated to the coupling moiety via a linker.

[0141] In some embodiments, the coupling portion is selected from protein tags. Such protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select appropriate protein tags (e.g., purification tags, detection tags, or tracer tags) according to the desired purpose.

[0142] In some embodiments, the coupling portion is selected from therapeutic agents, such as agents for treating diseases caused by bone metabolism-related disorders and / or parathyroid dysfunction (e.g., hyperthyroidism or hypothyroidism).

[0143] In some embodiments, the coupling moiety is selected from other bioactive peptides.

[0144] Pharmaceutical Composition

[0145] In another aspect, the present invention provides pharmaceutical compositions comprising the polypeptide construct of the present invention, a modified PTH protein, a modified PTH peptide, an isolated nucleic acid molecule, a carrier, a host cell, or a conjugate, and pharmaceutically acceptable carriers and / or excipients.

[0146] In some embodiments, the pharmaceutical composition further comprises additional pharmaceutically active agents.

[0147] In some embodiments, the additional pharmaceutically active agent is selected from agents for treating diseases caused by bone metabolism-related disorders and / or parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism).

[0148] In some embodiments, in the pharmaceutical composition, the polypeptide construct of the present invention, the modified PTH protein, the modified PTH peptide, the isolated nucleic acid molecule, the carrier, the host cell, or the conjugate, and the additional pharmaceutically active agent may be provided as separate components or as mixed components. Therefore, the polypeptide construct of the present invention, the modified PTH protein, the modified PTH peptide, the isolated nucleic acid molecule, the carrier, the host cell, or the conjugate, and the additional pharmaceutically active agent may be administered in combination (e.g., simultaneously, separately, or sequentially).

[0149] In some embodiments, the pharmaceutically acceptable carrier and / or excipient may comprise a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0150] The pharmaceutical compositions of the present invention may include, in "therapeutic effective amounts" or "preventative effective amounts," the polypeptide constructs of the present invention, modified PTH proteins, modified PTH peptides, isolated nucleic acid molecules, carriers, host cells, or conjugates. "Preventative effective amounts" refer to amounts sufficient to prevent, stop, or delay the onset of disease. "Therapeutic effective amounts" refer to amounts sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Therapeutic effective amounts may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments, etc.

[0151] Therapeutic applications

[0152] In another aspect, the present invention provides the use of the polypeptide constructs of the present invention, modified PTH proteins, modified PTH peptides, isolated nucleic acid molecules, carriers, host cells, conjugates, or pharmaceutical compositions for the preparation of medicaments for the prevention and / or treatment in subjects of diseases associated with RANKL and / or PTH1R.

[0153] In some embodiments, the disease is selected from: bone metabolism-related diseases, diseases caused by parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism), and any combination thereof.

[0154] In some implementations, the disease is selected from: osteoporosis (such as postmenopausal osteoporosis), osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, degenerative lumbar spondylolisthesis, degenerative intervertebral disc disease, bone injury, hypoparathyroidism, hyperparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria, and any combination thereof.

[0155] In some implementations, the disease is osteoporosis or osteopenia.

[0156] In some implementations, the subject is a mammal, such as a human.

[0157] In some embodiments, the polypeptide construct, modified PTH protein, modified PTH peptide, isolated nucleic acid molecule, carrier, host cell, conjugate, or pharmaceutical composition is administered alone or in combination with other pharmaceutically active agents (e.g., simultaneously, separately, or sequentially).

[0158] In some embodiments, the additional pharmaceutically active agent is selected from agents for treating diseases caused by bone metabolism-related disorders and / or parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism).

[0159] In another aspect, the present invention provides a method for preventing and / or treating diseases associated with RANKL and / or PTH1R in a subject, the method comprising: administering to a subject in need an effective amount of a polypeptide construct of the present invention, a modified PTH protein, a modified PTH peptide, an isolated nucleic acid molecule, a vector, a host cell, a conjugate, or a pharmaceutical composition.

[0160] In some embodiments, the disease is selected from: bone metabolism-related diseases, diseases caused by parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism), and any combination thereof.

[0161] In some implementations, the disease is selected from: osteoporosis (such as postmenopausal osteoporosis), osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, degenerative lumbar spondylolisthesis, degenerative intervertebral disc disease, bone injury, hypoparathyroidism, hyperparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria, and any combination thereof.

[0162] In some implementations, the disease is osteoporosis or osteopenia.

[0163] In some implementations, the subject is a mammal, such as a human.

[0164] In some embodiments, the polypeptide construct, modified PTH protein, modified PTH peptide, isolated nucleic acid molecule, carrier, host cell, conjugate, or pharmaceutical composition is administered alone or in combination with other pharmaceutically active agents (e.g., simultaneously, separately, or sequentially).

[0165] In some embodiments, the additional pharmaceutically active agent is selected from agents for treating diseases caused by bone metabolism-related disorders and / or parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism).

[0166] The polypeptide constructs, modified PTH proteins, modified PTH peptides, isolated nucleic acid molecules, carriers, host cells, conjugates, or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The polypeptide constructs, modified PTH proteins, modified PTH peptides, isolated nucleic acid molecules, carriers, host cells, conjugates, or pharmaceutical compositions of the present invention should be sterile and stable under manufacturing and storage conditions. A preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating an appropriate amount of the polypeptide construct of the present invention, modified PTH protein, modified PTH peptide, isolated nucleic acid molecule, carrier, host cell, conjugate, or pharmaceutical composition into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, preservatives, stabilizers, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0167] The polypeptide constructs, modified PTH proteins, modified PTH peptides, isolated nucleic acid molecules, carriers, host cells, conjugates, or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the polypeptide constructs, modified PTH proteins, modified PTH peptides, isolated nucleic acid molecules, carriers, host cells, conjugates, or pharmaceutical compositions of the present invention are administered by intravenous injection or bolus.

[0168] Methods for modifying PTH peptides or PTH proteins, or peptide constructs containing PTH peptides or PTH proteins.

[0169] In another aspect, the present invention provides a method for modifying a PTH peptide or PTH protein or a peptide construct containing a PTH peptide or PTH protein, the method comprising performing one or more of the following modifications on the PTH peptide or PTH protein or the peptide construct:

[0170] (i) The amino acid residue (e.g., asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by an amino acid residue other than the asparagine residue, preferably by a glutamic acid residue, a glycine residue or a serine residue;

[0171] (ii) The amino acid residue (e.g., serine residue) at the position corresponding to the 17th position of SEQ ID NO:31 is replaced by an amino acid residue other than serine residue, glycine residue and proline residue, preferably replaced by glutamic acid residue;

[0172] (iii) Increase glycosylation sites (e.g., N-glycosylation sites).

[0173] In some embodiments, the PTH polypeptide or PTH protein or polypeptide construct, after modification, includes the characteristic sequence NXS (or T), where N represents asparagine, X represents any amino acid other than proline, S represents serine, and T represents threonine.

[0174] In some embodiments, the PTH polypeptide or PTH protein or polypeptide construct can undergo N-glycosylation modification of the amino acid residue (e.g., asparagine residue) at the position corresponding to position 33 of SEQ ID NO:31 after modification.

[0175] In some embodiments, the amino acid residue (e.g., valine residue) at the position corresponding to position 35 of SEQ ID NO:31 of the PTH polypeptide, PTH protein, or polypeptide construct is replaced with a serine residue or a threonine residue.

[0176] In some embodiments, the amino acid residue (e.g., valine residue) at position 35 of SEQ ID NO:31 is replaced with a serine residue, and the amino acid residue (e.g., phenylalanine residue) at position 34 of SEQ ID NO:31 is replaced with an alanine residue.

[0177] In some embodiments, the method includes modifying the PTH peptide, PTH protein, or peptide construct as follows:

[0178] (i) an amino acid residue (e.g., an asparagine residue) at position 16 of SEQ ID NO:31 is replaced by a glutamic acid residue, a glycine residue, or a serine residue; and / or,

[0179] (ii) An amino acid residue (e.g., a serine residue) at position 17 of SEQ ID NO:31 is replaced by a glutamic acid residue.

[0180] In some embodiments, the method includes modifying the PTH peptide, PTH protein, or peptide construct with one or more of the following:

[0181] (i) An amino acid residue (e.g., an asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by a glutamic acid residue, a glycine residue or a serine residue;

[0182] (ii) An amino acid residue (e.g., a serine residue) at position 17 of SEQ ID NO:31 is replaced by a glutamic acid residue;

[0183] (iii) N-glycosylation modification of an amino acid residue (e.g., an asparagine residue) at the position corresponding to position 33 of SEQ ID NO:31.

[0184] In some embodiments, the method includes modifying the PTH peptide, PTH protein, or peptide construct with one or more of the following:

[0185] (i) An amino acid residue (e.g., an asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by a glutamic acid residue, a glycine residue or a serine residue;

[0186] (ii) An amino acid residue (e.g., a serine residue) at position 17 of SEQ ID NO:31 is replaced by a glutamic acid residue;

[0187] (iii) The amino acid residue (e.g., a valine residue) at position 35 of SEQ ID NO:31 is replaced with a serine or threonine residue. In some embodiments, the method includes modifying the PTH polypeptide, PTH protein, or polypeptide construct with one or more of the following modifications:

[0188] (i) An amino acid residue (e.g., an asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by a glutamic acid residue, a glycine residue or a serine residue;

[0189] (ii) An amino acid residue (e.g., a serine residue) at position 17 of SEQ ID NO:31 is replaced by a glutamic acid residue;

[0190] (iii) The amino acid residue (e.g., valine residue) at position 35 of SEQ ID NO:31 is replaced with a serine residue, and the amino acid residue (e.g., phenylalanine residue) at position 34 of SEQ ID NO:31 is replaced with an alanine residue.

[0191] In some implementations, the method is used for one or more of the following:

[0192] (i) Improve the stability of the PTH peptide, the PTH protein, or the peptide construct;

[0193] (ii) to prolong the in vivo half-life of the PTH polypeptide, the PTH protein, or the polypeptide construct;

[0194] (iii) Modulate (e.g., enhance or reduce) the binding activity of the PTH peptide, the PTH protein, or the peptide construct to PTH1R;

[0195] (iv) Modulate (e.g., enhance or reduce) the activation activity of the PTH peptide, the PTH protein, or the peptide construct for PTH1R.

[0196] In some embodiments, the modified PTH peptide or PTH protein or peptide construct has altered properties.

[0197] In some embodiments, compared to the original PTH peptide or PTH protein or peptide construct, the modified PTH peptide or PTH protein construct has one or more of the following characteristics:

[0198] (i) Improved stability;

[0199] (ii) altering (e.g., enhancing or reducing) PTH1R binding activity;

[0200] (iii) Altering (e.g., enhancing or reducing) PTH1R activation activity;

[0201] (iv) Extended half-life;

[0202] (v) Enhanced pharmacological activity.

[0203] Terminology Definition

[0204] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0205] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0206] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0207] As used herein, the term "receptor activator of nuclear factor κB ligand (RANKL)" is a type II transmembrane protein that is a ligand for the NF-κB (RANK) receptor. RANKL is expressed in osteoblasts, osteocytes, and immune cells. It binds to NF-κB and induces the differentiation of monocyte / macrophage lineage cells into osteoclasts, further leading to the maturation of osteoclast precursors. It is crucial for osteoclast maturation, bone modeling and remodeling, and lymph node (LN) development. Exemplary amino acid sequences of RANKL are available from public databases such as GenBank and UniProt.

[0208] As used herein, the term "parathyroid hormone (PTH)" refers to a basic single-chain polypeptide hormone secreted by the chief cells of the parathyroid gland. PTH regulates calcium and phosphorus metabolism in vertebrates, specifically increasing blood calcium levels and decreasing blood phosphorus levels. Its primary target organs are bone and kidney, and it can be used to treat bone metabolic diseases. An exemplary amino acid sequence of PTH can be found in SEQ ID NO:31 or obtained from public databases (e.g., GenBank, UniProt).

[0209] In this document, when referring to the amino acid sequence of PTH, the sequence shown in SEQ ID NO:31 is used for description. However, those skilled in the art will understand that mutations or variations can be naturally generated or artificially introduced into the amino acid sequence of PTH without affecting its biological function. Therefore, in this invention, the term "PTH" and similar expressions should include all such sequences, including, for example, the sequence shown in SEQ ID NO:31 and its natural or artificial variants (e.g., variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity). Furthermore, when describing residue positions or sequence fragments of PTH, it includes not only the residue positions or sequence fragments of SEQ ID NO:31, but also the corresponding residue positions or corresponding sequence fragments in its natural or artificial variants.

[0210] In this document, the term "PTH peptide" (also referred to as "PTH polypeptide," the two terms are used interchangeably) has the meaning commonly understood by those skilled in the art, generally referring to an active fragment of a full-length PTH protein (e.g., wild-type PTH protein or modified PTH protein), or, in some embodiments, the PTH peptide may also be used to refer to the full-length PTH protein (e.g., wild-type PTH protein or modified PTH protein) itself. Generally, PTH peptides typically possess the biological activities (e.g., PTH1R binding activity and / or PTH1R activation activity) of the full-length PTH protein from which they are derived (e.g., wild-type PTH protein or modified PTH protein). In this invention, non-limiting examples of PTH peptides include: PTH 3-16 PTH 3-17 PTH 3-27 PTH 3-33 PTH 3- 34 PTH 3-35 PTH 3-40 PTH 3-50 PTH 3-60 PTH 3-70 PTH 3-80 PTH 3-84PTH 1-16 PTH 1- 17 PTH 1-27 PTH 1-33 PTH 1-34 PTH 1-35 PTH 1-40 PTH 1-50 PTH 1-60 PTH 1-70 PTH 1- 80 and PTH 1-84 .

[0211] As used herein, the term "peptide construct" can comprise one or more peptide or protein components, each of which may independently possess different biological activities or functions and be linked in a covalent and / or non-covalent manner (e.g., covalently linked by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalently linked by hydrogen bonds). In this invention, the individual peptide or protein components in the "peptide construct" may reside in the same peptide chain or may reside in different peptide chains; that is, the "peptide construct" may comprise only one peptide chain or two or more peptide chains, which are covalently and / or non-covalently linked (e.g., covalently linked by covalent bonds comprising peptide bonds, isopeptide bonds, and / or disulfide bonds, and / or non-covalently linked by hydrogen bonds).

[0212] The twenty standard amino acids discussed herein were prepared in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine may be represented by A or Ala.

[0213] As used herein, the term "antigen-binding domain" refers to a molecular fragment derived from an antibody that specifically binds to a target antigen. The term "antibody," as used herein, refers to an immunoglobulin-derived molecule that specifically binds to a target antigen via at least one antigen-binding site located in its variable region. A "complete antibody" typically consists of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0214] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residue in the variable region of an antibody responsible for antigen binding. The nanobody contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by various numbering systems. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems.

[0215] As used herein, the term “frame region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0216] As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "Fab" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; the term "F(ab')2" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region; the term "Fab'" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (consisting of the VH and CH1 domains); and the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody.

[0217] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0218] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0219] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0220] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0221] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0222] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. The "binding rate constant" (k...) a or k on ) and "dissociation rate constant" (k dis or k off Both can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K. D(See Davies et al., Annual Rev Biochem, 1990; 59:439-473). K can be measured by any effective method. D k on and k dis The dissociation constant can be measured in Biacore using surface plasmon resonance (SPR) in some implementations. Alternatively, it can be measured using bioluminescence interferometry or Kinexa.

[0223] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0224] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0225] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Adjuvants include, but are not limited to, inorganic adjuvants (such as aluminum hydroxide and vanadium), biological adjuvants (such as Mycobacterium tuberculosis, BCG, Corynebacterium tumefaciens, Bordetella pertussis, Gram-negative bacillus endotoxins, B subunits of cholera toxin, muramyl dipeptides, and cytokines), synthetic adjuvants (such as double-chain polyadenylates and uridine monophosphate), oil formulations (such as Freund's complete adjuvant and peanut oil emulsions), and nano-adjuvants. Ionic strength enhancers include, but are not limited to, sodium chloride. Reagents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Reagents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, and sorbic acid. Stabilizers have the meaning commonly understood by those skilled in the art as being able to stabilize the desired activity of an active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% NaCl), glucose solutions (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solutions), Ringer's solutions, and any combination thereof.

[0226] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (e.g., survival without treatment).

[0227] As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject (e.g., a human) suffers from a bone metabolism-related disease (such as osteoporosis) or a disease caused by parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism) (such as hypoparathyroidism, hyperparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria), or is at risk of suffering from such a disease.

[0228] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing a disease (e.g., a bone metabolism-related disease or a disease caused by parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism)) means an amount sufficient to prevent, stop, or delay the onset of said disease; an effective amount for treating a disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0229] Beneficial effects of the invention

[0230] This invention provides a polypeptide construct (also referred to herein as a bifunctional fusion protein) comprising an antigen-binding domain capable of specifically binding to RANKL and a PTH peptide, which possesses one or more of the following beneficial effects:

[0231] 1) The polypeptide construct of the present invention adopts an asymmetric structural mode, which has significantly superior stability (e.g., it has better stability than constructs with other structural modes), and solves the problem of PTH peptide degradation during the production process.

[0232] 2) The structural pattern of the polypeptide construct of this invention significantly improves the half-life of the PTH terminus, thereby enhancing its activity in regulating bone metabolism. Furthermore, this invention introduces a glycosylation site at the PTH peptide terminus of the polypeptide construct, further extending the half-life of the PTH terminus.

[0233] 3) This invention improves the stability, drug-likeness, and in vivo pharmacodynamic activity of PTH peptides by designing specific mutations at the PTH peptide terminus.

[0234] 4) The polypeptide construct of the present invention can reduce the potential toxic side effects of PTH1R by reducing the activation activity of PTH1R at the PTH peptide terminus on cells expressing PTH1R alone. At the same time, by utilizing the targeting of RANKL, it can achieve conditional activation of PTH1R in cells expressing both PTH1R and RANKL, thereby achieving targeted activation of PTH peptide and thus improving the role and targeted activation activity of the polypeptide construct in regulating bone metabolism. Attached Figure Description

[0235] Figures 1A-1D: Schematic diagrams of bifunctional fusion protein molecular structures. Figure 1A shows a bifunctional molecule in structure 1 form; Figure 1B shows a bifunctional molecule in structure 2 form; Figure 1C shows a bifunctional molecule in structure 3Mol-1 form, with no glycosylation site introduced at the polypeptide terminus; Figure 1D shows a bifunctional molecule in structure 3Mol-2 form, with one glycosylation site introduced at the polypeptide terminus.

[0236] Figures 2A-2F: Mass spectrometry analysis and identification of bifunctional fusion proteins. Figure 2A shows the mass spectrometry analysis results of the PTH fusion fraction of molecule 0010; Figure 2B shows the mass spectrometry analysis results of the PTH fusion fraction of molecule 0004; Figure 2C shows the mass spectrometry identification results of the PTH fusion fraction of molecule 0005; Figure 2D shows the mass spectrometry analysis results of the PTH fusion fraction of molecule 0059; Figure 2E shows the mass spectrometry analysis results of the PTH fusion fraction of molecule 0060; and Figure 2F shows the mass spectrometry analysis results of the PTH fusion fraction of molecule 0061.

[0237] Figures 3A-3B: PTH1R activation assay of bifunctional fusion protein in Saos-2 cells.

[0238] Figures 4A-4B: PTH1R activation assay of the bifunctional fusion protein in RANKL-Saos-2 double-positive cells.

[0239] Figure 5: Results of detection of the neutralizing activity of the bifunctional fusion protein against sRANKL.

[0240] Figure 6: Affinity test results of bifunctional fusion proteins (positive molecules, 0059, 0060 and 0061) with sRANKL.

[0241] Figures 7A-7B: Affinity test results of bifunctional fusion protein (positive molecule, 0101) with sRANKL.

[0242] Figure 8: Animal weight 0-6 weeks post-surgery.

[0243] Figure 9: BMD values ​​of the femur and tibia in animals 5 weeks post-surgery. One-way ANOVA vs. sham group. Mean±sem, *P<0.05**P<0.01.

[0244] Figures 10A-10B: CT scan analysis of femoral tissue, one-way ANOVA vs. OVX-PBS group. Mean±sem, n=5-6*P<0.05**P<0.01.

[0245] Figures 11A-11B: CT analysis of isolated tibia, one-way ANOVA vs. OVX-PBS group. Mean±sem, n=5-6*P<0.05**P<0.01.

[0246] Figure 12: Animal weight 0-6 weeks post-surgery.

[0247] Figure 13: Tibial BMD values ​​of animals 5 weeks post-surgery.

[0248] Figure 14: Animal weights after grouping.

[0249] Figure 15: Uterine weight of animals 10 weeks post-surgery.

[0250] Figure 16: Femoral bone mass parameters. One-way ANOVA vs. OVX-solvent group. Mean±sem, n=5-6*P<0.05**P<0.01.

[0251] Figures 17A-17C: Femoral structural parameters. One-way ANOVA vs. OVX-solvent group. Mean±sem, n=5-6*P<0.05**P<0.01.

[0252] Figure 18: Tibial bone mass parameters. One-way ANOVA vs. OVX-solvent group. Mean±sem, n=5-6*P<0.05**P<0.01.

[0253] Figures 19A-19C: Tibial structural parameters. One-way ANOVA vs. OVX-solvent group. Mean±sem, n=5-6*P<0.05**P<0.01.

[0254] Figure 20: Three-point biomechanical testing of the femur. One-way ANOVA vs. OVX-solvent group. *P<0.05,**P<0.01.

[0255] Figures 21A-21G: HE staining analysis of bone sections. Figures 21A-F are HE-stained images. Mouse tibial tissue underwent paraffin preparation, H&E staining, and whole-section scanning. Taking Figure 21A (sham-operated group) as an example, HALO statistical analysis of the trabecular bone area was performed on a 1mm-2mm region of bone marrow parallel to the longitudinal axis of the tibia, starting from the midpoint of the tibial growth plate. The arrows indicate the trabeculae drawn using the Halo software. Figure 21G is a molecular plot showing the number of trabeculae after HE staining. One-way ANOVA vs. OVX-solvent group. Mean±sem, n=5-6*P<0.05**P<0.01.

[0256] Figure 22: Experimental endpoint, serum TRACP-5b ELISA results.

[0257] Figure 23: Experimental endpoint, serum osteocalcin ELISA results.

[0258] Figures 24A-24B: Experimental endpoint, mouse blood biochemical analysis. Figure 23A shows the detection of aspartate aminotransferase (AST) and creatinine, and Figure 23B shows the detection of urea and blood calcium.

[0259] Figures 25A-25B: PTH1R activation assay of the modified bifunctional fusion protein on Saos-2 cells.

[0260] Figures 26A-26B: PTH1R activation assay of the modified bifunctional fusion protein on RANKL-Saos-2 double-positive cells.

[0261] Figure 27: PTH1R activation experiment of the modified bifunctional fusion protein on HEK293 cells overexpressing mouse PTH1R.

[0262] Figure 28: Results of PTH1Rβ-arrestin recruitment experiments of the modified bifunctional fusion protein.

[0263] Figure 29: Affinity test results of the modified bifunctional fusion protein with sRANKL.

[0264] Figure 30: Results of neutralizing activity of the modified bifunctional fusion protein against sRANKL.

[0265] Figures 31A-31D: Blood biochemical analysis of mice during drug administration and at the experimental endpoint. Figure 31A shows blood calcium detection, Figure 31B shows aspartate aminotransferase (AST) detection, Figure 31C shows urea detection, and Figure 31D shows creatinine detection.

[0266] Figures 32A-32C: In vivo experimental endpoint mouse femur isolated CT results.

[0267] Figure 33: Three-point biomechanical analysis of mouse femur.

[0268] Figure 34: Blood biochemistry (blood calcium and phosphorus) analysis of mice in the model animals during drug administration and at the experimental endpoint.

[0269] Figures 35A-35B: In vivo experimental endpoint mouse femur isolated CT results.

[0270] Figure 36: Three-point biomechanical analysis of mouse femur.

[0271] Figure 37: CT analysis of isolated mouse iliac bone.

[0272] Figure 38: Ex vivo CT analysis of mouse L5 vertebra.

[0273] Figures 39A-39B: ELISA results of mouse serum at the experimental endpoint. Figure 39A shows the detection of mouse serum bone formation markers (osteocalcin), and Figure 39B shows the detection of mouse serum bone resorption markers (TRACP-5b).

[0274] Figure 40: In vivo CT results of mice at different time points after a single dose.

[0275] Figures 41A-41B: Results of serum bone turnover marker detection in mice after a single dose. Figure 41A shows the detection of serum bone formation marker (osteocalcin) in mice, and Figure 41B shows the detection of serum bone resorption marker (TRACP-5b) in mice.

[0276] Figures 42A-42D: Results of ex vivo CT scans. Figure 42A shows the ex vivo CT analysis of the femur, Figure 42B shows the ex vivo CT analysis of the tibia, Figure 42C shows the ex vivo CT analysis of the iliac bone, and Figure 42D shows the biomechanical analysis of the femur.

[0277] Figures 43A-43B: Blood drug concentration analysis after a single dose in the OVX mouse model. Figure 43A shows the serum concentration curve of antibody terminal molecules after a single dose, and Figure 43B shows the serum concentration curve of intact molecules after a single dose.

[0278] Figures 44A-44B: Blood drug concentration analysis after a single dose in cynomolgus monkeys. Figure 44A shows the serum concentration curve of antibody-terminated molecules after a single dose, and Figure 44B shows the serum concentration curve of intact molecules after a single dose.

[0279] Figure 45: Schematic diagram of the three-point bending experiment in a mouse femoral closed fracture model.

[0280] Figure 46: Micro-CT scan results of a mouse femoral closed fracture model.

[0281] Figure 47: Results of the three-point bending experiment in a mouse femoral closed fracture model.

[0282] Sequence information

[0283] Information about the sequences involved in this invention is described in the table below:

[0284] Table 1: Sequence Description Detailed Implementation

[0285] The invention will now be described in the following non-limiting embodiments.

[0286] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0287] Preparation example: Preparation of fusion proteins

[0288] 1.1 Structural composition of the fusion protein

[0289] Due to the inherent instability of PTH, mass spectrometry analysis was performed to identify the production status of PTH fusion proteins with different structural forms during the design of PTH fusion protein structures, and finally, a suitable fusion protein structure was selected.

[0290] 1.1.1 Candidate fusion protein structure

[0291] 1. Structure 1: The symmetrical structure of the "2+2" PTH-linker-heavy chain fusion protein, its molecular structure is shown in Figure 1A. The fusion protein consists of four peptide chains:

[0292] (1) The peptide chain IA contains, from the N-terminus to the C-terminus, the light chain variable region of the anti-RANKL antibody and the light chain constant region.

[0293] (2) The peptide chain IB contains PTH peptide, linker, heavy chain variable region of anti-RANKL antibody, heavy chain CH1 region, hinge region and Fc segment from N-terminus to C-terminus.

[0294] (3) Peptide chain IC, which has the same composition as peptide chain IB;

[0295] (4) Peptide chain ID, which has the same composition as peptide chain IA.

[0296] Specifically, the structural / sequence information of the exemplary "2+2" PTH-linker-heavy chain fusion proteins 0004 and 0010 involved in the embodiments of this application is shown in Table 2 below. The structure of 0010 is consistent with the description in PCT / US2006 / 044199. 0010 uses the adapter GGGAP to link PTH and the anti-RANKL antibody, and the heavy chain constant region isotype of the anti-RANKL antibody is IgG2. The difference between 0004 and 0010 is that 0004 uses the adapter (G4S)3 to link PTH and the anti-RANKL antibody, and the heavy chain constant region isotype of the anti-RANKL antibody is IgG4.

[0297] Table 2: Structure / Sequence Information of Exemplary Fusion Proteins 0004 and 0010

[0298] 2. Structure 2: The symmetrical structure of the "2+2" PTH-linker-light chain fusion protein, its molecular structure is shown in Figure 1B. The fusion protein consists of four peptide chains:

[0299] (1) The peptide chain IA contains PTH peptide, linker, light chain variable region of anti-RANKL antibody and light chain constant region from N-terminus to C-terminus.

[0300] (2) The peptide chain IB contains, from the N-terminus to the C-terminus, the heavy chain variable region, the heavy chain CH1 region, the hinge region, and the Fc segment of the anti-RANKL antibody.

[0301] (3) Peptide chain IC, which has the same composition as peptide chain IB;

[0302] (4) Peptide chain ID, which has the same composition as peptide chain IA.

[0303] Specifically, the structure / sequence information of the exemplary "2+2" PTH-linker-light chain fusion protein 0005 involved in the embodiments of this application is shown in Table 3 below. Structure 2 is consistent with the description in PCT / US2017 / 014836.

[0304] Table 3: Structure / Sequence Information of Exemplary Fusion Protein 0005

[0305] 3. Structure 3: The asymmetric structure of the "1+1" PTH-linker-Fc fusion, its molecular structure is shown in Figures 1C-1D. The Mol-1 fusion protein (Figure 1C) consists of three peptide chains:

[0306] (1) The peptide chain IA contains, from the N-terminus to the C-terminus, the light chain variable region of the anti-RANKL antibody and the light chain constant region.

[0307] (2) The peptide chain IB contains, from the N-terminus to the C-terminus, the heavy chain variable region of the anti-RANKL antibody, the heavy chain CH1 region, the hinge region, and the Fc segment containing amino acid modifications that can form the Hole structure.

[0308] (3) The peptide chain IC contains PTH peptide, linker, hinge region and Fc segment with amino acid modification that can form Knob structure from N-terminus to C-terminus.

[0309] In this structure, peptide chain IB and peptide chain IC are paired in a knot-in-hole configuration.

[0310] The Mol-2 fusion protein (Figure 1D) is based on the Mol-1 fusion protein, with a glycosylation site introduced at the end of the teriparatide.

[0311] Specifically, the structure / sequence information of the exemplary fusion proteins 0059, 0060, 0061, and 0101 involved in the embodiments of this application is shown in Table 4 below.

[0312] Table 4: Structure / Sequence Information of Exemplary Fusion Proteins 0059, 0060, 0061, and 0101

[0313] 1.1.2 Mass spectrometry analysis and identification of candidate fusion proteins

[0314] The reduced molecular weights of the analyte molecules 0010 (sequence from patent WO 2007 / 059136 A2, heavy chain sequence from SEQ ID NO:10, light chain sequence from SEQ ID NO:12), 0004 (PTH-linker3-HC), 0005 (PTH-linker3-LC), 0059 (PTH-linker1-Fc), 0060 (PTH-linker2-Fc), and 0061 (PTH-linker 3-Fc) were determined using reversed-phase chromatography-tandem mass spectrometry. These values ​​were compared with the theoretical molecular weights to assess the sample fragmentation.

[0315] The experimental method is as follows:

[0316] (1) Sample preparation: Molecular weight reduction. Take 50 μg of sample, dilute with water to 0.5 mg / mL, add 2 μL of 0.5 M DTT, and incubate at 37 °C for 1 h. Then add to a sample vial for analysis;

[0317] (2) The samples were analyzed using a BioResolveRP column (450A, 2.7μm, manufacturer: Waters, catalog number: 186008946) on a Thermo Orbitrap Exploris 240 mass spectrometer. The relevant detection parameters are shown in the instrument settings (Tables 5 and 6).

[0318] Table 5: Liquid Chromatography Parameters

[0319] Table 6: Mass Spectrometry Parameters

[0320] (3) Experimental Results: The results were analyzed using Thermo BioPharma Finder 5.0 software. As shown in Figures 2A-2F, the symmetrical structures of structures 1 and 2, as identified by mass spectrometry, exhibited severe PTH terminus breakage during the production process, posing a serious challenge to the future druggability of these molecules. However, no PTH terminus breakage was detected in the asymmetric molecule of structure 3 during protein production. This indicates that the "1+1" asymmetric structure can improve the stability of the PTH peptide, making the fusion protein more stable during production and thus giving it better druggability.

[0321] Therefore, in subsequent embodiments, structure 3 was selected for the construction of the fusion protein.

[0322] 1.2 Plasmid construction, cell transfection, and protein expression

[0323] The bispecific recombinant protein expression plasmid was synthesized by Baiying Biotechnology after codon optimization based on the protein sequence, cloned into the pcDNA3.1 plasmid, and the sequence was verified to be correct by sequencing. The obtained expression plasmid was centrifuged to determine the concentration, and the different components of the plasmid were mixed in an appropriate ratio.

[0324] Take an appropriate amount of cells, centrifuge to remove the supernatant, add an appropriate amount of electroporation buffer to the cells, mix well, and then add plasmids. After thorough mixing, add the suspension to an electroporation tube and place the tube in an electroporator for electroporation. After electroporation, aliquot the cells from the electroporation tube into shake flasks containing culture medium and incubate statically for 40 min. After incubation, place the shake flasks in a 37°C, 110 rpm, 8% CO2 incubator for 24 h, then add feed / sodium butyrate and continue culturing for 6–7 days.

[0325] 1.3 Protein purification

[0326] 1.3.1 Protein A affinity chromatography column purification

[0327] 1) Equilibration chromatography column: 1xPBS, flow rate 1ml / min, 20cv;

[0328] 2) Sample loading: Retention time 4 min;

[0329] 3) Washing: 1xPBS, flow rate 1ml / min, 20cv;

[0330] 4) Elution: Sodium acetate buffer (pH 3.4), retention time 5 min, collect in separate tubes, approximately 1000 μl per tube, and read the absorbance value at 280 nm using a NanoDrop instrument.

[0331] 5) Dialysis: Aspirate high-concentration protein into a dialysis bag and place it in a beaker containing 10 mM NaAc and pH 5.5 for dialysis.

[0332] 1.3.2 Ion column purification

[0333] (1) Experimental materials:

[0334] Solution A: 50mM NaAc, pH 5.0

[0335] Solution B: 50mM NaAc + 500mM NaCl, pH 5.0

[0336] 50mM NaAc, pH 5.0, centrifuge tubes of various sizes, CaptoS pre-packed columns

[0337] (2) Experimental method: cation exchange

[0338] 1) Sample pretreatment: Sample: Solution A = 1:3 dilution;

[0339] 2) Equilibration chromatography column: Solution A: 50mM NaAc pH5.0, flow rate 1ml / min, 20cv;

[0340] 3) Sample loading: Retention time 4 min;

[0341] 4) Washing: Solution A: 50mM NaAc pH5.0, flow rate 1ml / min, 10cv;

[0342] 5) Elution: 0-100% solution B, linear elution, retention time 6 min, collect the eluent according to the OD280 absorption peak, 1 ml per tube;

[0343] 6) Take samples from separate tubes and send them to the SEC for testing. Aspirate the qualified proteins into the dialysis bag and place them in a beaker containing 10mM NaAc and pH 5.5 for dialysis.

[0344] 1.3.3 Basic Protein Quality Control

[0345] (1) Concentration detection

[0346] Methods: The absorbance of sample A280 was read using a Thermo Nanodrop One spectrophotometer. The protein concentration was then calculated using the formula: Protein concentration = A280 reading / extinction coefficient. The results are shown in Table 8.

[0347] (2) Purity detection (SDS-PAGE, SEC-HPLC)

[0348] SDS-PAGE: Protein samples were analyzed using protein precast gels (made in-house by Baiying Biotechnology) with gradients of 4%-18%.

[0349] SC-HPLC: SEC experiments were performed using an LC-20AT high-performance liquid chromatograph and a gel chromatography column (manufacturer: SRT-C). Specific experimental parameters are shown in Table 7. Results are shown in Table 8.

[0350] Table 7: SEC Experimental Parameters

[0351] (3) Endotoxin detection

[0352] Endotoxin detection was performed on the samples using horseshoe crab reagent (sensitivity 0.25 EU / ml), and the results are shown in Table 8.

[0353] Table 8: Results of endotoxin detection in fusion protein

[0354] The results showed that the purified bifunctional fusion protein had high concentration, high purity, and low endotoxin content, and was used for subsequent experiments.

[0355] The positive molecules used in all the following examples are anti-RANKL positive control antibodies, the sequences of which are derived from patent document AU2019213305B2. The heavy chain sequence corresponds to SEQ ID NO:2 in that patent, and the light chain sequence corresponds to SEQ ID NO:4 in that patent.

[0356] Example 1: Detection of the activation activity of bifunctional molecules on PTH1R in Saos-2 and RANKL-Saos-2 dual-positive cell lines

[0357] 1. Detection of the activation activity of bifunctional molecules on PTH1R in the Saos-2 cell line

[0358] The activation activity of bifunctional molecules on PTH1R in Saos-2 cells (Beina Biotechnology, BNCC338485) was detected using the cAMP-Gs Hirange Kit (Cisbio, 62AM6PEC).

[0359] (1) Cell preparation: After digestion, the cells were centrifuged, resuspended in cell dilution buffer (0.5% BSA in DMEM + 0.5mM IBMX) and counted. The cell viability was >90%. The cell density was adjusted to 4×10⁶ cells / year using cell dilution buffer. 5 / ml.

[0360] (2) Experimental steps:

[0361] 1) Add 5 μl of test sample to a 384-well plate and centrifuge for 1 minute. Add 5 μl of cells from cell dilution buffer to the experimental plate, bringing the final cell density to 2000 cells / well. Centrifuge for 1 minute.

[0362] 2) Incubate at 37°C for 30 minutes to allow cells to fully bind to the test substances. Dilute cAMP-d2 and Anti-cAMP-Cryptate using the lysis and detection buffer provided in the kit, add 5 μl of the diluted cAMP-d2 and Anti-cAMP-Cryptate to the experimental plate respectively, and centrifuge for 1 minute;

[0363] 3) Incubate at room temperature for 60 minutes. Read the results using a multi-functional microplate reader (PE, Envisio).

[0364] (3) Experimental results: The data were processed using Graphpad. The processing results are shown in Figures 3A-3B and summarized in Tables 9-10.

[0365] 2. Detection of the PTH1R activation activity of bifunctional molecules in the RANKL-Saos-2 cell line.

[0366] The activation activity of bifunctional molecules on PTH1R in RANKL-Saos-2 cells (Jiman Biotechnology, BNCC338485) was detected using the cAMP-Gs Hirange Kit (Cisbio, 62AM6PEC).

[0367] (1) Cell preparation: After digestion, the cells were centrifuged, resuspended in cell dilution buffer (0.5% BSA in DMEM + 0.5mM IBMX) and counted. The cell viability was >90%. The cell density was adjusted to 4×10⁶ cells / year using cell dilution buffer. 5 / ml.

[0368] (2) Experimental steps:

[0369] 1) Add 5 μl of test sample to a 384-well plate and centrifuge for 1 minute. Add 5 μl of cells from cell dilution buffer to the experimental plate, bringing the final cell density to 2000 cells / well. Centrifuge for 1 minute.

[0370] 2) Incubate at 37°C for 30 minutes to allow cells to fully bind to the test substances. Dilute cAMP-d2 and Anti-cAMP-Cryptate using the lysis and detection buffer provided in the kit, add 5 μl of the diluted cAMP-d2 and Anti-cAMP-Cryptate to the experimental plate respectively, and centrifuge for 1 minute;

[0371] 3) Incubate at room temperature for 60 minutes. Read the results using a multi-functional microplate reader (PE, Envisio).

[0372] (3) Experimental results: The data were processed using Graphpad. The processing results are shown in Figures 4A-4B and summarized in Tables 9-10.

[0373] Table 9: Activation activity of bifunctional molecules in Saos-2 and RANKL-Saos-2 cells

[0374] As shown in Table 9, the tested molecules 0061, 0060, 0059, and 0101 all exhibited PTH1R activation activity on Saos-2 cells. Compared to Saos-2 cells, the activation activity of each molecule on PTH1R was significantly enhanced on RANKL-Saos-2 cells, indicating that the molecules of the present invention have higher selectivity for the RANKL-Saos-2 double-positive cell line, thereby bringing good bone tissue targeted activation activity.

[0375] Compared to the performance of PTH1-34 in the RANKL-Saos-2 double-positive cell line, as shown in Table 10, PTH1-34 exhibited higher activity in Saos-2 cells and did not show selectivity for the RANKL-Saos-2 double-positive cell line. Due to the broad expression profile of PTH1R, particularly its high expression in tissues such as the kidney and intestine, the high activation activity of the PTH1-34 peptide poses a risk of hypercalcemia during use, potentially leading to nephrotoxicity and other side effects. The high selectivity of the test molecules 0061, 0060, 0059, and 0101 of this invention results in lower toxicity.

[0376] Table 10: Activation activity of PTH 1-34 in Saos-2 and RANKL-Saos-2 cells

[0377] Example 2: Detection of the neutralizing activity of the anti-RANKL terminus of a bifunctional molecule against sRANKL

[0378] Raw264.7 cells (Chinese Academy of Sciences, SCSP-5036) differentiate into osteoclasts under the induction of sRANKL (Acro, RAL-H5240). Cell differentiation was analyzed by detecting the expression level of TRACP-5b in the cells. The neutralizing activity of sRANKL was assessed by detecting the inhibitory effect of the molecule on cell differentiation.

[0379] (1) Experimental steps:

[0380] 1) Day 1: Raw 264.7 cells were collected into EP tubes using a cell scraper for counting and diluted to 2×10⁻⁶. 4 / ml, add 500μl (20,000 cells) to each well of a 48-well plate and incubate overnight in an incubator;

[0381] 2) Day 2: Aspirate the culture medium from the wells. According to the experimental design, dilute sRANKL to 200 ng / ml with complete culture medium. Dilute the anti-RANKL positive control (sequence from patent document AU2019213305B2, the heavy chain sequence of which corresponds to SEQ ID NO:2 in the patent document, and the light chain sequence of which corresponds to SEQ ID NO:4 in the patent document, which will also be used as positive molecules in subsequent examples) and the sample. The initial concentration is 6 μg / ml. Then, dilute 8 concentrations 1:3 (dilution of all reagents to twice the working concentration). Add 250 μl of sample and 250 μl of sRANKL to each well of the 48-well plate. Add 500 μl of complete culture medium to the Blank control well and 250 μl of sRANKL solution to the negative control sRANKL well. Then, add complete culture medium to make up to 500 μl, that is, the final concentration is 100 ng / ml sRANKL.

[0382] 3) Day 3: Cells were cultured normally in a 37°C CO2 incubator for 2 days;

[0383] 4) Repeat Day 2 and Day 3;

[0384] 5) Day 6: Sample collection;

[0385] 6) Extract cellular RNA and perform qPCR to detect the expression level of TRACP-5B in cells.

[0386] (2) Experimental Results: Data were processed using GraphPad, and the results are shown in Figure 5. Both the detected molecules and the positive control molecules were able to inhibit the expression of TRACP-5B in RAW264.7 cells. This indicates that the tested molecules 0061 and 0059 can inhibit the ability of sRANKL to induce RAW264.7 cells to differentiate into osteoclasts, and their inhibitory activity is comparable to that of the positive control molecules. This suggests that the tested molecules inhibit bone resorption.

[0387] Example 3: Detection of the affinity between the anti-RANKL terminus and sRANKL of a bifunctional molecule using SPR (Cytiva, Biacore molecular interaction analysis system, model: 8K).

[0388] The analyte molecule was captured using an anti-human capture chip (Cytiva, catalog number: 29234600, lot number: 10315802), and sRANKL (ACRO, catalog number: RAL-H5240, Lot: C172P1-21CGF1-19Z) was used as the analytical stream through the chip. Kinetic analysis was performed based on the acquired data.

[0389] 1. Experimental Methods

[0390] (1) Sample preparation: The positive molecules (anti-RANKL positive control antibody, sequence from patent document AU2019213305B2, its heavy chain sequence corresponds to SEQ ID NO:2 in the patent document, and its light chain sequence corresponds to SEQ ID NO:4 in the patent document), 0059, 0060, 0061, and 0101 molecules were diluted to 1 μg / mL with HBS-EP+Buffer and set aside. The analyte sRANKL was diluted to 50 nM with HBS-EP+Buffer, and serially diluted 2-fold to 5 concentration points and set aside.

[0391] (2) Install the chip: Replace the Running Buffer with HBS-EP+Buffer, put in the S-series CM5 sensor chip, and rinse twice using the change solution command.

[0392] (3) Preparation of the conjugated chip (Amine Coupling kit, cytiva, catalog number: BR100633, batch number: 32274): The CM5 chip (cytiva, catalog number: 29149603, batch number: 10326703) was pre-activated using EDC / NHS for 7 min, and then conjugated with 25 μg / mL anti-Human antibody (flow rate: 10 μl / min, conjugation time: 7 min). After the conjugation was completed, the chip was blocked with Ethanolamine for 7 min. The conjugated chip was then ready for use.

[0393] (4) Editing program on-machine testing

[0394] 1) Capture ligand: analyte, concentration: 1 μg / mL, flow rate: 10 μl / min, capture time: 60 s, in flow cell 2, detection temperature: 25℃;

[0395] 2) Analyte setup: Analyte: sRANKL, starting concentration 100 nM, 2-fold serial dilution, 5 concentration points, set 12.5 nM & 0 nM as repeat concentrations, flow rate: 30 μl / min, binding time: 180 s, dissociation time: 420 s, in flow cell 1 & 2;

[0396] 3) Regeneration: Regeneration solution: 3M MgCl2, flow rate: 20μl / min, regeneration time: 60s, in flow cell 1 & 2.

[0397] 2. Experimental Results: The obtained experimental data were analyzed by dynamic fitting using Biacore Insight Evaluation Software 3.0.12. The results are shown in Figures 6A-6D and Tables 11-12.

[0398] Table 11: Affinity of anti-RANKL terminus of bifunctional molecules 0059, 0060 and 0061 to sRANKL

[0399] Conclusion: The tested molecules 0059, 0060, and 0061 exhibited affinity for sRANKL comparable to that of anti-RANKL positive molecules. Furthermore, the affinity for the sRANKL terminus was not affected by the length of the PTH-side linker.

[0400] Table 12: Affinity of the 0101 anti-RANKL terminus of bifunctional molecules to sRANKL

[0401] Conclusion: The bifunctional molecule 0101 has an affinity for sRANKL comparable to that of anti-RANKL positive molecules.

[0402] Example 4: Bifunctional Molecular Efficacy Experiment 1

[0403] The efficacy experiment was conducted using a bilateral oophorectomy (OVX) mouse model, and the abbreviations involved are shown in Table 13.

[0404] Table 13: List of Abbreviations

[0405] Basic information about the animal experiments is shown in Table 14.

[0406] Table 14: Basic Information

[0407] (1) Experimental method:

[0408] Thirty animals were included, with 5 undergoing sham surgery and 25 undergoing bilateral ovariectomy to establish the model. At week 5 post-surgery, the tibias of all live animals were scanned using Micro-CT, and bone mineral density (BMD) was analyzed at the scanned sites. Based on the tibial BMD values ​​at week 5 post-surgery, animals were enrolled at week 6 post-surgery to ensure consistent BMD values ​​across groups. The specific groupings were as follows: G1 (sham surgery group) consisted of 5 animals; G2-G3 each consisted of 5 animals, with G2 serving as the negative control group (model group); G3 was treated with a positive molecule (10 mg / kg); G4 was treated with a positive molecule (10 mg / kg) + PTH 1-34 (0.1 mg / kg); and G5 was treated with 0061 (10 mg / kg).

[0409] From the day of enrollment, positive molecule (10 mg / kg) and 0061 (10 mg / kg) were administered once a week for a total of 4 times. PTH 1-34 (0.1 mg / kg) was administered once daily for a total of 28 times.

[0410] At the end of the experiment (10 weeks post-operation), the uterus of groups G1-G5 was harvested and weighed only. The left femur and left tibia of groups G1-G5 were harvested for ex vivo CT scans (analytical parameters were: BMD, Tb.BMD, Tb.TMD, BV / TV, Tb.Th, Tb.Sp, Conn.D, Ct.BV, Ct.Th, Tb.BS / TV of the left femur; BMD, Tb.BMD, Tb.TMD, BV / TV, Tb.Th, Ct.BV, Ct.Th, Tb.BS / TV of the left tibia).

[0411] (2) Experimental results:

[0412] After modeling, the weight of animals in the ovariectomy group was significantly higher than that in the sham group (Figure 8). Five weeks post-surgery, the tibial BMD value of the surgical group was significantly lower than that of the sham group (Figure 9), indicating that the animals had progressed to osteoporosis.

[0413] Among all the indicators, the lower the trabecular separation (Tb.Sp) value, the better the effect; for the other indicators, the higher the value, the better the effect.

[0414] Enrollment and drug administration began 6 weeks post-surgery. Four weeks after administration (the experimental endpoint), CT scans showed that 0061, positive molecule, and the combination of positive molecule and PTH1-34 all improved tibial and femoral structure and bone mass parameters. Furthermore, 0061 was more effective than the positive molecule and the combination of positive molecule and PTH1-34 (see Figures 10A-10B: femoral bone mass and bone structure parameters; Figures 11A-11B: tibial bone mass and bone structure parameters).

[0415] Results analysis:

[0416] Compared with the Sham group (sham surgery group), the surgical control group showed a decreasing trend in BV / TV, indicating that bone synthesis in the trabeculae was less than decomposition, the number of trabeculae was significantly reduced, and BMD was significantly reduced, indicating that bone density in the medullary cavity was reduced, suggesting that osteoporosis occurred in the model group.

[0417] Compared with the model group, the bone mineral density of the positive molecule, the positive molecule combined with PTH1-34, and the 0061 administration group all increased, indicating that the drugs can increase bone mass and improve osteoporosis. Moreover, in terms of bone mass improvement, the combination therapy is better than the positive molecule alone, and the efficacy of 0061 is better than that of the positive molecule combined with PTH1-34.

[0418] Furthermore, 0061 can significantly improve various bone structure parameters, and its efficacy is significantly better than that of positive molecules combined with PTH1-34. For example, 0061 molecules can significantly increase the number of trabeculae, increase the thickness of cancellous bone and cortical bone, decrease trabecular separation, increase the number of trabecular connections, increase the surface area of ​​cancellous bone, and increase the volume of cortical bone, indicating that 0061 molecule treatment can not only increase bone mass but also improve bone structure, thereby achieving the effect of improving osteoporosis.

[0419] Example 5: Bifunctional Molecular Efficacy Experiment 2

[0420] Basic information about the animal experiments is shown in Table 15.

[0421] Table 15: Basic Information

[0422] (1) Experimental method:

[0423] Eighty animals were included, with 6 undergoing sham surgery and 74 undergoing bilateral ovariectomy to establish the model. At week 5 post-surgery, all live animals were scanned using Micro-CT to analyze bone mineral density (BMD) in the tibia. Based on the tibial BMD values ​​at week 5, animals were enrolled at week 6 post-surgery to ensure consistent BMD values ​​across groups. The specific groupings were as follows: G1 (sham surgery group) consisted of 6 animals; G2–G9 each consisted of 6 animals. G2 served as the negative control group (model group); G3 served as the positive control (10 mg / kg); G4–G5 served as the 0061 (3 mg / kg, 10 mg / kg) treatment group; G6–G7 served as the 0059 (3 mg / kg, 10 mg / kg) treatment group; and G8–G9 served as the 0101 (3 mg / kg, 10 mg / kg) treatment group.

[0424] Students were enrolled 6 weeks post-surgery, and received medication on the day of enrollment, once a week for a total of 4 weeks. Five days prior to enrollment, 160 μL of non-anticoagulated blood was collected. 24 hours and 72 hours after medication, 160 μL of non-anticoagulated blood was collected again. After each blood collection, the supernatant (at least 80 μL) was collected by centrifugation, and aspartic acid (AST), creatinine (CREA), urea (UREA), and serum calcium were measured using a biochemical analyzer.

[0425] The experiment ended 10 weeks post-surgery. 500-800 μL of non-anticoagulated blood was collected from the hearts of all animals. After centrifugation, the supernatant (at least 250 μL) was collected, and 80 μL of serum was taken. Aspartic acid (AST), creatinine (CREA), urea (UREA), and serum calcium were measured using a biochemical analyzer. The remaining serum was used to detect osteocalcin (Immutopic 60-1305 24960) and TRACP-5b (Immunodiagnostic Systems SB-TR103 J56372) using ELISA.

[0426] The left femur, left tibia, right tibia, lumbar vertebrae 1-5, and ilium were harvested, and the muscles were removed and fixed with formalin. The right tibia was used for HE detection to analyze the number of trabeculae, while the left femur and left tibia were used for ex vivo CT scans (analysis parameters included: left femur BMD, Tb.BMD, Tb.TMD, Tb.BV / TV, Tb.N, Tb.Th, Ct.Th, Tb.Sp, Ct.BV, Tb.BS / TV; left tibia BMD, Tb.BMD, Tb.TMD, Tb.BV / TV, Tb.Th, Conn.D, Tb.Sp).

[0427] The right femur (muscle removed) was wrapped in gauze soaked in pre-cooled saline and subjected to three-point biomechanical testing.

[0428] The uterus was removed and weighed only.

[0429] (2) Experimental results:

[0430] Among all the indicators, a lower trabecular separation (Tb.Sp) value indicates better results. For the other indicators, higher values ​​indicate better results.

[0431] After modeling, the weight of animals in the ovariectomy group was significantly higher than that in the sham surgery group (Figure 12).

[0432] Five weeks post-surgery, the tibial BMD value of the surgical group was significantly lower than that of the sham-operated group (Figure 13), indicating that the animals had progressed to osteoporosis.

[0433] After grouping, the animals in each group were weighed twice a week. The weight of the animals in the surgical group was higher than that of the sham-operated group (Figure 14).

[0434] Ten weeks after surgery (four weeks of drug administration), the uterus was removed and weighed. The uterine weight of mice in each OVX treatment group was significantly reduced, indicating that after ovariectomy, estrogen levels decreased and the uterus atrophied (Figure 15).

[0435] Five weeks post-surgery, compared with the Sham group (sham surgery group), the tibial BMD of the model group animals was significantly reduced. Drug administration began six weeks post-surgery, and four weeks after administration (the experimental endpoint), CT scans showed that 0061, 0059, and 0101 could improve the tibial and femoral structure and bone mass parameters, and their effects were superior to those of positive molecules (see Figures 16-19).

[0436] Compared with the Sham group (sham surgery group), the model group showed a decreasing trend in Tb.BV / TV, indicating that bone synthesis in the trabeculae was less than bone breakdown, the number of trabeculae was significantly reduced, and BMD was significantly decreased, indicating decreased bone density in the medullary cavity, suggesting osteoporosis in the model group. Compared with the model group, the BMD in the positive control drug group was significantly increased, indicating that the positive control drug could increase bone mass and improve osteoporosis.

[0437] In the 0059, 0061, and 0101 treatment groups, tibial and femoral bone mass parameters, such as Tb.BMD, Tb.TMD, and BMD, were significantly increased, indicating that 0059, 0061, and 0101 can all increase bone mass. At a dose of 3 mpk, the three test molecules showed significantly better bone mass enhancement than the positive molecule at a dose of 10 mpk. Furthermore, the bone structure of the tibia and femur was also significantly improved in the 0059, 0061, and 0101 treatment groups. The test molecules were significantly more effective than the positive molecule at 10 mpk in increasing the number of trabeculae, increasing trabecular and cortical bone thickness, and reducing intertrabecular separation. This indicates that the three test molecules, 0059, 0061, and 0101, are superior to the positive molecule at 10 mpk in both bone mass enhancement and bone structure improvement. This further demonstrates that the 0059, 0061, and 0101 molecules are significantly more effective than the positive molecule at 10 mpk in improving osteoporosis.

[0438] The three-point mechanical analysis results showed that the tested molecule could significantly increase the toughness and strength of bone, and the effect was better than that of the positive molecule (anti-RANKL positive control antibody) (see Figure 20).

[0439] HE staining analysis of bone sections showed that positive molecules and 0061, 0059, and 0101 could increase the number of trabeculae (Fig. 21A-Fig. 21G).

[0440] Serum osteocalcin (OC) and serum TRACP-5b (tartrate-resistant acid phosphatase 5b) are indicators related to bone synthesis and resorption, respectively. Four weeks after administration of the positive molecule (at the end of the experiment), serum TRACP-5b levels decreased, suggesting inhibition of bone resorption (Figure 22). 0061, 0059, and 0101 increased serum osteocalcin concentration (Figure 23) and decreased TRACP-5b concentration (Figure 22), suggesting increased bone synthesis and inhibition of bone resorption. Furthermore, at the experimental endpoint, the blood biochemical analysis results of the positive molecule, as well as 0059, 0061, and 0101, showed that serum aspartate aminotransferase (AST), creatinine (CREA), urea (UREA), and serum calcium (Ca) were all within physiological ranges (Figures 24A-24B), indicating good safety of the tested molecules.

[0441] Example 6: Drugability Assessment of Bifunctional Molecules

[0442] This experiment tested the high-temperature stability and freeze-thaw stability of bifunctional molecules.

[0443] 1. Instruments and equipment used:

[0444] Environmental testing chamber: Memmert HPPeco110;

[0445] Ultra-low temperature freezer: Alphavita medical ultra-low temperature storage box;

[0446] High-performance liquid chromatograph: Waters e2695 high-performance liquid chromatograph;

[0447] Thermo Scientific Vanquish;

[0448] Mass spectrometer: Thermo Orbitrap Exploris 240;

[0449] Column: Acquity UPLC Peptide CSH C18 1.7μm, 2.1 x 150mm (Manufacturer: Waters); BioResolve SEC mAb Column 2.5μm, 7.8 x 300mm (Manufacturer: Waters);

[0450] Proteases: Lys-C enzyme (manufacturer: Wako, catalog number: 121-05061), Trypsin enzyme (manufacturer: Promega, catalog number: V511C);

[0451] Capillary electrophoresis apparatus: SCIEX PA800 plus.

[0452] 2. Experimental Methods:

[0453] (1) High temperature stability test:

[0454] Test conditions: Temperature 40℃, humidity 75%.

[0455] Purity was then analyzed by size exclusion chromatography (SEC-HPLC), and purity and stability were detected by capillary electrophoresis (CE-SDS) and post-translational modification detection-peptide mapping-reversed-phase tandem mass spectrometry (chromatography-mass spectrometry).

[0456] (2) Freeze-thaw stability test:

[0457] Test conditions: six freeze-thaw cycles.

[0458] The purity was then determined by SEC-HPLC and CE-SDS.

[0459] To improve the drug-likeness and stability of the bifunctional molecule constructed in this application, the PTH terminus was optimized. Since deamination of asparagine may affect protein stability, this patent investigates the effect of three asparagine residues (N1O, N16, and N33) at the PTH terminus on peptide stability, specifically examining their deamination behavior in an accelerated assay at 40°C.

[0460] Liquid chromatography-mass spectrometry was used to detect the T0 (start point) and accelerated at 40℃ for 4 weeks (40℃-4W) to determine the proportion of molecules in the bifunctional molecule that underwent deamination at the corresponding amino acid site. The results are shown in Table 16, where the values ​​represent the proportion of molecules in the bifunctional molecule that underwent deamination at the corresponding amino acid site.

[0461] Table 16: Accelerated Tests at 40℃ for 0060, 0101, 0124, and 0135

[0462] N / A indicates that no relevant experimental data was detected.

[0463] As shown in Table 16, among the three asparagine sites N10, N16, and N33, the deamination rates at N10 and N33 are relatively low, while the deamination rate at N16 is relatively high. Therefore, mutating the asparagine at the N16 position is expected to significantly reduce the deamination reaction of the peptide and improve its stability. Therefore, the N16 position was subsequently modified.

[0464] The N16 deamination reaction was not detected at the N16 position in the 0124 molecule obtained by N16G mutation based on the 0060 molecule, and the N16S mutation obtained by N16S mutation based on the 0101 molecule (see the specific sequence in Example 7 below). The deamination rate of the entire polypeptide molecule was greatly reduced.

[0465] After being placed at 40°C for 4 hours, 0124 showed no increase in polymers as detected by size exclusion chromatography (SEC), but a 0.9% increase in fragments as detected by non-reducing CE-SDS. For 0135, after being placed at 40°C for 4 hours, SEC showed a 0.2% increase in polymers, and non-reducing CE-SDS showed a 1.4% increase in fragments. The relatively small or no increase in polymers and / or fragments indicates that 0124 and 0135 are not prone to forming polymers or breaking into fragments at 40°C, demonstrating their high stability.

[0466] After six freeze-thaw cycles, the purity of the two molecules showed no significant change.

[0467] The results above show that the high-temperature stability of the mutated molecule is significantly improved, while it also has extremely high freeze-thaw stability.

[0468] Example 7: Bifunctional Molecule Modification

[0469] 0060 and 0101 were selected as parental molecules for modification. While removing the deamination site, the activity of the PTH terminus was adjusted by different point mutations at key sites. Based on molecule 0060, molecules 0117 (S17E), 0123 (N16E), 0124 (N16G), and 0126 (N16S) were obtained. Based on molecule 0101, candidate molecules 0132 (N16E&F34A&V35S) and 0135 (N16S&F34A&V35S) were obtained. The sequence composition of the modified bifunctional molecules is shown in Table 17 below.

[0470] Table 17: Structural / Sequence Information of Modified Bifunctional Molecules

[0471] Example 8: Activation activity of the modified molecule on PTH1R in Saos-2 and RANKL-Saos-2 dual-positive cell lines

[0472] The experimental procedures were as described in Example 1. The activation activity of the modified molecule on PTH1R in the Saos-2 cell line is shown in Figures 25A-25B and Table 18. The activation activity of PTH1R on the RANKL-Saos-2 double-positive cell line is shown in Figures 26A-26B and Table 18.

[0473] Table 18: Activation activity of the modified bifunctional molecule in Saos-2 and RANKL-Saos-2 cells

[0474] As shown in Figures 25-26 and Table 18, the deamination-modified mutant molecules 0126 (N16S), 0123 (N16E), 0124 (N16G), and 0135 (N16S&F34A&V35S) of N16 all exhibited PTH1R activation activity in both Saos-2 and RANKL-Saos-2 double-positive cell lines. Compared to the activation activity of the tested molecules on PTH1R in Saos-2 cells, the activation activity of 0126 (N16S), 0123 (N16E), 0124 (N16G), and 0135 (N16S&F34A&V35S) in RANKL-Saos-2 double-positive cells was significantly increased, at least 10-fold, indicating that the modified molecules have good bone tissue-targeting activation activity.

[0475] Example 9: Detection of the cross-activity of the modified bifunctional molecule with mPTH1R

[0476] The activation activity of bifunctional molecules on PTH1R in 293-mouse PTH1R-overexpressing cell lines was detected using the cAMP-Gs Hirange Kit (Cisbio, 62AM6PEC).

[0477] (1) Cell preparation: After digestion, the cells were centrifuged, resuspended in cell dilution buffer (0.5% BSA in DMEM + 0.5mM IBMX) and counted. The cell viability was >90%. The cell density was adjusted to 4×10⁶ cells / year using cell dilution buffer. 5 / ml.

[0478] (2) Experimental steps:

[0479] 1) Add 5 μl of test sample to a 384-well plate and centrifuge for 1 minute. Add 5 μl of cells from cell dilution buffer to the experimental plate, bringing the final cell density to 2000 cells / well. Centrifuge for 1 minute.

[0480] 2) Incubate at 37°C for 30 minutes to allow cells to fully bind to the test substances. Dilute cAMP-d2 and Anti-cAMP-Cryptate using the lysis and detection buffer provided in the kit, add 5 μl of the diluted cAMP-d2 and Anti-cAMP-Cryptate to the experimental plate respectively, and centrifuge for 1 minute;

[0481] 3) Incubate at room temperature for 60 minutes. Read the results using a multi-functional microplate reader (PE, Envisio).

[0482] (3) Experimental results: The data were processed using Graphpad, and the results are shown in Figure 27 and Table 19.

[0483] Table 19: Cross-activity of the modified molecule with mPTH1R

[0484] The tested molecule exhibited good cross-activity with mouse PTH1R. The modified molecule showed similar activity on mouse PTH1R as it did on Saos-2 cells, both demonstrating activation activity against mouse PTH1R.

[0485] Example 10: Detection of the effect of the modified molecule on the recruitment signal of PTH1Rβ-arrestin

[0486] Activation of PTH1R affects multiple downstream signaling pathways, increasing bone metabolism through the cAMP / PKA pathway, promoting both bone formation and bone resorption. It also inhibits RANKL expression through the β-arrestin (cAMP delayed signaling pathway), thereby suppressing osteoclast differentiation. The regulatory role of the candidate molecule in bone metabolism was further verified by examining its effect on β-arrestin recruitment in the HEK293T-human PTH1R (Cat#GS003-Hu-PTH1R, Lot#20231107) cell line.

[0487] (1) Experimental method:

[0488] 1) Day 0

[0489] Cell culture plate preparation: 96-well opaque white-walled plates (corning 3917#), 0.1 mg / mL poly-L-lysine, diluted 10 times with PBS, 100 μL / well, incubated at 4 degrees Celsius for at least 4 hours.

[0490] Cell culture medium preparation: Take 500 mL of DMEM basal medium and add 55 mL of fetal bovine serum.

[0491] Cell seeding: Before seeding, remove poly-L-lysine residues, rinse once with 100 μl PBS, and air dry at room temperature; Digest 293T-Human PTH1R cells with 0.05% trypsin at 37°C for 2 min, neutralize with 2 volumes of complete culture medium, centrifuge at 1000 rpm at 25°C for 5 min, resuspend cells in culture medium, stain with trypan blue and count cells. Cell density was 3.97E6 / mL, viability 98%. Adjust cell density to 8 × 10⁶ cells / mL using culture medium. 5 Add cells to 96-well opaque white-walled plates at 100 μL / well according to the plate map, and incubate overnight in a cell culture incubator (37°C, 5% CO2) to allow them to adhere.

[0492] 2) Day 1

[0493] Sample preparation and dilution: The samples were diluted with 1X stimulation buffer and set aside.

[0494] Sample incubation: Remove the 96-well white-walled permeable plate, remove 50 μL of cell culture medium, add the diluted samples of each concentration to the 96-well plate, 50 μL / well, cover and seal the plate, and incubate at room temperature for 30 min.

[0495] Cell fixation: Remove 70 μL of compound solution, then add 30 μL of 1X Stabilization buffer and incubate at room temperature for 15 min;

[0496] Cleaning: Remove liquid (60 μL) from the wells, then wash three times with 1X wash buffer at 100 μL / well, and then remove all liquid.

[0497] d2+Eu incubation: Dilute 50X d2 and 50X Eu with 1X detection buffer and mix them 1:1. Add 100μL / well to a 96-well white-walled bottom plate, cover with a sealing film, and incubate at room temperature in the dark for 24h.

[0498] 3) Day 2

[0499] Reading: After 24 hours of incubation, Envision reads the values ​​of two wavelengths, 665nm and 620nm, from the HTRF module.

[0500] (3) Experimental results: Graphpad was used to process the data. The results are shown in Figure 28 and Table 20.

[0501] Table 20: PTH1Rβ-arrestin recruitment of the modified bifunctional fusion protein

[0502] Compared to the negative control, 0060, 0123, 0124, 0126, 0135, and 0132 all exhibited activation activity against human PTH1R, and could activate the PTH1R β-arrestin recruitment signaling pathway.

[0503] Example 11: Using SPR to detect the affinity between the anti-RANKL end and sRANKL of the modified molecule.

[0504] The molecules to be tested were 0060, 0123, 0124, 0126, 0101, 0132, and 0135. The experimental steps were the same as those described in Example 4. The results are shown in Figure 29 and Table 21.

[0505] Table 21: Affinity of the anti-RANKL terminus of the modified molecule to sRANKL

[0506] Conclusion: The affinity of the test molecule for sRANKL was not significantly different from that for anti-RANKL positive antibodies.

[0507] Example 12: Detection of the neutralizing activity of the anti-RANKL terminus of the modified molecule against sRANKL

[0508] The molecules to be tested were 0124 and 0135. The experimental steps were as described in Example 3, and the results are shown in Figure 30 and Table 22 below.

[0509] Table 22: Inhibitory activity of the modified molecule against sRANKL-induced differentiation of RAW264.7 cells into osteoclasts

[0510] The results showed that the modified molecules 0124 and 0135 had comparable inhibitory activity against sRANKL-induced differentiation of RAW264.7 cells into osteoclasts to positive molecules.

[0511] Example 13: Molecular Efficacy Experiment 1 after Modification

[0512] (1) Experimental method:

[0513] The basic information for the animal experiments was consistent with that in Examples 5 and 6. 54 animals were used; 6 underwent sham surgery, and 25 underwent bilateral ovariectomy to establish the model. Animals were grouped according to their weight at week 5 post-surgery to ensure consistent weight across groups (Table 23). The specific groupings were as follows: G1 (sham surgery group) consisted of 6 animals; G2–G9 each consisted of 6 animals. G2 served as the negative control group (model group); G3 was treated with 0060 (3 mg / kg); G4 with 0117 (3 mg / kg); G5 with 0123 (3 mg / kg); G6 with 0124 (3 mg / kg); G7 with 0126 (3 mg / kg); G8 with 0132 (3 mg / kg); and G9 with 0135 (3 mg / kg).

[0514] Table 23: Grouping Information

[0515] Students were enrolled 5 weeks post-surgery, and received medication on the day of enrollment, once a week for a total of 4 weeks. Five days prior to enrollment, and 48 hours after each dose (days 2, 9, 16, and 23 after enrollment), 200 μL of non-anticoagulated blood was collected. After each blood collection, the supernatant (at least 80 μL) was collected by centrifugation, and aspartic acid (AST), creatinine (CREA), urea (UREA), and serum calcium were measured using a biochemical analyzer.

[0516] The experiment ended 9 weeks after the surgery.

[0517] 1) Collect 500-800 μL of non-anticoagulated blood from the hearts of all animals, centrifuge, and take the supernatant (at least 250 μL). Take 80 μL of serum and use a biochemical analyzer to detect aspartic acid (AST), creatinine (CREA), urea (UREA), and blood calcium.

[0518] 2) Harvest the left femur, left tibia, right tibia, lumbar vertebrae 1-5, and ilium, removing muscle and fixing with formalin. The right tibia is used for HE analysis to determine the number of trabeculae, and the left femur is used for ex vivo CT scans (analysis parameters include left femoral BMD, Tb.BMD, Tb.TMD, Tb.Th, Ct.Th, BV / TV, Tb.Sp, Conn.D, Tb.N, Ct.BV, and Tb.BS / TV). Among these parameters, a lower trabecular separation (Tb.Sp) value indicates better results. Higher values ​​for the other parameters indicate better results.

[0519] 3) Take the right femur (muscle removed), wrap it with gauze soaked in pre-cooled saline, and perform three-point biomechanical testing.

[0520] 4) Remove the uterus and weigh it only.

[0521] (2) Experimental results:

[0522] During the administration process, all test molecules showed a temporary increase in serum calcium 48 hours after administration. Blood biochemistry tests at the experimental endpoint showed that serum calcium returned to normal levels. Other biochemical indicators, such as aspartate aminotransferase (AST), creatinine, and urea, were all within the physiological range (Figures 31A-31D).

[0523] The experimental endpoint was achieved through CT scans of the femur outside the body. Compared with the Sham group (sham surgery group), the surgical control group showed a decreasing trend in Tb.BV / TV, indicating that bone synthesis in the trabeculae was less than decomposition, the number of trabeculae was significantly reduced, and BMD was significantly reduced, indicating that bone density in the medullary cavity was reduced, suggesting that osteoporosis occurred in the model group.

[0524] Compared with the model group, molecules 0060, 0117, 0123, 0124, 0126, 0132, and 0135 significantly improved various bone structure parameters (BV / TV, Tb.sp, Tb.Th, Tb.N, Conn.D, Ct.Th, Ct.BV, Tb.BS / TV) and bone mass parameters (Tb.BMD, Tb.TMD, BMD), indicating that drug treatment can increase bone mass and improve osteoporosis. Specifically, in terms of increasing bone mass and improving bone structure, at the same dose, molecules 0117, 0123, 0124, and 0135 were more effective than molecules 0126 and 0132. In terms of improving bone mineral density, cortical bone thickness and volume, bone volume fraction, and trabecular bone connectivity density, the modified molecules achieved comparable or better results than the unmodified molecules (Figures 32A-32C).

[0525] Three-point biomechanical analysis of the femur showed that the Sham group had higher stiffness and toughness than the model group, while the drug-treated group showed significant improvements in the maximum load and fracture load of the bone, suggesting that the test molecule can improve the biomechanics of bone (Figure 33).

[0526] Example 14: Pharmacodynamic Experiment Based on Molecular Weight Effect Relationship after Modification

[0527] (1) Experimental method:

[0528] Seventy animals were purchased and divided into two groups, GA and GB, according to their weight. Six animals in the GA group underwent sham surgery, and 64 animals in the GB group underwent bilateral ovariectomy to create a model.

[0529] Animals were enrolled and administered medication at week 5 post-surgery. The specific groupings were as follows: the GA group was G1, and the GB group was evenly distributed among groups G2-G9 according to body weight. Group G1 (sham surgery group) consisted of 6 animals, and groups G2-G9 each consisted of 6 animals. G2 served as the negative control group (the modeling group received solvent), group G3 was treated with anti-RANKL positive molecules (10 mg / kg), groups G4-G6 were treated with 0124 (0.3 mg / kg, 1 mg / kg, 3 mg / kg), and groups G7-G9 were treated with 0135 (0.3 mg / kg, 1 mg / kg, 3 mg / kg).

[0530] The medication was administered on the day of enrollment and once a week for a total of four weeks.

[0531] Blood collection: 200 μL of non-anticoagulated blood was collected 5 days before enrollment and 2, 9, 16 and 23 days after enrollment and drug administration. After each blood collection, the supernatant (at least 80 μL) was centrifuged and used for blood calcium and phosphorus detection.

[0532] The experiment ended 9 weeks after the surgery.

[0533] 1) Blood collection: On the day of the experiment, collect non-anticoagulated blood from the heart, collecting as much as possible (>800μL). Centrifuge and collect the supernatant (>300μL), aliquoting it into two tubes for storage. One tube of serum (at least 80μL) will be used for serum calcium (Ca) and serum phosphorus (P) detection (blood biochemistry). The remaining serum will be used for Osteocalcin and TRAcP-5B detection (ELISA).

[0534] 2) Ex vivo femoral CT: The left femur was harvested, the muscle was removed and fixed in formalin, and an ex vivo CT scan was performed (analysis parameters were left femoral Tb.N, BV / TV, Tb.Th, Tb.SP, Tb.BMD, Ct.BV, Ct.Th, Tb.BS / TV, Tb.TMD).

[0535] 3) Femoral biomechanics test: Take a piece of the right femur (muscle removed), wrap it in gauze and soak it in pre-cooled saline solution for bone biomechanics test.

[0536] 4) Ex vivo CT scan of iliac bone and L5-lumbar vertebrae: Iliac bones and vertebrae from groups G1-G3 and G7-G9 were removed, muscles were removed and fixed with formalin, and ex vivo CT scans were performed (iliac bone analysis parameters were Tb.TMD, Tb.BMD, Tb.BMC (bone mineral content), Tb.BV / TV, Tb.Th; L5-lumbar vertebra analysis parameters were Tb.TMD, Tb.TMC, Tb.BMD, Tb.BMC, Tb.BV / TV, Tb.Th).

[0537] (2) Experimental Results

[0538] During administration, O124 and O135 molecules showed a transient increase in serum calcium 48 hours after administration. At doses of 1 mg / kg and 0.3 mg / kg, the fluctuations in serum calcium were within the physiological range for mice, while changes in serum phosphorus showed no statistically significant difference. End-point blood biochemistry analysis indicated that serum calcium returned to normal levels (Figure 34).

[0539] The experimental endpoint was the femoral CT results (Figures 35A-35B): Compared with the Sham group (sham surgery group), the surgical control group showed a decreasing trend in Tb.BS / TV, indicating that bone synthesis in the trabeculae was less than decomposition, the number of trabeculae was reduced, and BMD was reduced, indicating that bone density in the medullary cavity was reduced, suggesting that osteoporosis occurred in the model group.

[0540] Compared with the model group, positive molecule, 0124 molecule, and 0135 molecule improved bone structure and bone mass parameters (Tb.BV / TV, Tb.Th, Tb.N, Tb.BS / TV, Tb.BMD, Tb.TMD, Tb.SP, Ct.Th, Ct.BV) of femoral cancellous and cortical bone in a dose-dependent manner. This indicates that treatment with positive molecule, 0124, and 0135 molecule can increase bone mass and improve osteoporosis, and 0124 and 0135 molecules are more effective than the 10 mpk positive molecule. In terms of increasing bone mass and improving bone structure, 0135 molecule is slightly more effective than 0124 molecule.

[0541] Three-point biomechanical analysis of the femur showed that the Sham group had higher bone stiffness and toughness than the model group. The drug-treated group showed improvements in maximum load and fracture load, suggesting that positive molecules, O124 molecules, and O135 molecules can improve bone biomechanics. This indicates that O124 and O135 molecule treatment can increase bone mass in the iliac and vertebrae, improving osteoporosis. Among these, O124 and O135 molecules showed superior improvement in bone mechanics compared to the positive molecule. Furthermore, the mechanical improvement of O135 molecules was superior to that of O124 molecules (Figure 36).

[0542] The results of CT scans of the iliac bone and vertebrae in the experimental groups (G1, G2, G3, G7, G8, G9) were as follows (Figures 37-38): Compared with the Sham group (sham surgery group), the surgical control group showed a decreasing trend in Tb.BV / TV, indicating that bone synthesis in the trabeculae was less than decomposition. The decreased BMD indicated that the bone density in the medullary cavity was reduced, suggesting that osteoporosis occurred in the model group.

[0543] Compared with the model group, the positive molecule and the 0135 molecule improved bone structure and bone mass parameters (Tb.BV / TV, Tb.Th, Tb.BMD, Tb.BMC, Tb.TMD, Tb.TMC) of the iliac bone and vertebrae in a dose-dependent manner. Furthermore, the 0135 molecule was more effective than the 10 mpk positive molecule.

[0544] The serum ELISA results at the experimental endpoint showed that 0124 and 0135 molecules increased serum osteocalcin concentration in a dose-dependent manner after 4 weeks of administration (end of the experiment), suggesting that 0124 and 0135 molecules can promote bone formation. The positive molecule group showed a decrease in serum osteocalcin concentration, indicating that the positive molecule could not promote bone formation (Figure 39A). After 4 weeks of administration, the positive molecule (10 mpk), 0124 molecule (3 mpk), and 0135 molecule (3 mpk) all reduced the levels of bone resorption markers in mouse serum, indicating decreased bone resorption, with 0135 molecule showing a better effect than 0124 molecule (Figure 39B).

[0545] Example 15: Pharmacodynamic evaluation of the modified molecule after a single dose in an OVX mouse model

[0546] (1) Experimental methods

[0547] Thirty-five animals were purchased and divided into two groups, GA and GB, according to their weight. Six animals in the GA group underwent sham surgery, while 29 animals in the GB group underwent bilateral ovariectomy to create a model. The day of the surgery was recorded as Day 0.

[0548] Animals were enrolled and administered medication at week 5 post-surgery. The specific groupings were as follows: the GA group was group G1, and the GB group was evenly distributed among groups G2-G6 according to body weight. Group G1 (sham surgery group) consisted of 6 animals, and each of groups G2-G6 consisted of 6 animals. Animals remaining after enrollment served as replacements. Group G2 was the negative control group (the modeling group received the solvent), groups G3-G4 were the positive molecule (1 mg / kg) treatment groups, and groups G5-G6 were the O135 (1 mg / kg) treatment groups.

[0549] The patient was given a single dose on the day of enrollment and observed for 4 weeks after administration.

[0550] Blood samples were collected 5 days before enrollment (5 weeks post-surgery) and 2, 7, 14, 21 and 28 days after drug administration.

[0551] Three animals were randomly selected from group G1, three animals were randomly selected from group G2, and 200 μL of blood (non-anticoagulated blood) was collected from groups G3 and G5. After centrifugation, the supernatant was collected and the serum osteocalcin content was detected by ELISA.

[0552] The remaining 3 animals in group G1, the remaining 3 animals in group G2, and the remaining 200 μL of blood (non-anticoagulated blood) were collected from groups G4 and G6. After centrifugation, the supernatant was collected and the serum TRACP-5b content was detected by ELISA.

[0553] Three mice randomly selected from groups G1 and G2, as well as groups G3 and G5, underwent in vivo left femoral CT scans at weeks 7 and 9 post-surgery to analyze femoral bone mineral density.

[0554] Three mice from the remaining groups G1 and G2, as well as mice from groups G4 and G6, underwent in vivo left femoral CT scans at weeks 6 and 8 post-surgery to analyze femoral bone mineral density.

[0555] (2) Experimental Results

[0556] In vivo CT results at different time points after drug administration (Figure 40) showed that compared with the sham surgery group, the TMD in the surgical control group showed a decreasing trend, indicating that bone synthesis was less than decomposition, suggesting that osteoporosis occurred in the model group.

[0557] Compared to the model group, one week after administration, both the positive molecule and the O135 molecule increased bone mass in the test groups after a single dose, and at the same dose, the O135 molecule showed a greater increase in bone mineral density than the positive control group. With prolonged administration, the efficacy of the drugs gradually weakened, and bone mineral density began to decrease. Compared to the O135 molecule, the decrease in bone mineral density was more significant in the positive molecule group. Four weeks after administration, the bone mineral density of the positive molecule group was lower than that of the surgical control group, while the bone mineral density of the O135 molecule group remained higher than that of the surgical control group. This suggests that at the same dose, the efficacy of the O135 molecule lasts longer than that of the positive molecule after a single dose.

[0558] During the experiment, serum bone turnover marker detection results showed (Figures 41A-41B) that in the 0135 molecule group, the bone formation marker (osteocalcin) (Figure 41A) experienced a brief increase on day 7 after administration, followed by a decline, while the bone formation marker in the positive molecule group decreased rapidly after administration. On days 2 and 7 after administration, the bone resorption marker (TRACP-5b) in both the 0135 molecule test group and the positive molecule group decreased rapidly, gradually recovering to baseline levels after 4 weeks of administration (Figure 41B). This suggests that the 0135 molecule can both promote bone formation and inhibit bone resorption, while the positive molecule mainly reduces bone resorption.

[0559] Example 16: Comparative experiment on the efficacy of the modified molecule in an OVX mouse model compared with positive molecule, and in combination with positive molecule and PTH.

[0560] (1) Experimental methods

[0561] Eighty animals were purchased and divided into two groups, GA and GB, according to their weight. Eight animals in the GA group underwent sham surgery, and 72 animals in the GB group underwent bilateral ovariectomy to create a model. The day of the surgery was recorded as Day 0.

[0562] Drug administration began 6 weeks post-surgery, with enrollment two days prior to administration. The specific groupings were as follows: the GA group was group G1, and the GB group was evenly distributed among groups G2-G8 according to body weight. Group G1 (sham surgery group) consisted of 8 animals, G2 (model group) of 8 animals, and G3-G8 each of 10 animals. Animals remaining after enrollment served as replacements. Specifically, G2 was the negative control group (the model group received the solvent), G3 was the positive control group (10 mg / kg), G4 was the PTH 1-34 (0.1 mg / kg) treatment group, G5 was the positive control group + PTH (10 + 0.1 mg / kg) combined treatment group, G6 was the O135 (2 mg / kg) treatment group, G7 was the O135 (1 mg / kg) treatment group, and G8 was the O135 (0.5 mg / kg) treatment group.

[0563] Medication was started 6 weeks post-surgery. Patients were observed for 4 weeks after administration, and the study concluded at 10 weeks.

[0564] The grouping and dosing regimens are shown in Table 24 below.

[0565] Table 24: Grouping and Dosing Regimens

[0566] Ex vivo CT scan

[0567] During the endpoint test, the left hip bone, left femur, and left tibia of the mouse were taken for CT scan analysis.

[0568] Bone biomechanics testing

[0569] Take a piece of the right femur (muscle removed), wrap it in gauze, and soak it in pre-cooled physiological saline for mechanical testing.

[0570] (2) Data collection and statistical analysis

[0571] Raw data from measurements and observations must be recorded. Analysis should be performed based on the raw data, and results should be expressed as mean ± standard error (Mean ± SEM). Statistical analysis should also be performed on the test results, with P < 0.05 considered statistically significant. Both statistical and biological significance should be considered in the results analysis.

[0572] (3) Experimental Results

[0573] The experimental endpoints were achieved via isolated CT scans of the tibia and femur (Figures 42A-42B). Compared to the model group, positive molecule, PTH 1-34, the combination of positive molecule and PTH, and 0135 molecule improved bone structure and bone mass parameters (Tb.BMD, Tb.Th, Ct.BV, Ct.Th) in a dose-dependent manner. This indicates that treatment with positive molecule, PTH 1-34, the combination of positive molecule and PTH, and 0135 molecule can increase bone mass and improve osteoporosis. Furthermore, the efficacy of 0135 molecule at 2 mpk was superior to that of the positive molecule, PTH 1-34, and the combination of positive molecule and PTH.

[0574] Three-point biomechanical analysis of the femur showed that the sham-operated group had higher bone stiffness and toughness than the model group. The drug-treated group showed improvements in maximum bone load and maximum load energy, indicating that positive molecule, PTH 1-34, the combination of positive molecule and PTH, and 0135 molecule can all improve bone biomechanics. Among these, 0135 molecule showed better improvement in bone mechanics than the positive molecule, PTH 1-34, and the combination of positive molecule and PTH. Furthermore, 0135 molecule showed better improvement in femoral biomechanics than 0124 molecule (Figure 42D).

[0575] The endpoint CT results of the iliac bone in the experimental group (Figure 42C): Compared with the model group, positive molecule, PTH 1-34, positive molecule + PTH combination therapy, and O135 molecule improved the bone structure and bone mass parameters (Tb.BV / TV, Tb.Th, Tb.BMC, Tb.BS / TV) of the tibia in a dose-dependent manner. The efficacy of 2mpk of O135 molecule was superior to that of positive molecule, PTH 1-34, and positive molecule + PTH combination therapy groups.

[0576] Example 17: Pharmacokinetic study of the modified molecule in the OVX mouse model

[0577] (1) Experimental methods

[0578] Animal selection: Twelve female huRANKL mice (provided by Biocytogen), with an estimated weight of 18-22g and an age of 9-12 weeks, were selected. Thirty-six mice were randomly divided into two groups of six each: a 0.124 molecule group and a 0.135 molecule group. A single subcutaneous administration of 1 mg / ml was administered.

[0579] Sample collection: Baseline blood samples were collected one day before administration as 0h data; whole blood was collected via facial vein at multiple time points after administration, including 3h, 10h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, 240h, 264h, 288h, 312h, and 336h, with a collection volume of 100μL at each time point.

[0580] Animal handling: 24 hours after drug administration, mice were euthanized by inhaling carbon dioxide to ensure compliance with laboratory animal ethics requirements.

[0581] Sample processing: After whole blood collection, immediately transfer it to a blank centrifuge tube and store it upright at room temperature for 0.5 hours. Then centrifuge at 2000 rcf centrifugation force at room temperature (RT) for 10 minutes. After centrifugation, use a pipette to aspirate the supernatant to obtain a serum sample. Store the serum sample at -40℃ until blood drug concentration analysis is performed.

[0582] The sandwich ELISA method was used to detect the samples: sRANKL (ACRO, catalog number RAL-H5240) and Goat Anti-Fab Fragment HRP conjugated antibody (Sigma, catalog number A0293) were used to detect and analyze the antibody concentration at the antibody terminus in the sample serum. sRANKL and biotin anti-PTH 1-7 antibody (sequence from patent) were also used.

[0583] US7318925B2, SEQ ID NO: 38), and SA-HRP (manufacturer: BD Pharmingen, catalog number: 554066) were used to detect and analyze the concentration of intact molecules in the sample serum.

[0584] PK parameter analysis: Using a non-compartmental model, (Version 8.2, Pharsight, Mountain View, CA) software was used to analyze the obtained plasma concentration-time data to obtain relevant PK parameters.

[0585] (2) Experimental results

[0586] Based on the results of this experiment, at a dosing dose of 1mpk: The half-life of the antibody-terminal molecules of 0124 and 0135 was significantly longer than that of the intact molecules. The Cmax and AUC inf of the antibody-terminal molecules were significantly higher than those of the intact molecules. The clearance rate of the intact molecules was higher than that of the antibody-terminal molecules. The Cmax and AUC inf of the 0135 intact molecules were significantly higher than those of the 0124 molecules (Figure 43A - 43B, Table 25).

[0587] Table 25: PK parameter analysis

[0588] Example 18: Pharmacokinetic study of the modified molecule in cynomolgus monkeys

[0589] (1) Experimental method

[0590] Female cynomolgus monkeys aged 2.4 - 4 years and weighing 2.5 - 4 kg were selected (Source of cynomolgus monkeys: Guangzhou Xiangguan Biotechnology Co., Ltd., use license: SCXK(Yue) 2023 - 0043) for single subcutaneous dosing pharmacokinetic analysis. The experimental grouping is shown in Table 26 below.

[0591] Table 26: Experimental grouping

[0592] Blood collection: 0 (before dosing), 0.5 h, 2 h, 6 h, 24 h (day 1), day 2, 4, 7, 10, 14, 17 after dosing.

[0593] Sample processing: After whole blood collection, it was immediately transferred to a blank centrifuge tube and stored upright at room temperature for 0.5 hours. Subsequently, it was centrifuged at a centrifugal force of 2000 rcf at room temperature (RT) for 10 minutes. After centrifugation, the supernatant was aspirated using a pipette to obtain serum samples, and the serum samples were frozen in a -40°C refrigerator until plasma concentration analysis. <了

[0594] The sandwich ELISA method was used to detect the samples: sRANKL (ACRO, catalog number RAL-H5240) and Goat Anti-Fab Fragment HRP conjugated antibody (Sigma, catalog number A0293) were used to detect and analyze the antibody concentration at the antibody end in the serum samples. sRANKL, SA anti-PTH 1-7 antibody, and intact molecule concentrations in the serum samples were also detected and analyzed.

[0595] PK parameter analysis: Using a non-room model, applying... The software (Version 8.2, Pharsight, Mountain View, CA) analyzes the acquired blood drug concentration-time data to derive relevant PK parameters.

[0596] (2) Experimental Results

[0597] At doses of 0.2, 1, and 3 mg / kg, the Cmax and AUC of the 0135 antibody terminal showed a linear increase, while the T1 / 2 increased with increasing dose (62.9 h, 146 h vs 265 h), and the CL were 0.812 mL / h / kg, 0.39 mL / h / kg, and 2.37 mL / h / kg, respectively. At a dose of 1 mg / kg, the Cmax of the 0135 molecule antibody terminal and the positive molecule were 10270 ng / mL vs 7140 ng / mL, the T1 / 2 was 146 h vs 235 h, and the CL was 0.39 mL / h / kg vs 0.368 mL / h / kg. Based on the results in monkeys and literature reports, the RANKL target has a significant target-mediated drug clearance effect on PK (Figure 44A, Table 27).

[0598] The half-life of the 0135 antibody terminus (63-265 h) is significantly longer than that of the intact molecule (35.6-56.4 h) (Figures 44A-44B, Table 27).

[0599] Table 27: PK Parameter Analysis

[0600] Example 19: Pharmacological evaluation of the modified molecule in a mouse model of closed femoral fracture

[0601] (1) Experimental methods

[0602] Thirty-eight huRANKL mice were procured, with 14 reserved. The experimental procedures designed for animal use in this study were approved by Pengli Biotechnology's IACUC (Laboratory Animal Management and Use Committee) before implementation. After arriving at Pengli, the animals underwent an acclimatization period of at least three days. Following the completion of the acclimatization period, all 38 mice underwent surgery.

[0603] Preoperatively, each animal will be anesthetized with 1.5-3% isoflurane, and oxygen flow can be maintained at 0.8-1.5 liters if necessary. The right hind limb of each animal will be shaved and cleaned with 2% iodine tincture. An incision will be made around the medial patella, dislocating the patella laterally to expose the femoral condyle. A guide hole will be made in the trochlear groove of the femur using a 26G sterile needle, and the needle will be inserted through the medullary canal until it reaches the inner surface of the lesser trochanter. A sterile Kirschner wire (0.6 mm in diameter, approximately 15 mm long) will be inserted into the intramedullary canal through the guide hole. The outer end of the wire will be trimmed and pushed flush with the trochlear surface. The patella will be returned to the bone groove, and the knee will be flexed several times to ensure smooth joint patency. Postoperatively, the surgical wound will be irrigated with sterile saline. The fascia will be sutured with absorbable sutures, and the skin incision will be sutured with skin clips. After closing the skin incision, the midshaft of the nailed femur was fractured at a vertical distance of 7 cm using a three-point bending device driven by a single 200g weight. This resulted in a highly repeatable transverse fracture with minimal fragmentation and the smallest angle of the intramedullary nail.

[0604] While the animal is still under anesthesia, the fracture will be confirmed by X-ray imaging. Administer the analgesic Painkiller (10 mg / kg, IM) and the antibiotic Gentamicin (20 mg / kg, IM). During the anesthesia recovery period, the animal is allowed free movement and full weight-bearing. After recovery from anesthesia, the animal will be allowed full weight-bearing and unrestricted movement. Food and water can be placed in the cage post-surgery to ensure the animal has access to them. In addition to general health monitoring, skin healing should be monitored daily. If pain persists, all animals will continue to receive analgesia and antibiotic treatment, including but not limited to Painkiller (20 mg / kg, IM) and Gentamicin (20 mg / kg, IM), once daily for 3 consecutive days. Animals with successful surgical modeling will be randomly divided into two groups based on body weight using Bio-Book (grouping shown in Table 28 below). Each group will consist of 12 animals, and weight balance between groups must be maintained. The first administration will begin on the second day post-surgery.

[0605] Table 28: Animal grouping and dosing regimens

[0606] (2) Detection indicators

[0607] (a)Weight

[0608] Weigh and record measurements once a week (during the adaptation and treatment periods).

[0609] (b) Imaging analysis

[0610] Postoperatively (Day 0), X-ray microscopy will be performed, followed by weekly imaging to monitor the healing process. Animals will be anesthetized with 1.5-3% isoflurane during imaging.

[0611] (c)Micro-CT

[0612] After the animals were euthanized following the last administration, femoral bones from the fractured side of 5 animals in each group were harvested, fixed in 10% neutral foramin for 48 hours, and then transferred to 70% ethanol for Micro-CT scanning (Skyscan 1276 Micro-CT, Bruker microCT, Kontich, Belgium). The scanning resolution was 8 μm. After scanning, the bone was reconstructed using NRecon software (Bruker microCT, Kontich, Belgium). CTAn (Bruker microCT, Kontich, Belgium) was used to analyze vBMD (bone mineral density), BMC (bone mineral content), BV (bone volume), TV (tissue volume), and BV / TV (bone volume fraction) at the fracture site.

[0613] (d) Bone biomechanics

[0614] After the animals were euthanized at the end of the experiment, the right femur of each of the seven animals in the group was harvested at the center of the fracture callus for bone biomechanical testing. All bone specimens requiring biomechanical testing were individually wrapped in gauze, immersed in physiological saline, covered with plastic film, placed in plastic bags, and stored at -20°C for bone biomechanical testing.

[0615] Three-point bending test: During the test, the right femur stored at -20℃ was thawed at room temperature, muscles and ligaments were removed, and it was rehydrated with physiological saline. The biomechanical properties of the femur were detected and analyzed using an 858 Mini Bionix II material testing system. The test adopted the three-point bending method, with the femur placed on an MTS (Mechanical Testing & Simulation) testing machine at a loading speed of 6 mm / min. The forces are shown in Figure 45. TestWork™ software (version 4.08E) automatically recorded the load (F) and deflection (d) changes at each time point.

[0616] Biomechanical testing parameters include maximum load (N) and stiffness (N / m).

[0617] (3) Statistical Analysis

[0618] Experimental data are expressed as mean ± standard error (mean ± SD). Data were analyzed using Grapgpad with appropriate statistical methods. p < 0.05 was considered statistically significant.

[0619] (4) Experimental Results

[0620] After the animals were euthanized following the last administration, femoral bones from the fractured side of 5 animals in each group were collected, fixed in 10% neutral forlin for 48 hours, and then transferred to 70% ethanol for micro-CT scanning. The results are shown in Figure 46. Compared with the solvent group (G1), TV, vBMD, BV, BV / TV, and BMC were all significantly increased in G2.

[0621] After the experiment, the animals were euthanized, and the right femur of each of the seven animals in each group was taken for bone biomechanical measurements at the center of the fracture callus. The results are shown in Figure 47. Compared with the solvent group (G1), the maximum load of the G2 group was significantly increased, while the stiffness showed no significant difference, but an increasing trend.

[0622] (5) Conclusion

[0623] This study evaluated the efficacy of local intramuscular injection of the test substance at different administration cycles in a mouse fracture model using Micro-CT and three-point bending tests. Micro-CT results showed that, compared with the solvent group, local intramuscular injection of O135 molecule starting on Day 1 significantly improved vBMD, BV / TV, BMC, and TV at the fracture site in mice.

[0624] The three-point bending results showed that, compared with the solvent group, local intramuscular injection of 0135 molecules at the beginning of Day 1 improved the maximum load and stiffness indices of the fracture site in mice, and the maximum load was significantly increased.

[0625] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A polypeptide construct comprising an antigen-binding domain specifically capable of binding to RANKL, a PTH peptide, and an Fc domain; wherein, The Fc domain includes a first Fc domain monomer and a second Fc domain monomer. The antigen-binding domain and the PTH peptide are each linked to one of the first and second Fc domain monomers.

2. The polypeptide construct of claim 1, wherein, The PTH peptide is selected from wild-type PTH peptide or its variants; Preferably, the wild-type PTH peptide is an active fragment of the wild-type PTH protein; Preferably, the wild-type PTH peptide possesses the biological activity of the wild-type PTH protein from which it is derived (e.g., PTH1R binding activity and / or PTH1R activation activity). Preferably, the wild-type PTH peptide comprises amino acid residues in the wild-type PTH protein at positions 1-16 corresponding to those in SEQ ID NO:31; Preferably, the wild-type PTH peptide comprises amino acid residues in the wild-type PTH protein at positions 1-16 (or 1-17, or 1-27, or 1-33, or 1-34, or 1-35, or 1-40, or 1-50, or 1-60, or 1-70, or 1-80, or 1-84) corresponding to positions in SEQ ID NO:

31. Preferably, the wild-type PTH peptide comprises amino acid residues in the wild-type PTH protein at positions 1-33, 1-34, or 1-40 of SEQ ID NO:

31.

3. The polypeptide construct of claim 2, wherein, The PTH peptide variant, compared to its derived wild-type PTH peptide, possesses one or more of the following characteristics: (i) In the PTH peptide variant, compared to the wild-type PTH peptide, the amino acid residue (e.g., asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by an amino acid residue other than the asparagine residue, preferably replaced by a glutamic acid residue, a glycine residue or a serine residue. (ii) In the PTH peptide variant, compared to the wild-type PTH peptide, the amino acid residue (e.g., serine residue) at the position corresponding to the 17th position of SEQ ID NO:31 is replaced by an amino acid residue other than serine residue, glycine residue and proline residue, preferably replaced by glutamic acid residue; (iii) Compared to the wild-type PTH peptide, the PTH peptide variant has an increased number of glycosylation sites (e.g., N-glycosylation sites); preferably, the PTH peptide variant comprises the characteristic sequence NXS (or T), where N represents asparagine, X represents any amino acid other than proline, S represents serine, and T represents threonine; preferably, the amino acid residue (e.g., asparagine residue) at position 33 of SEQ ID NO:31 in the PTH peptide variant is capable of N-glycosylation modification; preferably, compared to the wild-type PTH peptide, the amino acid residue (e.g., valine residue) at position 35 of SEQ ID NO:31 in the PTH peptide variant is replaced by a serine residue or a threonine residue; preferably, compared to the wild-type PTH peptide, in the PTH peptide variant, the amino acid residue (e.g., valine residue) at position 35 of SEQ ID NO:31 is replaced by a serine residue or a threonine residue; The amino acid residue at position 34 of NO:31 (e.g., phenylalanine residue) is replaced by an alanine residue.

4. The polypeptide construct according to any one of claims 1-3, wherein, The PTH polypeptide (e.g., the wild-type PTH peptide or the PTH polypeptide variant) has an amino acid sequence as shown in any one of SEQ ID NO: 7-9, 11-16, 31.

5. The polypeptide construct according to any one of claims 1-4, wherein, The antigen-binding domain is linked to the first Fc domain monomer, and the PTH peptide is linked to the second Fc domain monomer; or, the antigen-binding domain is linked to the second Fc domain monomer, and the PTH peptide is linked to the first Fc domain monomer.

6. The polypeptide construct of claim 5, wherein, The first and second Fc domain monomers each independently contain one or more amino acid modifications that promote heterodimerization of the first and second Fc domain monomers.

7. The polypeptide construct of claim 6, wherein, The first Fc domain monomer contains amino acid modifications that can form a hole structure, and the second Fc domain monomer contains amino acid modifications that can form a knob structure. The hole structure can pair with the knob structure to form a heterodimeric Fc domain.

8. The polypeptide construct according to any one of claims 5-7, wherein, The first Fc domain monomer and / or the second Fc domain monomer are derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4); Preferably, compared to the wild-type Fc domain monomer from which it is derived, the first Fc domain monomer and / or the second Fc domain monomer each independently have amino acid modifications capable of forming hole or knob structures, altered (e.g., enhanced or reduced) effector functions, prolonged half-life (e.g., enhanced FcRn binding activity), and / or altered (e.g., enhanced, reduced, or eliminated) protein A binding activity. Preferably, the first Fc domain monomer and / or the second Fc domain monomer are derived from human immunoglobulin IgG4, and the first Fc domain monomer contains substitution mutations T366S, L368A, and / or Y407V; and / or the second Fc domain monomer contains substitution mutation T366W. Preferably, the first Fc domain monomer and / or the second Fc domain monomer are derived from human immunoglobulin IgG4, and the first Fc domain monomer contains substitution mutations: (i) F234A, L235A, and / or, (ii) T366S, L368A, Y407V; and / or, the second Fc domain monomer contains substitution mutations: (i) F234A, L235A, and / or, (ii) T366W; Preferably, one of the first Fc domain monomer and the second Fc domain monomer further comprises substitution mutations H435R and / or Y436F.

9. The polypeptide construct according to any one of claims 5-8, wherein, The first Fc domain monomer contains the amino acid sequence shown in SEQ ID NO:20, and the second Fc domain monomer contains the amino acid sequence shown in SEQ ID NO:

32.

10. The polypeptide construct according to any one of claims 1-9, wherein, The antigen-binding domain and the PTH peptide are each connected to the Fc domain monomer (e.g., the first Fc domain monomer or the second Fc domain monomer) via a hinge region. Preferably, the hinge region is derived from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4); Preferably, the hinge region is selected from the hinge region of wild-type human immunoglobulin and its variants; Preferably, the hinge region is derived from human immunoglobulin IgG4 and contains the substitution mutation S228P; Preferably, the hinge region comprises an amino acid sequence as shown in SEQ ID NO:

10.

11. The polypeptide construct according to any one of claims 1-10, wherein, The antigen-binding domain is selected from Fab, Fab', F(ab')2, scFab, VHH, Fv, Fv and scFv linked by disulfide bonds. Preferably, the antigen-binding domain is Fab.

12. The polypeptide construct according to any one of claims 1-10, wherein, The antigen-binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL), which together form an antigen-binding domain that specifically binds to RANKL.

13. The polypeptide construct of claim 12, wherein, The VH includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1; the VL includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:

3. Preferably, the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

14. The polypeptide construct of claim 12 or 13, wherein, The VH includes HCDR1 as shown in SEQ ID NO:34, HCDR2 as shown in SEQ ID NO:35, and HCDR3 as shown in SEQ ID NO:36; and / or, the VL includes LCDR1 as shown in SEQ ID NO:37, LCDR2 as shown in SEQ ID NO:38, and LCDR3 as shown in SEQ ID NO:

39. The CDRs are defined by the Kabat numbering system; Preferably, the VH contains the amino acid sequence shown in SEQ ID NO:1, and / or the VL contains the amino acid sequence shown in SEQ ID NO:

3.

15. The polypeptide construct according to any one of claims 12-14, comprising peptide chain IA, peptide chain IB, and peptide chain IC; wherein, The peptide chain IA includes the VL and the light chain constant region, the peptide chain IB includes the VH, the heavy chain CH1 region, the hinge region and the first Fc domain monomer (or the second Fc domain monomer), and the peptide chain IC includes the PTH peptide, the hinge region and the second Fc domain monomer (or the first Fc domain monomer). Preferably, the peptide chain IA includes the VL and a light chain constant region from the N-terminus to the C-terminus, the peptide chain IB includes the VH, a heavy chain CH1 region, a hinge region, and a first Fc domain monomer (or a second Fc domain monomer) from the N-terminus to the C-terminus, and / or the peptide chain IC includes the PTH peptide, a hinge region, and a second Fc domain monomer (or a first Fc domain monomer) from the N-terminus to the C-terminus.

16. The polypeptide construct of claim 15, wherein, The adjacent domains of the peptide chain IA are optionally connected by or without a connector, the adjacent domains of the peptide chain IB are optionally connected by or without a connector, and / or the adjacent domains of the peptide chain IC are optionally connected by or without a connector. Preferably, each of the peptide connectors is independently the same or different peptide connectors (e.g., rigid peptide connectors or flexible peptide connectors); preferably, each of the peptide connectors is independently selected from peptide connectors containing one or more glycine (G) and / or serine (S), for example, having (G) m S) n The peptide linker with the structure shown is wherein m and n are each independently an integer not less than 0, for example, each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, each peptide linker independently comprises an amino acid sequence as shown in any one of SEQ ID NO:17-19.

17. The polypeptide construct of claim 15 or 16, wherein, The peptide chain IC comprises, from N-terminus to C-terminus: the PTH peptide, a peptide linker, a hinge region, and a second Fc domain monomer (or the first Fc domain monomer), wherein the peptide linker comprises an amino acid sequence as shown in any one of SEQ ID NO:17-19.

18. The polypeptide construct according to any one of claims 15-17, wherein, The light chain constant region is the constant region of the human immunoglobulin κ or λ light chain; Preferably, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:

4.

19. The polypeptide construct according to any one of claims 15-18, wherein, The heavy chain CH1 region is the heavy chain CH1 region of human immunoglobulins (such as IgG1, IgG2, IgG3 or IgG4); Preferably, the heavy chain CH1 region contains an amino acid sequence as shown in SEQ ID NO:

2.

20. The polypeptide construct according to any one of claims 15-19, wherein, The polypeptide construct comprises: The peptide chain IA contains the amino acid sequence shown in SEQ ID NO:6, the peptide chain IB contains the amino acid sequence shown in SEQ ID NO:5, and the peptide chain IC contains the amino acid sequence shown in any one of SEQ ID NOs:21-30.

21. A modified PTH protein, compared to its wild-type derivative, possesses one or more of the following characteristics: (i) In the modified PTH protein, compared to the wild-type PTH protein, the amino acid residue (e.g., asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by an amino acid residue other than the asparagine residue, preferably replaced by a glutamic acid residue, a glycine residue or a serine residue. (ii) In the modified PTH protein, compared to the wild-type PTH protein, the amino acid residue (e.g., serine residue) at the position corresponding to the 17th position of SEQ ID NO:31 is replaced by an amino acid residue other than serine residue, glycine residue and proline residue, preferably replaced by glutamic acid residue.

22. The modified PTH protein of claim 21, having an increased number of glycosylation sites (e.g., N-glycosylation sites) compared to the wild-type PTH protein; preferably, the modified PTH protein comprises the characteristic sequence NXS (or T), wherein, N represents asparagine, X represents any amino acid other than proline, S represents serine, and T represents threonine; preferably, the modified PTH protein has an amino acid residue (e.g., an asparagine residue) at position 33 of SEQ ID NO:31 that is N-glycosylated; preferably, compared to the wild-type PTH protein, the modified PTH protein has an amino acid residue (e.g., a valine residue) at position 35 of SEQ ID NO:31 that is replaced by a serine residue or a threonine residue; preferably, compared to the wild-type PTH protein, in the modified PTH protein, the amino acid residue (e.g., a valine residue) at position 35 of SEQ ID NO:31 is replaced by a serine residue, and the amino acid residue (e.g., a phenylalanine residue) at position 34 of SEQ ID NO:31 is replaced by an alanine residue.

23. A modified PTH peptide, which is an active fragment of the modified PTH protein as described in claim 21 or 22; Preferably, the modified PTH peptide has an amino acid sequence different from that of the wild-type PTH peptide; Preferably, the modified PTH peptide possesses the biological activity of the modified PTH protein from which it is derived (e.g., PTH1R binding activity and / or PTH1R activation activity). Preferably, the modified PTH peptide comprises amino acid residues in the modified PTH protein at positions 3-16 corresponding to those in SEQ ID NO:31; Preferably, the modified PTH peptide comprises amino acid residues in the modified PTH protein at positions 3-16 (or 3-17, or 3-27, or 3-33, or 3-34, or 3-35, or 3-40, or 3-50, or 3-60, or 3-70, or 3-80, or 3-84, or 1-16, or 1-17, or 1-27, or 1-33, or 1-34, or 1-35, or 1-40, or 1-50, or 1-60, or 1-70, or 1-80, or 1-84) corresponding to positions in SEQ ID NO:

31. Preferably, the modified PTH peptide comprises amino acid residues in the modified PTH protein at positions 1-33, 1-34, or 1-40 of SEQ ID NO:

31. Preferably, the modified PTH peptide has an amino acid sequence as shown in any one of SEQ ID NO:9, 11-16.

24. A polypeptide construct comprising the modified PTH peptide of claim 23 and an Fc domain; Preferably, the polypeptide construct further comprises an antigen-binding domain capable of specifically binding to RANKL.

25. An isolated nucleic acid molecule encoding a polypeptide construct according to any one of claims 1-20, a modified PTH protein according to claim 21 or 22, a modified PTH peptide according to claim 23, or a polypeptide construct according to claim 24.

26. A vector comprising the nucleic acid molecule of claim 25; preferably, the vector is a cloning vector or an expression vector.

27. A host cell comprising the nucleic acid molecule of claim 25 or the vector of claim 26.

28. A method for preparing the polypeptide construct according to any one of claims 1-20, or the modified PTH protein according to claim 21 or 22, or the modified PTH peptide according to claim 23, or the polypeptide construct according to claim 24, comprising the following steps: Host cells are cultured under conditions that allow protein expression, and the polypeptide construct or modified PTH protein or modified PTH peptide is recovered from the cultured host cell culture; wherein, The host cell contains a nucleotide sequence encoding the polypeptide construct or a modified PTH protein or a modified PTH peptide.

29. A conjugate comprising the polypeptide construct of any one of claims 1-20, the modified PTH protein of claim 21 or 22, the modified PTH peptide of claim 23, or the polypeptide construct of claim 24, and a coupling moiety; Preferably, the coupling portion is selected from purified tags and therapeutic agents.

30. A pharmaceutical composition comprising a polypeptide construct according to any one of claims 1-20, a modified PTH protein according to claim 21 or 22, a modified PTH peptide according to claim 23, a polypeptide construct according to claim 24, an isolated nucleic acid molecule according to claim 25, a carrier according to claim 26, a host cell according to claim 27, or a conjugate according to claim 29; and a pharmaceutically acceptable carrier and / or excipient.

31. Use of the polypeptide construct of any one of claims 1-20, the modified PTH protein of claim 21 or 22, the modified PTH peptide of claim 23, the polypeptide construct of claim 24, the isolated nucleic acid molecule of claim 25, the carrier of claim 26, the host cell of claim 27, the conjugate of claim 29, or the pharmaceutical composition of claim 30 for the preparation of a medicament for the prevention and / or treatment in a subject of diseases associated with RANKL and / or PTH1R; Preferably, the disease is selected from: bone metabolism-related diseases, diseases caused by parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism), and any combination thereof; Preferably, the disease is selected from: osteoporosis (such as postmenopausal osteoporosis), osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, degenerative lumbar spondylolisthesis, degenerative intervertebral disc disease, bone injury, hypoparathyroidism, hyperparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria and any combination thereof; Preferably, the disease is osteoporosis or osteopenia; Preferably, the subject is a mammal, such as a human; Preferably, the polypeptide construct, modified PTH protein, modified PTH peptide, isolated nucleic acid molecule, carrier, host cell, conjugate, or pharmaceutical composition is administered alone or in combination with other pharmaceutically active agents (e.g., simultaneously, separately, or sequentially).

32. A method for preventing and / or treating diseases associated with RANKL and / or PTH1R in a subject, the method comprising administering to a subject in need an effective amount of the polypeptide construct of any one of claims 1-20, the modified PTH protein of claim 21 or 22, the modified PTH peptide of claim 23, the polypeptide construct of claim 24, the isolated nucleic acid molecule of claim 25, the carrier of claim 26, the host cell of claim 27, the conjugate of claim 29, or the pharmaceutical composition of claim 30; Preferably, the disease is selected from: bone metabolism-related diseases, diseases caused by parathyroid dysfunction (e.g., hyperparathyroidism or hypoparathyroidism), and any combination thereof; Preferably, the disease is selected from: osteoporosis (such as postmenopausal osteoporosis), osteopenia, osteogenesis imperfecta, transplant-related bone loss, autoimmune-induced bone loss, disuse-induced bone loss, degenerative lumbar spondylolisthesis, degenerative intervertebral disc disease, bone injury, hypoparathyroidism, hyperparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria and any combination thereof; Preferably, the disease is osteoporosis or osteopenia; Preferably, the subject is a mammal, such as a human; Preferably, the polypeptide construct, modified PTH protein, modified PTH peptide, isolated nucleic acid molecule, carrier, host cell, conjugate, or pharmaceutical composition is administered alone or in combination with other pharmaceutically active agents or therapies (e.g., simultaneously, separately, or sequentially).

33. A method for modifying a PTH peptide or PTH protein or a peptide construct containing a PTH peptide or PTH protein, the method comprising performing one or more of the following modifications on the PTH peptide or PTH protein or the peptide construct: (i) The amino acid residue (e.g., asparagine residue) at the position corresponding to the 16th position of SEQ ID NO:31 is replaced by an amino acid residue other than the asparagine residue, preferably by a glutamic acid residue, a glycine residue or a serine residue; (ii) The amino acid residue (e.g., serine residue) at the position corresponding to the 17th position of SEQ ID NO:31 is replaced by an amino acid residue other than serine residue, glycine residue and proline residue, preferably replaced by glutamic acid residue; (iii) Increase glycosylation sites (e.g., N-glycosylation sites); Preferably, the PTH polypeptide, PTH protein, or polypeptide construct, after modification, contains the characteristic sequence NXTS (or T), wherein, N represents asparagine, X represents any amino acid except proline, S represents serine, and T represents threonine. Preferably, the PTH polypeptide, PTH protein, or polypeptide construct can undergo N-glycosylation modification at the amino acid residue (e.g., asparagine residue) at the position corresponding to position 33 of SEQ ID NO:31 after modification. Preferably, the amino acid residue (e.g., valine residue) at the position corresponding to the 35th position of SEQ ID NO:31 in the PTH polypeptide, PTH protein, or polypeptide construct is replaced with a serine residue or a threonine residue. Preferably, the amino acid residue (e.g., valine residue) at the position corresponding to the 35th position of SEQ ID NO:31 is replaced with a serine residue, and the amino acid residue (e.g., phenylalanine residue) at the position corresponding to the 34th position of SEQ ID NO:31 is replaced with an alanine residue. Preferably, the modified PTH peptide, PTH protein, or peptide construct has altered properties; for example, compared to the unmodified form, the modified PTH peptide, PTH protein, or peptide construct has enhanced stability, altered (e.g., enhanced or reduced) PTH1R binding activity, altered (e.g., enhanced or reduced) PTH1R activating activity, prolonged half-life, and / or improved pharmacodynamic activity.

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