Regulators of the parathyroid hormone receptor (PTHR1)

Abstract

Synthetic parathyroid hormones (sPTHs) are provided that can modulate the activity of parathyroid hormone receptors, such as parathyroid hormone receptor 1 (PTHR1). Also provided are methods of using these sPTHs to modulate PTHR activity in cells, e.g., cells in an organism. The present invention provides a polypeptide that specifically binds to a parathyroid hormone receptor (PTHR), the polypeptide comprising synthetic parathyroid hormone (sPTH).

Description

Technical Field

[0001] [Related Applications] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 884,703, filed Aug. 9, 2019. The entire disclosure of the above application is incorporated herein by reference.

[0002] Incorporation by Reference of Material in ASCII Text File The Sequence Listing contained in the following ASCII text file, which is submitted concurrently with this application, is incorporated herein by reference. File name: 57081028001_Sequence_Listing.txt; created on Aug. 7, 2020, size 57 KB.

Background Art

[0003] The parathyroid hormone receptor (PTHR or PTH1R, HGNC:9608, human gene ID:5745, HomoloGene:267) is a G protein-coupled receptor (GPCR) that is highly expressed in bone and kidney and controls calcium ion homeostasis. PTHR has two natural ligands, parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). Alterations in the biology of PTHR are involved in many important diseases, including osteoporosis and other diseases of calcium homeostasis dysregulation. Diseases caused by inactivating mutations of PTHR include, for example, Blomstrand lethal chondrodysplasia, Ollier disease, familial primary failure of tooth eruption, and Eiken syndrome. Diseases caused by ligand imbalance include brachydactyly type E and hypoparathyroidism. Osteoporosis, an imbalance between bone resorption and bone formation processes, is also controlled by signaling through PTHR.

[0004] Therapeutic peptides based on the native ligands of PTHR are produced recombinantly and are approved as drugs for the treatment of osteoporosis: teriparatide (corresponding to amino acids 1 - 34 of PTH, Eli Lilly) and abaloparatide (amino acids 1 - 34 of PTHrP, Radius Health).

[0005] Despite their FDA approval and clinical use, both native ligands have serious side effects. Both teriparatide and abaloparatide are sold with a black box label warning of the risk of osteosarcoma, and treatment with these drugs for more than two years over a patient's lifetime is not recommended. Furthermore, long - term exposure to teriparatide often causes heterotopic bone resorption in patients, offsetting the desirable results of treatment. Additionally, these compounds do not address other diseases that require, for example, antagonist or inverse agonist activity of PTHR.

Summary of the Invention

Means for Solving the Problems

[0006] The invention disclosed herein is based in part on the discovery that the polypeptides of the invention specifically bind to the parathyroid hormone receptor (PTHR). Accordingly, the invention generally relates to compositions (e.g., polypeptides, pharmaceutical compositions) and methods useful for modulating PTHR - mediated signal transduction in cells.

[0007] Polypeptides that specifically bind to PTHR are provided herein. In one aspect, the invention provides a polypeptide that specifically binds to PTHR, where the polypeptide comprises synthetic parathyroid hormone (sPTH).

[0008] In some embodiments, sPTH is a) 1 - 3 polar residues, and 1 - 14 amino acid substitutions relative to SEQ ID NO: 7; b) at least 4 polar residues, at least 1 charged residue, and 1 - 14 amino acid substitutions relative to SEQ ID NO: 7; c) At least 5 polar residues, at least 2 charged residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7; d) At least 5 polar residues, valine at position 2, and 1 to 13 amino acid substitutions relative to SEQ ID NO: 7; or e) At least 6 polar residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7 comprising a 14 - amino acid sequence.

[0009] In some embodiments, sPTH a) Up to 13 polar residues, and 1 to 32 amino acid substitutions relative to SEQ ID NO: 8; b) At least 14 polar residues, valine at position 2, and 1 to 31 substitutions relative to SEQ ID NO: 8; c) At least 14 polar residues, and 22 to 28 amino acid substitutions relative to SEQ ID NO: 8; d) Positions 1 - 14 of SEQ ID NO: 8, and 1 to 18 amino acid substitutions at positions 15 - 32 relative to SEQ ID NO: 5 comprising a 32 - amino acid sequence.

[0010] In some embodiments, sPTH comprises an amino acid sequence having at least about 20% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 108 - 160. In some embodiments, sPTH comprises an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 108 - 160.

[0011] In some embodiments, sPTH comprises an amino acid sequence having at least about 20% sequence identity to an amino acid sequence selected from SEQ ID NO: 9, 10, 19, 20, 31, 35, 41, 88, 101 - 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 - 138, 140 - 143, 145, 147 - 152, 154, 155, and 157 - 160. In some embodiments, sPTH comprises an amino acid sequence selected from SEQ ID NO: 9, 10, 19, 20, 31, 35, 41, 88, 101 - 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 - 138, 140 - 143, 145, 147 - 152, 154, 155, and 157 - 160.

[0012] In some embodiments, sPTH comprises an amino acid sequence having at least about 20% sequence identity to SEQ ID NO: 5.

[0013] In some embodiments, sPTH comprises the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the amino acid sequence shown in SEQ ID NO: 6 is at the N-terminus of sPTH. In some embodiments, sPTH comprises an amino acid sequence having at least about 55% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 102 - 106 and SEQ ID NO: 132 - 155.

[0014] In some embodiments, sPTH comprises the amino acid sequence of SEQ ID NO: 107.

[0015] In some embodiments, sPTH is an agonist of PTHR. In some embodiments, sPTH is a biased agonist of PTHR. In some embodiments, sPTH is an inverse agonist of PTHR. In some embodiments, sPTH is an antagonist of PTHR.

[0016] In some embodiments, the polypeptide of the present invention is a fusion protein.

[0017] In another aspect, the present invention provides a polynucleotide encoding a polypeptide disclosed herein, a vector comprising such a polynucleotide, and a host cell comprising such a polynucleotide or vector.

[0018] In another aspect, the present invention provides a method of modulating PTHR signaling in mammalian cells, comprising contacting the mammalian cells with an effective amount of a polypeptide disclosed herein or a composition (e.g., a pharmaceutical composition) comprising a polypeptide disclosed herein.

[0019] In another aspect, the present invention provides a method of treating a subject in need thereof (e.g., a subject having osteoporosis), comprising administering to the subject an effective amount of a polypeptide disclosed herein or a composition (e.g., a pharmaceutical composition) comprising a polypeptide disclosed herein.

[0020] In another aspect, the present invention provides a method of modulating PTHR signaling in a subject in need thereof (e.g., a subject having osteoporosis), comprising administering to the subject an effective amount of a polypeptide disclosed herein or a composition (e.g., a pharmaceutical composition) comprising a polypeptide disclosed herein.

[0021] The foregoing will be apparent from the following more particular description of exemplary embodiments, as illustrated in the accompanying drawings wherein like reference numerals refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

Figure 1

Figure 2

Figure 3

BRIEF DESCRIPTION OF THE INVENTION

[0023] The following is a description of exemplary embodiments.

[0024] There is a need for novel ligands for PTHR that can regulate PTHR activity. The present invention provides, among other things, synthetic parathyroid hormone (sPTH) that can regulate the activity of PTHR, as well as conjugates (e.g., including heterologous moieties), fusion proteins, and compositions comprising sPTH and nucleic acids encoding sPTH, and methods of using sPTH (e.g., for regulating PTHR activity (e.g., for treating disorders of calcium homeostasis and / or PTHR signaling)).

[0025] [PTH AND RELATED PEPTIDES] Parathyroid hormone (PTH) (HGNC:9606), the natural ligand of PTHR, is a secreted peptide hormone that binds to PTHR. The peptide is composed of a signal sequence and a cleaved propeptide fold, leaving an 84 - amino acid active molecule (amino acids 32 - 115). The bioactive sequence of PTH is provided as SEQ ID NO:1 (Table 1). A review of the biology of PTHR and its role in disease can be found in Cheloha et al., Nature Reviews Endocrinology 11(12):712 - 24(2015).

[0026] Parathyroid hormone - related peptide (PTHrP) (HGNC:9607), the second natural ligand of PTHR, is a secreted peptide hormone that binds to PTHR. The peptide is composed of a signal peptide, a propeptide, and a long chain that is cleaved by endoproteolysis into three major secreted forms called PTHrP[1 - 36], PTHrP[38 - 94], and osteostatin (PTHrP[107 - 139]). The complete sequence of PTHrP is provided as SEQ ID NO:2 (Table 1).

[0027] From the experimental results, PTH[1-14], which is the first 14 amino acids at the N-terminus of PTH, binds to the receptor and promotes signal transduction. On the other hand, PTH[15-34], which is the last 20 amino acids at the C-terminus, is known to bind to the extracellular domain of PTHR and confer a certain degree of receptor specificity (Pioszak & Xu, Proc Natl Acad Sci U S A. 105(13):5034-39(2008); Pioszak et al., J Biol Chem. 284(41):28382-91(2009)).

[0028] The sequence of the therapeutic peptide teriparatide corresponding to amino acids 1-34 of the natural ligand PTH is provided as SEQ ID NO: 3 (Table 1).

[0029] The sequence of the therapeutic peptide abaloparatide is provided as SEQ ID NO: 4 (Table 1) and corresponds to the first 20 amino acids of the natural ligand PTHrP. Residues 21-34 have 50% diversity with respect to the last 14 amino acids at the C-terminus of PTHrP, including the unnatural amino acid 2-aminoisobutyric acid (designated as "X").

[0030] The structure of PTHR was recently elucidated in a complex with the peptide ligand LA-PTH, which is a hybrid of PTH and PTHrP. See Zhao LH et al., Science 364:148-53(2019). The sequence of 32-mer LA-PTH is provided as SEQ ID NO: 5, and the 14-mer at the N-terminus of LA-PTH is provided as SEQ ID NO: 6 (Table 1).

[0031] The consensus sequences encompassing the aforementioned PTH and PTHrP sequences are provided as SEQ ID NO: 7 and SEQ ID NO: 8 (Table 1).

[0032] [Table 1]

[0033] [sPTH provided by the present invention] In one aspect, the present invention provides a polypeptide that specifically binds to PTHR, where the polypeptide comprises synthetic parathyroid hormone (sPTH).

[0034] "Synthetic parathyroid hormone" or "sPTH" or "sPTH provided by the present invention" etc. refers to a polypeptide that binds to PTHR and contains an amino acid sequence similar in size to the biologically active portion of the mature PTH peptide, and does not contain, consist of, or essentially consist of the amino acid sequence of wild-type PTH (SEQ ID NO: 1), wild-type PTHrP (SEQ ID NO: 2), teriparatide (FORTEO®; SEQ ID NO: 3), abaloparatide (TYMLOS®; SEQ ID NO: 4), or "long-acting" PTH (N-terminal 32-mer (SEQ ID NO: 5) or N-terminal 14-mer (SEQ ID NO: 6)).

[0035] In certain embodiments, the sPTH provided by the present invention does not contain, consist of, or essentially consist of the consensus amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, and / or SEQ ID NO: 107. In some embodiments, the sPTH of the present invention does not contain, consist of, or essentially consist of the amino acid sequences disclosed in U.S. Patent No. 6,921,750, U.S. Patent No. 7,803,770, and / or International Publication No. 2000 / 010596 pamphlet. In some embodiments, the sPTH of the present invention contains, essentially consists of, or consists of amino acids that are included in the consensus sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 107 and satisfy one or more criteria shown in Table 2. In some embodiments, sPTH contains the amino acid sequence of SEQ ID NO: 7. In some embodiments, sPTH contains the amino acid sequence of SEQ ID NO: 8. In some embodiments, sPTH contains the amino acid sequence of SEQ ID NO: 107.

[0036]

Table 2

[0037] "Polar" amino acid residues include basic, acidic, and hydrophilic but uncharged canonical and non-canonical amino acids. In certain embodiments, the polar residues are canonical amino acids, such as basic (K, R, H), acidic (D, E), or hydrophilic but uncharged (S, T, N, Q, C). In some embodiments, the polar residues are non-canonical amino acids.

[0038] "Charged" residues include basic and acidic canonical and non-canonical amino acids. In some embodiments, the charged residues are canonical amino acids, such as basic (K, R, H) or acidic (D, E). In some embodiments, the charged residues are non-canonical amino acids.

[0039] In some embodiments, the sPTH of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NOs: 9-106 and 108-159 (Table 3), or a variant thereof.

[0040] [Table 3]

[0041] [Table 4]

[0042] [Table 5]

[0043] [Table 6]

[0044] "Variant of sPTH" polypeptides (e.g., "variant" with respect to sPTH) etc. include, for example, amino acid sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid substitutions (more in the case of longer sequences, such as 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 etc.) when compared with a reference sequence (e.g., one of SEQ ID NOs: 9 to 107 or 108 to 160). In certain embodiments, the variant of sPTH provided by the present invention includes up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, for example, 1 to 3 amino acid substitutions. Various amino acid substitutions of the variant of sPTH provided by the present invention, including substitutions with non-canonical amino acids, are possible. Non-limiting examples of non-canonical amino acids include D-isomers of any canonical amino acid (e.g., D-alanine), selenocysteine, pyrrolysine, β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine, citrulline, homocitrulline, homoserine, norleucine, norvaline, and ornithine. In some embodiments, the amino acid substitution is a conservative amino acid substitution. "Conservative substitution" with respect to the reference sequence means that the value of a given amino acid substitution is 0 or more in BLOSUM62. In some embodiments, the amino acid substitution is a highly conservative amino acid substitution. "Highly conservative substitution" with respect to the reference sequence means that the value of a given amino acid substitution is 1 or more (e.g., in some embodiments, 2 or more) in BLOSUM62.

[0045] In some embodiments, the "variant of sPTH" polypeptide comprises an amino acid sequence that is at least about 50% identical to the sPTH provided by the present invention (for example, the sPTH of SEQ ID NOs: 9-106 or 108-160 (Table 3)). For example, the variant is at least about 50%, about 60%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any one or more of the sPTHs of SEQ ID NOs: 9-106 and 108-160 (Table 3).

[0046] As used herein, the term "sequence identity" refers to the degree to which two nucleotide sequences or two amino acid sequences have the same residue at the same position when the sequences are aligned to achieve the maximum level of identity expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as the reference sequence and is compared with the test sequence. The sequence identity between the reference sequence and the test sequence is expressed as the percentage of positions over the full length of the reference sequence where the reference sequence and the test sequence share the same nucleotide or amino acid to achieve the maximum level of identity. As an example, at alignment to achieve the maximum level of identity, if the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the full length of the reference sequence, the two sequences are considered to have 70% sequence identity.

[0047] Alignment of the arrays for comparison to achieve the maximum level of identity can be readily performed by one of ordinary skill in the art using an appropriate alignment method or algorithm. In some cases, the alignment may include gaps introduced to provide the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (generally, see Ausubel et al., Current Protocols in Molecular Biology).

[0048] When using a sequence comparison algorithm, a test sequence and a reference sequence are entered into a computer, coordinates are then specified as necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the specified program parameters. A commonly used tool for determining percent sequence identity is the Protein Basic Local Alignment Search Tool (BLASTP) available from the National Center for Biotechnology Information of the National Library of Medicine of the National Institutes of Health, U.S.A. (Altschul et al., 1990).

[0049] In some embodiments, sPTH is a) 1 to 3 polar residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO:7; b) at least 4 polar residues, at least 1 charged residue, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7; c) at least 5 polar residues, at least 2 charged residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7; d) at least 5 polar residues, valine at position 2, and 1 to 13 amino acid substitutions relative to SEQ ID NO: 7; or a) at least 6 polar residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7 comprising a 14-amino acid sequence.

[0050] In some embodiments, sPTH is a) at most 13 polar residues, and 1 to 32 amino acid substitutions relative to SEQ ID NO: 8; b) at least 14 polar residues, valine at position 2, and 1 to 31 substitutions relative to SEQ ID NO: 8; c) at least 14 polar residues, and 22 to 28 amino acid substitutions relative to SEQ ID NO: 8; d) positions 1 to 14 of SEQ ID NO: 8, and 1 to 18 amino acid substitutions at positions 15 to 32 relative to SEQ ID NO: 5 comprising a 32-amino acid sequence.

[0051] In some embodiments, sPTH comprises an amino acid sequence having at least about 20% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9-106 and SEQ ID NOs: 108-160. For example, having at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9-106 and SEQ ID NOs: 108-160; having about 20-97%, about 25-97%, about 25-95%, about 30-95%, about 30-90%, about 35-90%, about 35-85%, about 40-85%, about 40-80%, about 45-75%, about 45-70%, about 50-70%, about 50-65%, or about 55-65% sequence identity; or having up to about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity. In some embodiments, sPTH comprises an amino acid sequence having about 50-97% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9-106 and SEQ ID NOs: 108-160.

[0052] In some embodiments, sPTH comprises at least one amino acid substitution with respect to an amino acid sequence selected from SEQ ID NOs: 9-106 and SEQ ID NOs: 108-160. For example, sPTH comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acid substitutions with respect to an amino acid sequence selected from SEQ ID NOs: 9-106 and SEQ ID NOs: 108-160; or about 1-32, about 1-28, about 1-24, about 1-20, about 1-15, about 1-10, about 1-5, about 2-32, about 2-28, about 2-24, about 2-20, about 2-15, about 2-10, about 2-5, about 3-32, about 3-28, about 3-24, about 3-20, about 3-15, about 3-10, about 3-5, about 4-32, about 4-28, about 4-24, about 4-20, about 4-15, about 4-10, or about 4-5 amino acid substitutions.

[0053] In some embodiments, sPTH comprises up to 32 amino acid substitutions relative to the amino acid sequences selected from SEQ ID NOs: 9-106 and SEQ ID NOs: 108-160. For example, sPTH comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 amino acid substitutions relative to the amino acid sequences selected from SEQ ID NOs: 9-106 and SEQ ID NOs: 108-160; or up to 1-32, 1-28, 1-24, 1-20, 1-15, 1-10, 1-5, 2-32, 2-28, 2-24, 2-20, 2-15, 2-10, 2-5, 3-32, 3-28, 3-24, 3-20, 3-15, 3-10, 3-5, 4-32, 4-28, 4-24, 4-20, 4-15, 4-10 or 4-5 amino acid substitutions.

[0054] In some embodiments, sPTH comprises an amino acid sequence selected from SEQ ID NOs: 9-106 and SEQ ID NOs: 108-160.

[0055] In some embodiments, sPTH comprises an amino acid sequence having at least about 20% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155, and 157-160. For example, having at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155, and 157-160; having about 20-97%, about 25-97%, about 25-95%, about 30-95%, about 30-90%, about 35-90%, about 35-85%, about 40-85%, about 40-80%, about 45-75%, about 45-70%, about 50-70%, about 50-65%, or about 55-65% sequence identity; or having up to about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity. In some embodiments, sPTH comprises an amino acid sequence having about 50-97% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155, and 157-160.

[0056] In some embodiments, sPTH comprises at least one amino acid substitution relative to an amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155 and 157-160. For example, sPTH comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155 and 157-160; or about 1-32, about 1-28, about 1-24, about 1-20, about 1-15, about 1-10, about 1-5, about 2-32, about 2-31, about 2-28, about 2-24, about 2-20, about 2-18, about 2-15, about 2-10, about 2-5, about 3-32, about 3-28, about 3-24, about 3-20, about 3-15, about 3-10, about 3-5, about 4-32, about 4-28, about 4-24, about 4-20, about 4-15, about 4-10 or about 4-5 amino acid substitutions.

[0057] In some embodiments, sPTH comprises up to 32 amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155, and 157-160. For example, sPTH comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155, and 157-160; or up to 1-32, 1-28, 1-24, 1-20, 1-15, 1-10, 1-5, 2-32, 2-31, 2-28, 2-24, 2-20, 2-18, 2-15, 2-10, 2-5, 3-32, 3-28, 3-24, 3-20, 3-15, 3-10, 3-5, 4-32, 4-28, 4-24, 4-20, 4-15, 4-10, or 4-5 amino acid substitutions.

[0058] In some embodiments, sPTH comprises an amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155, and 157-160.

[0059] In some embodiments, sPTH comprises an amino acid sequence having at least about 20% sequence identity to an amino acid sequence selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 - 138, 140 - 143, 145, 147 - 152, 154, 155, and 157 - 160. For example, having at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity to an amino acid sequence selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 - 138, 140 - 143, 145, 147 - 152, 154, 155, and 157 - 160; having about 20 - 97%, about 25 - 97%, about 25 - 95%, about 30 - 95%, about 30 - 90%, about 35 - 90%, about 35 - 85%, about 40 - 85%, about 40 - 80%, about 45 - 75%, about 45 - 70%, about 50 - 70%, about 50 - 65%, or about 55 - 65% sequence identity; or having up to about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity. In some embodiments, sPTH comprises an amino acid sequence having about 50 - 97% sequence identity to an amino acid sequence selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 - 138, 140 - 143, 145, 147 - 152, 154, 155, and 157 - 160.

[0060] In some embodiments, sPTH comprises at least one amino acid substitution with respect to the amino acid sequence selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155 and 157-160. For example, sPTH comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acid substitutions; or about 1-32, about 1-28, about 1-24, about 1-20, about 1-15, about 1-10, about 1-5, about 2-32, about 2-31, about 2-28, about 2-24, about 2-20, about 2-18, about 2-15, about 2-10, about 2-5, about 3-32, about 3-28, about 3-24, about 3-20, about 3-15, about 3-10, about 3-5, about 4-32, about 4-28, about 4-24, about 4-20, about 4-15, about 4-10 or about 4-5 amino acid substitutions with respect to the amino acid sequence selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155 and 157-160.

[0061] In some embodiments, sPTH comprises up to 32 amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 - 138, 140 - 143, 145, 147 - 152, 154, 155, and 157 - 160. For example, sPTH comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 - 138, 140 - 143, 145, 147 - 152, 154, 155, and 157 - 160; or up to 1 - 32, 1 - 28, 1 - 24, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 2 - 32, 2 - 31, 2 - 28, 2 - 24, 2 - 20, 2 - 18, 2 - 15, 2 - 10, 2 - 5, 3 - 32, 3 - 28, 3 - 24, 3 - 20, 3 - 15, 3 - 10, 3 - 5, 4 - 32, 4 - 28, 4 - 24, 4 - 20, 4 - 15, 4 - 10, or 4 - 5 amino acid substitutions.

[0062] In some embodiments, sPTH comprises an amino acid sequence selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 - 138, 140 - 143, 145, 147 - 152, 154, 155, and 157 - 160.

[0063] In some embodiments, sPTH consists of 28 - 36 amino acids, such as 18, 29, 30, 31, 32, 33, 35, or 36 amino acids. In some embodiments, sPTH consists of 30 - 34 amino acids. In some embodiments, sPTH consists of 32 amino acids.

[0064] In some embodiments, sPTH comprises an amino acid sequence having at least about 20% sequence identity to SEQ ID NO:5. For example, having at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity to SEQ ID NO:5; having about 20-97%, about 25-97%, about 25-95%, about 30-95%, about 30-90%, about 35-90%, about 35-85%, about 40-85%, about 40-80%, about 45-75%, about 45-70%, about 50-70%, about 50-65%, or about 55-65% sequence identity to SEQ ID NO:5; or having at most about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity to SEQ ID NO:5. In some embodiments, sPTH comprises an amino acid sequence having about 50-97% sequence identity to SEQ ID NO:5.

[0065] In some embodiments, sPTH comprises at least one amino acid substitution relative to SEQ ID NO:5. For example, sPTH comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acid substitutions relative to SEQ ID NO:5; or about 1-32, about 1-28, about 1-24, about 1-20, about 1-15, about 1-10, about 1-5, about 2-32, about 2-31, about 2-28, about 2-24, about 2-20, about 2-18, about 2-15, about 2-10, about 2-5, about 3-32, about 3-28, about 3-24, about 3-20, about 3-15, about 3-10, about 3-5, about 4-32, about 4-28, about 4-24, about 4-20, about 4-15, about 4-10, or about 4-5 amino acid substitutions relative to SEQ ID NO:5.

[0066] In some embodiments, sPTH comprises up to 32 amino acid substitutions relative to SEQ ID NO:5. For example, sPTH comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 amino acid substitutions relative to SEQ ID NO:5; or up to 1-32, 1-28, 1-24, 1-20, 1-15, 1-10, 1-5, 2-32, 2-31, 2-28, 2-24, 2-20, 2-18, 2-15, 2-10, 2-5, 3-32, 3-28, 3-24, 3-20, 3-15, 3-10, 3-5, 4-32, 4-28, 4-24, 4-20, 4-15, 4-10, or 4-5 amino acid substitutions relative to SEQ ID NO:5.

[0067] In some embodiments, sPTH comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the amino acid sequence set forth in SEQ ID NO:6 is at the N-terminus of sPTH.

[0068] In some embodiments, sPTH comprises an amino acid sequence having at least about 55% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 102-106 and SEQ ID NOs: 132-155. For example, having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%; about 55-97%, about 60-97%, about 60-95%, about 65-95%, about 65-90%, about 70-90%, about 70-85%, or about 80-85%; or up to about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 97% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 102-106 and SEQ ID NOs: 132-155. In some embodiments, sPTH comprises an amino acid sequence having about 50-97% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 102-106 and SEQ ID NOs: 132-155. In some embodiments, sPTH comprises an amino acid sequence having about 75-97% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 102-106 and SEQ ID NOs: 132-155. In some embodiments, sPTH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 102-106 and SEQ ID NOs: 132-155.

[0069] In some embodiments, at least the amino acid substitutions are conservative substitutions. In some embodiments, at least the amino acid substitutions are highly conservative substitutions. In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the amino acid substitutions are highly conservative substitutions.

[0070] In some embodiments, sPTH binds to PTHR with an affinity that is at least about 5% higher than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. For example, sPTH binds to PTHR with an affinity that is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, or about 70% higher than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0071] In some embodiments, sPTH binds to PTHR with an affinity that is at least about 5% lower than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. For example, sPTH binds to PTHR with an affinity that is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, or about 70% lower than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0072] In some embodiments, it is about 5% or less lower than a polypeptide having an amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. For example, sPTH binds to PTHR with an affinity that is about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 50% or less, about 60% or less, or about 70% or less lower than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0073] In some embodiments, the sPTH or variant thereof provided by the present invention includes post - translational modifications such as acetylation, amidation, formylation, glycosylation, hydroxylation, methylation, myristoylation, phosphorylation, deamidation, prenylation (e.g., farnesylation, geranylation, etc.), ubiquitination, ribosylation, or sulfation, or one or more post - translational modifications resulting from any combination of the foregoing.

[0074] The sPTH polypeptide of the present invention can function as an agonist (e.g., a biased agonist), an inverse agonist, or an antagonist of one or more PTHR signaling pathways. In some embodiments, one or more PTHR signaling pathways are Gα S -adenylyl cyclase - cAMP - protein kinase A (PKA) pathway (G s pathway), Gα q -phospholipase C (PLC)β - inositol trisphosphate - cytoplasmic Ca 2+ -protein kinase C pathway (G q pathway), Gα 12 / 13 -phospholipase D - transforming protein RhoA pathway (G 12 / 13 pathway), β - arrestin - extracellular signal - regulated kinase 1 / 2 (ERK1 / 2) pathway (β - arrestin pathway), and combinations thereof. In some embodiments, the PTHR signaling pathway is Gα S -adenylyl cyclase - cAMP - protein kinase A (PKA) pathway (G s pathway). In some embodiments, the PTHR signaling pathway is Gα q -phospholipase C (PLC)β - inositol trisphosphate - cytoplasmic Ca 2+ -protein kinase C pathway (G q pathway). In some embodiments, the PTHR signaling pathway is Gα 12 / 13 -phospholipase D - transforming protein RhoA pathway (G 12 / 13 pathway). In some embodiments, the PTHR signaling pathway is β - arrestin - extracellular signal - regulated kinase 1 / 2 (ERK1 / 2) pathway (β - arrestin pathway). Additional examples of PTHR signaling pathways are described in Cheloha RW et al., Nat Rev Endocrinol. 11(12):712 - 24(2015), the content of which is incorporated by reference in its entirety.

[0075] In some embodiments, the sPTH polypeptide of the present invention functions as an agonist of one or more PTHR signaling pathways. An "agonist of PTHR" binds and activates PTHR signaling. The agonists of PTHR provided by the present invention can be used, inter alia, to increase bone mass, increase bone formation, and / or reduce fractures in subjects (e.g., human patients) having or at increased risk of osteoporosis.

[0076] In some embodiments, the sPTH polypeptide that is an agonist of PTHR has improved activity (e.g., about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold or greater activity) compared to a reference PTH polypeptide (e.g., PTH1-34). In certain embodiments, the sPTH polypeptide that is an agonist of PTHR has equivalent or similar activity (e.g., about 90, 95, 97, 98, 99 or 100% activity) relative to a reference PTH polypeptide (e.g., PTH1-34). Non-limiting examples of activity include, for example, signaling activity via the Gs pathway, Gq pathway, G12 / 13 pathway, β-arrestin pathway, and combinations thereof.

[0077] In some embodiments, the sPTH polypeptide that is an agonist of PTHR has a lower binding constant (K D ) value (e.g., about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50% or less) compared to a reference PTH polypeptide (e.g., PTH1-34). In certain embodiments, the sPTH polypeptide that is an agonist of PTHR has an equivalent or similar K D value (e.g., about 96%, about 97%, about 98%, about 99%, about 100%, about 102%, about 105%, or about 110%) compared to a reference PTH polypeptide (e.g., PTH1-34). As used herein, "binding constant," also referred to as "equilibrium dissociation constant" or "affinity constant," "K DThe term " " is a measure of the degree of reversible association between two molecular species (e.g., an antibody and a target protein), and includes both actual binding affinity and apparent binding affinity. Binding affinity can be determined using methods known in the art, including, for example, measurements of surface plasmon resonance, such as biolayer interferometry (Octet, ForteBio) or surface plasmon resonance (Biacore) systems and assays. A reference comparing various surface technologies for measuring binding affinity and kinetics is Yang et al., Analytical Biochemistry 508:78-96 (2016), the content of which is incorporated herein by reference in its entirety.

[0078] In some embodiments, the sPTH polypeptide, which is an agonist of PTHR, comprises more than 14 amino acids (e.g., 32 amino acids, 34 amino acids), wherein the 14 amino acids at the N-terminus of the polypeptide correspond to SEQ ID NO: 6.

[0079] Specific examples of the sPTH polypeptide that is an agonist of PTHR include SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101-103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155, and 157-160. See, for example, Example 6 herein.

[0080] In some embodiments, the sPTH polypeptide of the present invention is selected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 136, SEQ ID NO: 143, SEQ ID NO: 145, and SEQ ID NO: 159. In some embodiments, the sPTH polypeptide of the present invention is SEQ ID NO: 145 or SEQ ID NO: 159. In some embodiments, the sPTH polypeptide of the present invention is selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 115, SEQ ID NO: 131, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 149, SEQ ID NO: 155, and SEQ ID NO: 157. In some embodiments, the sPTH polypeptide of the present invention is selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 115, SEQ ID NO: 131, SEQ ID NO: 136, SEQ ID NO: 143, SEQ ID NO: 155, and SEQ ID NO: 157. In some embodiments, the sPTH polypeptide of the present invention is selected from the group consisting of SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 158, and SEQ ID NO: 160. In some embodiments, the sPTH polypeptide of the present invention is selected from the group consisting of SEQ ID NO: 101, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 158, and SEQ ID NO: 160.

[0081] In some embodiments, the sPTH polypeptide of the invention functions as a biased agonist of one or more PTHR signaling pathways. A "biased agonist of PTHR" binds to PTHR and preferentially activates a distinct subset of intracellular signaling responses that are normally activated by the native ligand or that induce changes in signal duration and intracellular localization. See, e.g., Cheloha RW et al., Nat Rev Endocrinol. 11(12):712 - 24(2015) for further description. In some embodiments, the sPTH polypeptide acting as a PTHR biased agonist induces selective activation of one pathway independent of one or more other pathways. For example, the sPTH polypeptide acting as a PTHR biased agonist induces selective activation of the β-arrestin pathway without inducing activation of one or more G protein-coupled signaling mechanisms (G s pathway, G q pathway, G 12 / 13 pathway, or any combination thereof as described above). Thus, the use of a PTHR biased agonist can selectively induce anabolic bone formation and reduce side effects in a subject (e.g., a human patient) having or at high risk of osteoporosis to improve the therapeutic effect. In some embodiments, the sPTH polypeptide of the invention is a variant of a reference biased agonist. Non-limiting examples of reference biased agonists include D-Trp12 (D-Trp at position 12) and Tyr34-PTH(7-34) (Tyr at position 34) on the PTH(7-34) scaffold. See, e.g., Bohinc BN and Gesty-Palmer D, Mini Rev Med Chem. 12(9):856 - 65(2012).

[0082] In some embodiments, the sPTH polypeptide of the present invention functions as an inverse agonist of one or more PTHR signaling pathways. An "inverse agonist of PTHR" is a polypeptide that can bind to PTHR and inhibit the constitutively active PTHR or reduce basal PTHR signaling. See, e.g., Cheloha RW et al., Nat Rev Endocrinol. 11(12):712-24(2015). In some embodiments, the sPTH polypeptide acting as a PTHR inverse agonist is used to inhibit and / or reverse the over-activation of PTHR due to gain-of-function mutations. In some embodiments, the sPTH polypeptide acting as a PTHR inverse agonist is used to treat and / or prevent one or more indications. Non-limiting examples of indications include Jansen type metaphyseal chondrodysplasia, hypercalcemia, hypercalciuria, nephrocalcinosis, and chronic kidney disease. See, e.g., Saito H et al., J Clin Endocrinol Metab. 49:20-28(2018).

[0083] In some embodiments, the sPTH polypeptide of the present invention functions as an antagonist of one or more PTHR signaling pathways. An "antagonist of PTHR" is a polypeptide that binds to PTHR and prevents changes in PTHR signaling by agonists or inverse agonists. See, e.g., Cheloha RW et al., Nat Rev Endocrinol. 11(12):712-24(2015). In some embodiments, the sPTH polypeptide acting as a PTHR antagonist is used to inhibit the over-activation of PTHR. In some embodiments, the sPTH polypeptide acting as a PTHR antagonist is used to treat and / or prevent one or more indications. Non-limiting examples of indications include hypercalcemia, hyperparathyroidism, parathyroid cancer, and metastatic bone disease. See, e.g., Mirza AM et al., AACR Annual Meeting 2017.

[0084] In some embodiments, PTHR signaling is evaluated by a luciferase assay, which can be performed according to Hattersly et al., Endocrinology 157(1):141-49(2016) or Kumar et al., 2007, which are hereby incorporated by reference in their entirety. The sPTH polypeptide-mediated effect on PTHR signaling (e.g., agonist activity or inverse agonist activity) can be evaluated, for example, by a FRET (Fluorescence Resonance Energy Transfer) assay. For example, luciferase-tagged β-arrestin and YFP-tagged GPCR are used. In some embodiments, the sPTH polypeptide-mediated effect (e.g., inverse agonist activity or antagonist activity) is evaluated by detecting a decrease in cAMP levels. In some embodiments (e.g., inverse agonist activity), the background cAMP level is determined using a constitutively active receptor, e.g., comprising one or more constitutive mutations. Non-limiting examples of constitutive mutations include H223R, T410P, or I458R or combinations thereof underlying Jansen type metaphyseal chondrodysplasia. In some embodiments (e.g., antagonist activity), the background cAMP level is determined using a native ligand. A detailed description of the assays can be found in Example 2B (antagonist activity) and Example 2C (inverse agonist activity).

[0085] In some embodiments, the sPTH polypeptide is conjugated to a heterologous moiety. The term "conjugated" refers to binding via a covalent or non-covalent interaction. Conjugation can be performed using any suitable linker. Non-limiting examples include peptide linkers, compound linkers, and chemical cross-linking agents.

[0086] In some embodiments, the heterologous moiety is a therapeutic agent, a diagnostic agent, or a combination thereof. In some embodiments, the heterologous moiety is polyethylene glycol (PEG), hexadecanoic acid, a hydrogel, a nanoparticle, a multimerization domain, and a carrier peptide.

[0087] In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is a polymer nanoparticle. In some embodiments, the polymer is an amphiphilic polymer. In other embodiments, the polymer is a hydrophobic or hydrophilic polymer. Non-limiting examples of polymers include poly(lactic acid)-poly(ethylene glycol), poly(lactic-co-glycolic acid)-poly(ethylene glycol), poly(lactic-co-glycolic) acid (PLGA), poly(lactic-co-glycolic acid)-d-α-tocopheryl polyethylene glycol succinate, poly(lactic-co-glycolic acid)-ethylene oxide fumarate, poly(glycolic acid)-poly(ethylene glycol), polycaprolactone-poly(ethylene glycol), or salts thereof. In some embodiments, the polymer nanoparticle comprises poly(lactic-co-glycolic) acid (PLGA).

[0088] [Fusion protein] In another aspect, the present invention provides a fusion protein comprising one or more of the SPTHs described herein.

[0089] The term "fusion protein" refers to a synthetic, semi-synthetic, or recombinant single protein molecule. A fusion protein can comprise all or part of two or more different proteins and / or polypeptides that are joined by a covalent bond (e.g., a peptide bond).

[0090] The fusion protein of the present invention can be produced recombinantly or synthetically using conventional methods and reagents well known in the art. For example, the fusion protein of the present invention can be produced recombinantly in a suitable host cell (e.g., bacteria) according to methods known in the art. See, for example, Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992; and Molecular Cloning: a Laboratory Manual, 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, a nucleic acid molecule containing a nucleotide sequence encoding the fusion protein described herein can be introduced and expressed in a suitable host cell (e.g., Escherichia coli (E. coli)), and the expressed fusion protein can be isolated / purified from the host cell (e.g., in inclusion bodies) using conventional methods and readily available reagents. For example, DNA fragments encoding different protein sequences (e.g., a photosensitive domain, a heterologous peptide component) can be ligated together in frame according to conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including an automated DNA synthesizer. Alternatively, PCR amplification of nucleic acid fragments can be performed using anchor primers that produce complementary overhangs between two consecutive nucleic acid fragments, followed by annealing and reamplification to generate chimeric nucleic acid sequences (see Ausubel et al., Current Protocols in Molecular Biology, 1992).

[0091] [Nucleic Acid, Expression Vector, Expression Host Cell] In another aspect, the present invention provides one or more polynucleotides encoding any one of the SPTH polypeptides or fusion proteins described herein. In some embodiments, the SPTH polypeptide or fusion protein of the present invention is encoded by a single polynucleotide. In some embodiments, the SPTH polypeptide or fusion protein of the present invention is encoded by a plurality of polynucleotides.

[0092] In some embodiments, the SPTH polynucleotide comprises a nucleotide sequence that is codon-optimized for a selected host cell.

[0093] In another aspect, the present invention provides an expression vector comprising any one or more of the polynucleotides described herein.

[0094] The term "expression vector" refers to a replicable nucleic acid that can express one or more proteins when the expression vector is transformed into a suitable expression host cell.

[0095] In some embodiments, the expression vector further comprises an expression control polynucleotide sequence operably linked to the polynucleotide, a polynucleotide sequence encoding a selectable marker, or both. In some embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In some embodiments, the expression control polynucleotide sequence comprises an inducible promoter sequence. The term "promoter" refers to a region of DNA to which RNA polymerase binds to initiate transcription of a gene. The term "operably linked" means that a nucleic acid is positioned in a recombinant polynucleotide, such as a vector, in such a way that it enables expression of the nucleic acid under the control of an element (e.g., a promoter) to which it is linked. The term "selectable marker element" refers to an element that confers a trait suitable for artificial selection. A selectable marker element can be a negative or positive selectable marker.

[0096] In another aspect, the present invention provides an expression host cell comprising any one or more of the polynucleotides or expression vectors described herein.

[0097] The term "expression host cell" refers to a cell useful for receiving, maintaining, replicating, and / or amplifying a vector.

[0098] Non-limiting examples of expression host cells include mammalian cells such as Chinese hamster ovary (CHO) cells, COS cells, human embryonic kidney (HEK), yeast cells such as Pichia pastoris cells, or bacterial cells such as DH5α.

[0099] [Composition] In another aspect, the present invention provides a composition comprising any one of the polypeptides or fusion proteins described herein. In some embodiments, the composition is a pharmaceutical composition.

[0100] In some embodiments, the composition (e.g., pharmaceutical composition) further comprises a pharmaceutically acceptable carrier, excipient, stabilizer, diluent, or tonicity agent (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents, or tonicity agents include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, or dextrin); chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-protein complexes); nonionic surfactants (e.g., Tween), PLURONICS® and polyethylene glycol (PEG).

[0101] In some embodiments, the compositions of the invention (e.g., pharmaceutical compositions) are formulated for an appropriate dosing schedule and route. Non-limiting examples of administration routes include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical, among others. In some embodiments, the compositions of the invention (e.g., pharmaceutical compositions) are stored in the form of an aqueous solution or a dry formulation (e.g., lyophilized).

[0102] In some embodiments, the composition is formulated to be administered by infusion (e.g., intravenous infusion). In some embodiments, the composition is formulated to be administered with a second therapeutic agent as combination therapy.

[0103] [Method for Preparing sPTH] The SPTH polypeptide, its conjugate, its fusion protein, and the compositions of the present invention can be produced by any suitable means known in the art. In some embodiments, methods of chemical synthesis are used. In some embodiments, methods of recombinant production / expression are used. In some embodiments, a combination of chemical synthesis and recombinant production / expression is used. For example, the SPTH polypeptide component is produced recombinantly and then the heterologous moiety is conjugated chemically. In some embodiments, the host cells of the present invention (e.g., containing the SPTH polynucleotide or expression vector of the present invention) are cultured under appropriate conditions to produce sPTH, which is then isolated from the host cells or the culture supernatant, thereby producing sPTH.

[0104] [Methods of using sPTH] In another aspect, the present invention provides a method of modulating PTHR signaling in a cell (e.g., a mammalian cell) comprising contacting the cell with an effective amount of a polypeptide, fusion protein, or composition described herein (e.g., a pharmaceutical composition).

[0105] In another aspect, the present invention provides a method of modulating PTHR signaling in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide, fusion protein, or composition described herein (e.g., a pharmaceutical composition).

[0106] In some embodiments, modulation of PTHR signaling involves agonist activity (e.g., biased agonist activity), inverse agonist activity, or antagonist activity of PTHR signaling, or a combination of the above. In some embodiments, modulation of PTHR signaling involves agonist activity of PTHR signaling. In some embodiments, modulation of PTHR signaling involves biased agonist activity of PTHR signaling. In some embodiments, modulation of PTHR signaling involves inverse agonist activity of PTHR signaling. In some embodiments, modulation of PTHR signaling involves antagonist activity of PTHR signaling.

[0107] In another aspect, the invention provides a method of treating a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition described herein.

[0108] In some embodiments, treating a subject in need thereof involves agonist activity (e.g., biased agonist activity), inverse agonist activity, or antagonist activity of PTHR signaling, or a combination of the above. In some embodiments, treating a subject in need thereof involves agonist activity of PTHR signaling. In some embodiments, treating a subject in need thereof involves biased agonist activity of PTHR signaling. In some embodiments, treating a subject in need thereof involves inverse agonist activity of PTHR signaling. In some embodiments, treating a subject in need thereof involves antagonist activity of PTHR signaling.

[0109] The terms "subject" or "patient" refer to an animal (e.g., a mammal). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is at least 50 years old. In some embodiments, the subject is a female. In some embodiments, the subject is in the perimenopausal, menopausal, or postmenopausal period. In some embodiments, the subject is in the perimenopausal period. In some embodiments, the subject is in the menopausal period. In some embodiments, the subject is in the postmenopausal period.

[0110] A subject treated according to the methods described herein can be a person diagnosed with a condition or at risk of developing such a condition. In some embodiments, the subject has the condition. In some embodiments, the subject has been diagnosed with the condition. In other embodiments, the subject is at risk of developing the condition. Exemplary conditions (indications) treatable by the methods provided by the present invention include those described in Tables 4-6 below or in the claims, or in the above-listed embodiments.

[0111] [Table 7]

[0112] [Table 8]

[0113] [Table 9]

[0114] [Table 10]

[0115] [Table 11]

[0116] In some embodiments, the subject has or is at risk of developing dysregulated calcium homeostasis.

[0117] In some embodiments, the subject has or is at risk of developing a disorder selected from osteoporosis, brachydactyly type A2 chondrodysplasia, familial idiopathic failure of tooth eruption, Eiken syndrome, Ollier disease, hypercalcemia, hyperparathyroidism, Jansen type metaphyseal chondrodysplasia, hypercalcemia, hypercalciuria, nephrocalcinosis, and combinations thereof.

[0118] In some embodiments, the subject has or is at risk of developing low bone density.

[0119] In some embodiments, the subject has or is at risk of developing a disorder selected from brachydactyly type lethal chondrodysplasia, Ollier disease, familial idiopathic failure of tooth eruption, Eiken syndrome, short digit type E, hypoparathyroidism, osteoporosis, and combinations thereof.

[0120] In some embodiments, the subject has or is at risk of developing osteoporosis.

[0121] In some embodiments, the subject has or is at high risk of developing a disorder selected from hypercalcemia, hyperparathyroidism, parathyroid cancer, metastatic bone disease, and combinations thereof. In some embodiments, hyperparathyroidism occurs as a complication of parathyroid cancer. In some embodiments, metastatic bone disease occurs with associated hypercalcemia.

[0122] In some embodiments, the subject has, or is at high risk of developing, a disease selected from Jansen type metaphyseal chondrodysplasia, hyperparathyroidism, hypercalcemia, hypercalciuria, nephrocalcinosis, chronic kidney disease, and combinations thereof. In some embodiments, chronic kidney disease occurs in association with, or as a result of, hypercalcemia, hypercalciuria, nephrocalcinosis, or combinations thereof.

[0123] Diagnosis can be performed by any method or technique known in the art. One of ordinary skill in the art will understand that a subject to be treated according to the present disclosure may have undergone standard tests or may have been identified as being at risk due to the presence of one or more risk factors associated with a disease or condition without testing.

[0124] The sPTH (and compositions, conjugates (e.g., including heterologous moieties supported by binding or other means (e.g., by covalent bonding)), and fusion proteins containing them; or nucleic acids, vectors, or cells encoding them) provided by the present invention can be used in a variety of situations to modulate PTHR signaling, e.g., as an agonist of PTHR, a biased agonist of PTHR, an inverse agonist of PTHR, or an antagonist of PTHR. Modulation of PTHR signaling can be in vitro, intracellularly, or in vivo, e.g., in a subject. Exemplary indications treatable by the methods provided by the present invention include those described in Tables 2-4 below or in the claims, or in the embodiments listed above.

[0125] In these methods, sPTH (composition, conjugate, fusion protein, or nucleic acid or cell) is provided to a subject in an effective amount by appropriate means. The terms "effective amount," "therapeutically effective amount," or "sufficient amount" refer to an amount sufficient to produce a beneficial or desired result, including an effect at the cellular, tissue, or clinical level, when administered to a subject, including a mammal (e.g., a human), and thus the "effective amount" or its synonyms will depend on the context in which it is applied. For example, in some embodiments, this is an amount of the composition sufficient to achieve a treatment response as compared to the response obtained without administration of the composition. The amount of a given composition described herein corresponding to such an amount will vary depending on various factors such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject (e.g., age, gender, weight) or host being treated, but can nevertheless be determined by one of ordinary skill in the art in a conventional manner. Also, as used herein, a "therapeutically effective amount" of the compositions of the present disclosure is an amount that produces a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of the compositions of the present disclosure can be readily determined by one of ordinary skill in the art by conventional methods known in the art. The dosing regimen and route of administration can be adjusted to provide an optimal therapeutic response.

[0126] In some embodiments, the amount is effective to promote calcium homeostasis, increase, maintain, or decrease a reduction in bone mass, reduce the risk of fracture, promote fracture healing, or a combination of the foregoing.

[0127] In some embodiments, the effective amount does not induce catabolic bone resorption.

[0128] As used herein, "treatment" and "treating" refer to the medical management of a subject aimed at improving, alleviating, stabilizing (i.e., not worsening), preventing or curing a disease, medical condition, or disorder (such as the specific indications exemplified herein). This term includes active treatment (treatment directed at improving a disease, medical condition, or disorder), causative treatment (treatment directed at the cause of a related disease, medical condition, or disorder), palliative treatment (treatment designed to relieve symptoms), prophylactic treatment (treatment directed at minimizing or partially or completely inhibiting the onset of a related disease, medical condition, or disorder); and adjunctive treatment (treatment utilized to supplement another therapy). Treatment includes a detectable or undetectable decrease in the degree of a disease or condition; prevention of the expansion of a disease or condition; delay or deceleration of the progression of a disease or condition; improvement or alleviation of a disease or condition; and remission (partial or complete). "Improving" or "alleviating" a disease or condition means that the degree and / or undesirable clinical symptoms of the disease, disorder, or condition are reduced, and / or the time course of progression is decelerated or lengthened as compared to the degree or time course in the absence of treatment. "Treatment" can also mean prolonging survival as compared to survival expected in the absence of treatment. Persons in need of treatment include those who already have the condition or disease, those who are prone to developing the condition or disease, or those in whom the condition or disease should be prevented.

[0129] The features of the sPTH (and conjugates, fusion proteins, and compositions containing them; or nucleic acids, vectors, or cells encoding them) provided by the present invention, or methods of using the same, may include one or more of the following recited embodiments.

[0130] Embodiment 1: A polypeptide that specifically binds to a parathyroid hormone receptor (PTHR), wherein the polypeptide comprises a synthetic parathyroid hormone (sPTH).

[0131] Embodiment 2: The sPTH is a) 1 to 3 polar residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 6; b) at least 4 polar residues, at least 1 charged residue, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7; c) at least 5 polar residues, at least 2 charged residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7; d) at least 5 polar residues, valine at position 2, and 1 to 13 amino acid substitutions relative to SEQ ID NO: 7; or e) at least 6 polar residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7 A polypeptide according to embodiment 1, comprising a 14 - amino - acid sequence, comprising:

[0132] Embodiment 3: sPTH is a) up to 13 polar residues, and 1 to 32 amino acid substitutions relative to SEQ ID NO: 8; b) at least 14 polar residues, valine at position 2, and 1 to 31 amino acid substitutions relative to SEQ ID NO: 8; c) at least 14 polar residues, and 22 to 28 amino acid substitutions relative to SEQ ID NO: 8; or d) positions 1 to 14 of SEQ ID NO: 8, and 1 to 18 amino acid substitutions at positions 15 to 32 of SEQ ID NO: 5 A polypeptide according to embodiment 1, comprising a 32 - amino - acid sequence, comprising:

[0133] Embodiment 4: A polypeptide according to embodiment 1, wherein sPTH comprises an amino acid sequence having at least about 20% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 108 - 160.

[0134] Embodiment 5: A polypeptide according to embodiment 1, wherein sPTH comprises an amino acid sequence having about 50 - 97% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 108 - 160.

[0135] Embodiment 6: A polypeptide according to embodiment 1, wherein sPTH comprises at least one amino acid substitution relative to an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 108 - 160.

[0136] Embodiment 7: The polypeptide according to Embodiment 1, wherein sPTH contains a maximum of 2 to 31 amino acid substitutions with respect to the amino acid sequences selected from SEQ ID NOs: 9 to 106 and SEQ ID NOs: 108 to 160.

[0137] Embodiment 8: The polypeptide according to Embodiment 1, wherein sPTH contains a maximum of 2 to 18 amino acid substitutions with respect to the amino acid sequences selected from SEQ ID NOs: 9 to 106 and SEQ ID NOs: 108 to 160.

[0138] Embodiment 9: The polypeptide according to Embodiment 1, wherein sPTH contains the amino acid sequences selected from SEQ ID NOs: 9 to 106 and SEQ ID NOs: 108 to 160.

[0139] Embodiment 10: The polypeptide according to Embodiment 1, wherein sPTH contains an amino acid sequence having at least about 20% sequence identity with respect to the amino acid sequences selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101 to 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155, and 157 to 160.

[0140] Embodiment 11: The polypeptide according to Embodiment 1, wherein sPTH contains an amino acid sequence having about 50 to 97% sequence identity with respect to the amino acid sequences selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101 to 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155, and 157 to 160.

[0141] Embodiment 12: The polypeptide according to Embodiment 1, wherein sPTH contains at least one amino acid substitution with respect to the amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101 to 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155 and 157 to 160.

[0142] Embodiment 13: The polypeptide according to Embodiment 1, wherein sPTH contains a maximum of 2 to 31 amino acid substitutions with respect to the amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101 to 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155 and 157 to 160.

[0143] Embodiment 14: The polypeptide according to Embodiment 1, wherein sPTH contains a maximum of 2 to 18 amino acid substitutions with respect to the amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101 to 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155 and 157 to 160.

[0144] Embodiment 15: The polypeptide according to Embodiment 1, wherein sPTH contains the amino acid sequence selected from SEQ ID NOs: 9, 10, 19, 20, 31, 35, 41, 88, 101 to 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155 and 157 to 160.

[0145] Embodiment 16: The polypeptide according to Embodiment 1, wherein sPTH consists of 28 to 36 amino acids.

[0146] Embodiment 17: The polypeptide according to Embodiment 1, wherein sPTH consists of 30 to 34 amino acids.

[0147] Embodiment 18: The sPTH is the polypeptide according to Embodiment 1, which consists of 32 amino acids.

[0148] Embodiment 19: The polypeptide according to any one of Embodiments 16 to 18, wherein the sPTH comprises an amino acid sequence having at least about 20% sequence identity to SEQ ID NO: 5.

[0149] Embodiment 20: The polypeptide according to Embodiment 19, wherein the sPTH comprises an amino acid sequence having about 50 - 97% sequence identity to SEQ ID NO: 5.

[0150] Embodiment 21: The polypeptide according to Embodiment 19, wherein the sPTH comprises at least one amino acid substitution with respect to SEQ ID NO: 5.

[0151] Embodiment 22: The polypeptide according to Embodiment 19, wherein the sPTH comprises 2 - 18 amino acid substitutions with respect to SEQ ID NO: 5.

[0152] Embodiment 23: The polypeptide according to Embodiment 19, wherein the sPTH comprises the amino acid sequence shown in SEQ ID NO: 6.

[0153] Embodiment 24: The polypeptide according to Embodiment 23, wherein the amino acid sequence shown in SEQ ID NO: 6 is at the N - terminus of the sPTH.

[0154] Embodiment 25: The polypeptide according to Embodiment 19, wherein the sPTH comprises an amino acid sequence having at least about 55% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 102 - 106 and SEQ ID NOs: 132 - 155.

[0155] Embodiment 26: The polypeptide according to Embodiment 19, wherein the sPTH comprises an amino acid sequence having about 75 - 97% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 102 - 106 and SEQ ID NOs: 132 - 155.

[0156] Embodiment 27: The polypeptide according to Embodiment 1, wherein sPTH contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 102 to 106 and SEQ ID NOs: 132 to 155.

[0157] Embodiment 28: The polypeptide according to Embodiment 1, wherein sPTH contains the amino acid sequence of SEQ ID NO: 107.

[0158] Embodiment 29: The polypeptide according to any one of Embodiments 6, 12, and 21, wherein at least one amino acid substitution is a conservative substitution.

[0159] Embodiment 30: The polypeptide according to Embodiment 29, wherein at least one amino acid substitution is a highly conservative substitution.

[0160] Embodiment 31: The polypeptide according to any one of Embodiments 2, 3, 7, 8, 13, 14, and 22, wherein the amino acid substitution is a conservative substitution.

[0161] Embodiment 32: The polypeptide according to Embodiment 31, wherein the amino acid substitution is a highly conservative substitution.

[0162] Embodiment 33: The polypeptide according to any one of Embodiments 1 to 32, wherein sPTH binds to PTHR with an affinity that is at least about 5% higher than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0163] Embodiment 34: The polypeptide according to any one of Embodiments 1 to 32, wherein sPTH binds to PTHR with an affinity that is at least about 5% lower than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0164] Embodiment 35: The polypeptide according to any one of Embodiments 1 to 32, wherein sPTH binds to PTHR with an affinity that is about 5% or less lower than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0165] Embodiment 36: The polypeptide according to any one of Embodiments 1 to 35, wherein sPTH is an agonist of PTHR.

[0166] Embodiment 37: The polypeptide according to Embodiment 36, wherein sPTH is a biased agonist of PTHR.

[0167] Embodiment 38: The polypeptide according to any one of Embodiments 1 to 35, wherein sPTH is an inverse agonist of PTHR.

[0168] Embodiment 39: The polypeptide according to any one of Embodiments 1 to 35, wherein sPTH is an antagonist of PTHR.

[0169] Embodiment 40: The polypeptide according to any one of Embodiments 1 to 39, wherein the polypeptide is conjugated to a heterologous moiety.

[0170] Embodiment 41: The polypeptide according to Embodiment 40, wherein the heterologous moiety is selected from the group consisting of polyethylene glycol (PEG), hexadecanoic acid, hydrogel, lipid nanoparticles, polymer nanoparticles, and heterologous polypeptide sequences, or combinations thereof.

[0171] Embodiment 42: The polypeptide according to Embodiment 41, wherein the polymer nanoparticles contain poly(lactic-co-glycolic) acid (PLGA).

[0172] Embodiment 43: The polypeptide according to Embodiment 40, wherein the heterologous polypeptide sequence contains a carrier polypeptide.

[0173] Embodiment 44: The polypeptide according to embodiment 43, wherein the carrier polypeptide is albumin or an Fc polypeptide.

[0174] Embodiment 45: A fusion protein comprising the polypeptide according to any one of embodiments 1 to 44.

[0175] Embodiment 46: A polynucleotide comprising a sequence encoding the polypeptide according to any one of embodiments 1 to 44 or the fusion protein according to embodiment 45.

[0176] Embodiment 47: An expression vector comprising the polynucleotide of embodiment 46.

[0177] Embodiment 48: A host cell comprising the polynucleotide according to embodiment 46 or the expression vector according to embodiment 47.

[0178] Embodiment 49: A composition comprising the polypeptide according to any one of embodiments 1 to 44 or the fusion protein according to embodiment 45.

[0179] Embodiment 50: The composition according to embodiment 49, further comprising one or more pharmaceutical excipients, diluents, or carriers.

[0180] Embodiment 51: A method of regulating PTHR signaling in mammalian cells, comprising contacting mammalian cells with an effective amount of the composition of embodiment 49 or 50.

[0181] Embodiment 52: A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the composition of embodiment 49 or 50.

[0182] Embodiment 53: A method of regulating PTHR signaling in a subject in need thereof, comprising administering to the subject an effective amount of the composition of embodiment 49 or 50.

[0183] Embodiment 54: The method according to embodiment 52 or 53, wherein the subject is a mammal.

[0184] Embodiment 55: The method according to embodiment 54, wherein the subject is human.

[0185] Embodiment 56: The method according to any one of embodiments 52 to 55, wherein the subject is an adult.

[0186] Embodiment 57: The method according to any one of embodiments 52 to 56, wherein the subject is at least 50 years old.

[0187] Embodiment 58: The method according to any one of embodiments 52 to 57, wherein the subject is female.

[0188] Embodiment 59: The method according to embodiment 58, wherein the subject is in the perimenopausal, menopausal, or postmenopausal period.

[0189] Embodiment 60: The method according to any one of embodiments 52 to 59, wherein the subject has or is at risk of developing dysregulated calcium homeostasis.

[0190] Embodiment 61: The method according to any one of embodiments 52 to 59, wherein the subject has or is at risk of developing a disease selected from osteoporosis, Blomstrand chondrodysplasia, familial primary failure of tooth eruption, Eiken syndrome, Ollier disease, hypercalcemia, hyperparathyroidism, Jansen metaphyseal chondrodysplasia, hypercalcemia, hypercalciuria, nephrocalcinosis, and combinations thereof.

[0191] Embodiment 62: The method according to embodiment 61, wherein sPTH is an agonist of PTHR.

[0192] Embodiment 63: The method according to embodiment 62, wherein sPTH is a biased agonist of PTHR.

[0193] Embodiment 64: The method according to any one of embodiments 52 to 63, wherein the subject has or is at risk of developing low bone density.

[0194] Embodiment 65: The method according to any one of Embodiments 52 to 59, wherein the subject has a disease selected from the group consisting of brachydactyly type lethal chondrodysplasia, Ollier disease, familial primary failure of tooth eruption, Eiken syndrome, brachydactyly type E, hypoparathyroidism, osteoporosis, and combinations thereof, or is at risk of developing the same.

[0195] Embodiment 66: The method according to any one of Embodiments 52 to 59, wherein the subject has osteoporosis or is at risk of developing the same.

[0196] Embodiment 67: The method according to any one of Embodiments 52 to 66, wherein the amount is effective in promoting calcium homeostasis, increasing, maintaining or decreasing the reduction of bone mineral content, reducing the risk of fracture, promoting fracture healing, or a combination of the foregoing.

[0197] Embodiment 68: The method according to any one of Embodiments 52 to 67, wherein the effective amount does not induce catabolic bone resorption.

[0198] Embodiment 69: The method according to any one of Embodiments 52 to 59, wherein the subject has a disease selected from the group consisting of hypercalcemia, hyperparathyroidism, parathyroid cancer, metastatic bone disease, and combinations thereof, or is at high risk of developing the same.

[0199] Embodiment 70: The method according to Embodiment 69, wherein hyperparathyroidism occurs as a complication of parathyroid cancer.

[0200] Embodiment 71: The method according to Embodiment 69 or 70, wherein metastatic bone disease occurs with associated hypercalcemia.

[0201] Embodiment 72: The method according to any one of Embodiments 52 to 59, wherein the subject has a disease selected from the group consisting of Jansen metaphyseal chondrodysplasia, hyperparathyroidism, hypercalcemia, hypercalciuria, nephrocalcinosis, chronic kidney disease, and combinations thereof, or is at high risk of developing the same.

[0202] Embodiment 73: The method according to Embodiment 72, wherein the chronic kidney disease occurs in association with or as a result of hypercalcemia, hypercalciuria, nephrocalcinosis, or a combination thereof.

Examples

[0203] Sequences predicted to bind and activate the PTHR were generated using computational models and elucidated structures of the PTHR, including a recent CryoEM report published in Zhao LH et al., Science 364, 148 - 53 (2019). Sequences predicted to optimally bind the receptor while retaining a structure similar to the native ligand. The family of sequences was generated as described in Table 2.

[0204] Example 1. Peptide Synthesis Peptides are produced using one of two methods known in the art: recombinant production from bacteria or chemical synthesis.

[0205] In the case of recombinant production, the DNA sequence of the peptide is encoded in a DNA vector suitable for bacterial production, such as a PET vector. The bacteria are transformed with the plasmid and grown in an appropriate growth medium. When the bacteria reach the logarithmic growth phase, translation of the plasmid is induced, for example, by IPTG. The protein is collected from the bacteria by lysis and recovery from inclusion bodies.

[0206] For solid - phase chemical synthesis, the standard method is described in Stawikowski and Fields, Curr Protoc Protein Sci 2002, Feb. Chapter 18.1, available at the following Uniform Resource Locator: www.ncbi.nlm.nih.gov / pmc / articles / PMC3564544.

[0207] Example 2. In vitro Testing of Receptor Activity Regarding Agonism, Antagonism, and Inverse Agonism To measure PTHR activity, standard methods such as cAMP accumulation assays as described, for example, in Hattersley G et al., Endocrinology, 157(1):141-49(2016) are used. These assays are performed at room temperature on cells transfected with plasmid DNA encoding the PTHR variant (sPTHR) provided by the present invention. Standard kits, for example, Promega's cAMP-Glo™ assay, are used to measure intracellular cAMP levels in a homogeneous bioluminescence high-throughput assay that monitors cAMP production (see, for example, Kumar M et al., Assay Drug Dev Technol. 5(2):237-45(2007). The assay is based on the principle that cyclic AMP (cAMP) stimulates protein kinase A (PKA) holoenzyme activity, thereby reducing the available ATP and decreasing light production in the coupled luciferase reaction.

[0208] The assay is performed according to the manufacturer's protocol. Cells are treated with peptides at various concentrations. The cells are then lysed with lysis buffer. After adding the PKA reaction mix, the cell lysate is incubated for 20 minutes. Next, the development buffer containing the Kinase-Glo reagent is added, and the plate is incubated for 10 minutes. Finally, luminescence is measured using a plate-reading luminometer.

[0209] a) Agonist activity The peptides produced are tested for receptor activation in a cell line expressing PTHR recombinantly, as described, for example, in Gardella et al., Endocrinology 137(9):3936-41(1996). Briefly, COS-7 cells are cultured in DMEM supplemented with 10% FBS. The cells are transfected with DEAE / dextran and plasmid DNA encoding PTHR cDNA (100 ng / well). The receptor assay is performed on intact COS-7 cells (500,000 cells / well) 3 days after transfection.

[0210] In order to test agonist activity, cells are treated with agonist peptides at various concentrations. Next, PTHR activation is measured using the cAMP accumulation assay described herein (e.g., the cAMP-Glo™ assay). Activation of PTHR by an agonist peptide is expected to result in the accumulation of cAMP within the cells, which is reflected as a higher luminescence reading than in control or untreated cells.

[0211] b) Antagonist activity The peptides produced are tested for their ability to inhibit receptor activation in cell lines recombinantly expressing PTHR as described in the agonist activity section. To test antagonist activity, cells are treated with antagonist peptides at various concentrations together with a fixed concentration of 1 nM of a validated agonist (e.g., PTH1-34) as described by Shimizu N et al., J Biol Chem. 280(3):1797-807 (2005).

[0212] Next, PTHR activity is measured using the cAMP accumulation assay described herein (e.g., the cAMP-Glo™ assay). Inhibition of PTHR activation by an antagonist peptide prevents the accumulation of cAMP within the cells, which is reflected as a lower luminescence reading than in cells treated with the validated agonist.

[0213] c) Inverse agonist activity The generated peptides are tested for their ability to reduce the basal receptor activity of cell lines recombinantly expressing gain-of-function mutants of PTHR, as described, for example, in Saito H et al., J Clin Endocrinol Metab. 103(7):2660-69 (2018). Briefly, HEK293 cells are seeded into 96-well plates at a density of 20,000 cells / well and cultured in DMEM supplemented with 10% FBS. The cells are transfected with DEAE / dextran and plasmid DNA (100 ng / well) encoding a gain-of-function mutant of PTHR (e.g., H223R). The receptor assay is performed on intact HEK293 cells (100,000 cells / well) 2 days after transfection.

[0214] The cells are treated with various concentrations of inverse agonist peptide. Next, mutant PTHR activation is measured using the cAMP accumulation assay described herein (e.g., the cAMP-Glo™ assay). A decrease in basal mutant PTHR activity is expected to lead to a decrease in intracellular cAMP, which is reflected as a lower luminescence readout than in control or untreated cells.

[0215] Example 3. In Vitro Testing of Receptor Activity for Biased Agonism To test PTHR biased β-arrestin signaling, for example, standard methods for measuring β-arrestin mobilization as described in Wang T et al., Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004 - 2017 Nov 20 are performed. Standard kits include, for example, the DiscoverX PathHunter β-arrestin assay. A CHO cell line that stably expresses PTHR extended at the carboxyl terminus with a peptide fragment of β-galactosidase and the corresponding deletion mutant of β-galactosidase fused to β-arrestin is used. This assay measures the level of reconstituted β-galactosidase in a homogeneous bioluminescence high-throughput assay. The assay is based on the principle that β-arrestin mobilization results in the reconstitution of active β-galactosidase, which catalyzes a bioluminescence reaction from an exogenous substrate to generate a bioluminescence reading.

[0216] The assay is performed according to the manufacturer's protocol. Cells are treated with peptides at various concentrations. Next, the cells are incubated with the provided Working Detection Solution in the dark at room temperature for 1 hour. Finally, luminescence is measured using a plate-reading luminometer.

[0217] An increase in β-arrestin mobilization to PTHR is expected to lead to an increase in reconstituted active β-galactosidase within the cell, which is reflected as a higher luminescence reading compared to control or untreated cells.

[0218] Example 4. In Vivo Activity Testing Peptides are evaluated for functional activity in a standard laboratory model of osteoporosis, the ovariectomized rat model. In this model, after surgical ovariectomy, bone resorption initially exceeds bone formation, resulting in bone loss. Soon afterwards, bone tissue repair reaches a steady state and a balance between resorption and formation is achieved. Statistically significant bone loss is seen at the proximal tibial metaphysis after 14 days, at the vertebral bodies after 60 days, and at the femoral neck after 30 days (see, for example, Lelovas et al., Comp Med 58(5):424-30(2008)).

[0219] Peptides are administered daily by subcutaneous injection to experimental rats (see, for example, Bernhardsson M et al., Acta Orthop 89(6):674-77(2018)). Bone density is measured non-invasively by densitometry and micro-computed tomography (MicroCT), and invasively by histomorphometry and mechanical strength evaluation including three-point bending, four-point bending, and torsion tests.

[0220] Peptides that signal via PTHR (e.g., agonists or biased signaling agonists of PTHR) are expected to lead to a reduction in bone loss in ovariectomized rats, and thus increase bone density and bone strength compared to untreated control animals.

[0221] Example 5. Treatment of human patients with osteoporosis Patients are diagnosed as having or being at risk of osteoporosis, e.g., postmenopausal women aged 49 - 86 years with a bone mineral density T-score of -2.5 or less and greater than -5.0 as evaluated by dual energy X-ray absorptiometry (see Miller PD et al., JAMA 316(7):722-33(2016)). Patients are prescribed treatment with a peptide as described herein, e.g., an agonist of PTHR or a biased signaling agonist of PTHR.

[0222] Methods for the administration of PTHR agonists and bone density assessment are described, for example, in Miller PD et al., JAMA 316(7):722-33(2016). The peptide is administered daily by subcutaneous injection, and the patient's bone mineral content is monitored by dual-energy X-ray absorptiometry.

[0223] Example 6. Bioluminescence Resonance Energy Transfer (BRET) Assay Synthesis: The peptide was synthesized by Genscript using standard synthetic methods and purified using TFA. The peptide was shipped lyophilized and resuspended in DMSO before use.

[0224] a) Measurement of β-arrestin mobilization HEK293T cells (CRL-3216, ATCC, VA) at a density of 2 million / mL were transfected with β-arrestin 1 or β-arrestin 2-Rluc cDNA (1 ng / well) (Life Technologies Corporation, CA) and Flag-PTHR1-YFP cDNA (199 ng / well) (Genwiz, New Jersey) using polyethyleneimine (0.8 μL / well) (Polysciences, PA). 18 - 24 hours after transfection, the luciferase substrate coelenterazine (NanoLight Technologies, AZ) was added to the cells, and baseline BRET measurements were taken after a 5-minute incubation. Next, serial dilutions of the peptide diluted in PBS (0.5 mM MgCl 2 + 0.1% BSA FAF) were added to the cells, and the BRET signal was measured every 2 minutes for 30 minutes using a hybrid multimode reader (Synergy Neo2, BioTek, VT). 75 peptides were tested in the β-arrestin mobilization assay.

[0225] b) Measurement of cAMP accumulation CHO-K1 / Ga15 / PTHR1 (Genscript USA, NJ) cells were cultured in 10 cm Petri dishes and harvested when the confluence of the cell culture reached 80 - 85%. A cell suspension (5 μL) was seeded into 384-well plates at a density of 3,000 cells / well. The cells were treated with test samples (5 μL) or an agonist (PTH1-34) at room temperature for 30 minutes. A cAMP-d2 working solution (5 μL) and an anti-cAMP cryptate working solution (5 μL) were added to the cells and incubated at room temperature for 60 minutes. The signal of the assay plate was read with a PheraStar (BMG LABTECH, NC), an HTRF-compatible reader.

[0226] c) Results To determine the ability to activate the PTHR receptor, β-arrestin mobilization and cAMP accumulation assays were performed on the designed peptides. PTH1-34 was the positive control in both assays. In the β-arrestin1 mobilization assay, many of the generated peptides were able to induce responses of similar magnitude compared to PTH1-34 (e.g., R1-101, R1-81, R2-13, R2-29, and R2-36), while two peptides showed higher levels of β-arrestin1 mobilization (R2-38 and R2-52) (Figure 1). Similarly, in the β-arrestin2 assay, many peptides showed similar activity to the control peptide (e.g., R2-34, R2-33, R2-31, R2-30, and R2-42), while nine peptides showed increased activity compared to the control (R2-48, R2-50, R2-8, R2-1, R1-101, R2-25, R2-36, R1-81, and R2-29) (Figure 2). There were also peptides that showed lower activity than the control peptide in both β-arrestin assays.

[0227] Differences in activity were also observed for the peptides designed in the cAMP accumulation assay. In this assay, more peptides showed increased activity (e.g., R2-14, R2-40, R2-45, R2-43, R2-44, R2-24, R2-41, R2-22, R1-41, R1-94, R2-51, R2-47, R2-37, R1-101, and R1-103) compared to the control peptide, while some peptides showed activity similar to the control peptide (e.g., R2-36, R2-35, R1-96, R2-27, and R2-25) (Figure 3).

[0228] For further details, see Tables 7 and 8.

[0229] [Table 12]

[0230] [Table 13]

[0231] [Table 14]

[0232] [Table 15]

[0233] [Table 16]

[0234] [Table 17]

[0235] [Table 18]

[0236] Treatment with the peptide is expected to lead to reduction of bone density loss, reduction of the risk of fracture, and improvement of osteoporosis in the patient.

[0237] For all numerical ranges that describe some parameters in this application, such as "about", "at least", "less than", and "more than", it should be understood that the description also necessarily includes any range limited by the recited values. Thus, for example, the recitation "at least 1, 2, 3, 4, or 5" also describes ranges such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, and 4 to 5, among others.

[0238] For all patents, applications, or other references cited herein, such as non-patent literature and reference sequence information, it should be understood that they are hereby incorporated by reference in their entirety for all purposes, in the same manner as the proposals cited. In case of any conflict between the document incorporated by reference and this application, this application shall prevail. All information related to the reference gene sequences disclosed in this application, such as gene IDs or accession numbers (referring typically to NCBI accession numbers), includes, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNAs (including exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (such as PubChem compounds, PubChem substances, or PubChem bioassay entries including annotations such as structures and assays), and are hereby incorporated by reference in their entirety into this specification.

[0239] The headings used in this application are for convenience only and do not affect the interpretation of this application.

[0240] The preferred features of each aspect provided by the present invention are applicable, with necessary modifications, to all other aspects of the present invention and are exemplified, without limitation, by the dependent claims, and also include combinations and permutations of the individual features (e.g., elements including numerical ranges and exemplary embodiments) of specific embodiments and aspects of the present invention including the examples. For example, the specific experimental parameters exemplified in the examples can be adapted for piecemeal use in the claimed invention without departing from the present invention. For example, with respect to the disclosed materials, the specific references to the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, but each is specifically contemplated and described herein. Thus, if classes of elements A, B, and C, and classes of elements D, E, and F are disclosed and an example of a combination of elements A-D is disclosed, each is individually and collectively contemplated even if not individually listed. Thus, in this example, the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are each specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the exemplary combination A-D. Similarly, these subsets or combinations are also specifically contemplated and disclosed. Thus, for example, the subgroups A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the exemplary combination A-D. This concept applies to all aspects of this application, including the elements of a composition of matter and the steps of a method of making or using a composition.

[0241] The foregoing aspects of the present invention recognized by one of ordinary skill in the art in accordance with the teachings herein can be claimed in any combination or permutation, so long as they are novel and non-obvious over the prior art. Thus, so long as an element is described in one or more references known to one of ordinary skill in the art, they can be excluded from the claimed invention, inter alia, by a negative recitation or disclaimer of the feature or combination of features.

[0242] The teachings of all patents, published applications, and references cited in this specification are hereby incorporated by reference in their entirety.

[0243] Exemplary embodiments have been particularly shown and described, but it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the embodiments encompassed by the appended claims. The present invention includes the following contents as embodiments. 〔Embodiment 1〕 A polypeptide that specifically binds to a parathyroid hormone receptor (PTHR), wherein the polypeptide comprises a synthetic parathyroid hormone (sPTH). 〔Embodiment 2〕 The sPTH is a) 1 to 3 polar residues and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7; b) at least 4 polar residues, at least 1 charged residue, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7; c) at least 5 polar residues, at least 2 charged residues, and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7; d) at least 5 polar residues, valine at position 2, and 1 to 13 amino acid substitutions relative to SEQ ID NO: 7; or e) at least 6 polar residues and 1 to 14 amino acid substitutions relative to SEQ ID NO: 7 The polypeptide according to Embodiment 1, comprising a 14 - amino acid sequence. 〔Embodiment 3〕 The sPTH is a) at most 13 polar residues and 1 to 32 amino acid substitutions relative to SEQ ID NO: 8; b) at least 14 polar residues, valine at position 2, and 1 to 31 substitutions relative to SEQ ID NO: 8; c) at least 14 polar residues and 22 to 28 amino acid substitutions relative to SEQ ID NO: 8; or d) positions 1 to 14 of SEQ ID NO: 8 and 1 to 18 amino acid substitutions at positions 15 to 32 relative to SEQ ID NO: 5 The polypeptide according to Embodiment 1, comprising a 32 - amino acid sequence. 〔Embodiment 4〕 The polypeptide according to Embodiment 1, wherein the sPTH comprises an amino acid sequence having at least 20% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 109 - 160. 〔Embodiment 5〕 The polypeptide according to Embodiment 1, wherein the sPTH comprises an amino acid sequence having 50 - 97% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 109 - 160. 〔Embodiment 6〕 The polypeptide according to Embodiment 1, wherein the sPTH comprises at least one amino acid substitution relative to an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 109 - 160. 〔Embodiment 7〕 The polypeptide according to Embodiment 1, wherein the sPTH comprises 2 to 31 amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 9 - 106 and SEQ ID NOs: 109 - 160. 〔Embodiment 8〕 The polypeptide according to embodiment 1, wherein the sPTH contains 2 to 18 amino acid substitutions with respect to the amino acid sequences selected from SEQ ID NOs: 9 to 106 and SEQ ID NOs: 109 to 160. [Embodiment 9] The polypeptide according to embodiment 1, wherein the sPTH contains the amino acid sequences selected from SEQ ID NOs: 9 to 106 and SEQ ID NOs: 109 to 160. [Embodiment 10] The polypeptide according to embodiment 1, wherein the sPTH contains an amino acid sequence having at least about 20% sequence identity with respect to the amino acid sequences selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155, and 157 to 160. [Embodiment 11] The polypeptide according to embodiment 1, wherein the sPTH contains an amino acid sequence having about 50 to 97% sequence identity with respect to the amino acid sequences selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155, and 157 to 160. [Embodiment 12] The polypeptide according to embodiment 1, wherein the sPTH contains at least one amino acid substitution with respect to the amino acid sequences selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155, and 157 to 160. [Embodiment 13] The polypeptide according to embodiment 1, wherein the sPTH contains about 2 to 31 amino acid substitutions with respect to the amino acid sequences selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155, and 157 to 160. [Embodiment 14] The polypeptide according to embodiment 1, wherein the sPTH contains about 2 to 18 amino acid substitutions with respect to the amino acid sequences selected from SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136 to 138, 140 to 143, 145, 147 to 152, 154, 155, and 157 to 160. [Embodiment 15] The polypeptide according to Aspect 1, wherein the sPTH comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 101, 103, 108, 115, 120, 121, 129, 131, 132, 134, 136-138, 140-143, 145, 147-152, 154, 155, and 157-160. 〔Aspect 16〕 The polypeptide according to Aspect 1, wherein the sPTH consists of 28 to 36 amino acids. 〔Aspect 17〕 The polypeptide according to Aspect 1, wherein the sPTH consists of 30 to 34 amino acids. 〔Aspect 18〕 The polypeptide according to Aspect 1, wherein the sPTH consists of 32 amino acids. 〔Aspect 19〕 The polypeptide according to any one of Aspects 16 to 18, wherein the sPTH comprises an amino acid sequence having at least 20% sequence identity to SEQ ID NO: 5. 〔Aspect 20〕 The polypeptide according to Aspect 19, wherein the sPTH comprises an amino acid sequence having 50 to 97% sequence identity to SEQ ID NO: 5. 〔Aspect 21〕 The polypeptide according to Aspect 19, wherein the sPTH comprises at least one amino acid substitution relative to SEQ ID NO: 5. 〔Aspect 22〕 The polypeptide according to Aspect 19, wherein the sPTH comprises 2 to 18 amino acid substitutions relative to SEQ ID NO: 5. 〔Aspect 23〕 The polypeptide according to Aspect 19, wherein the sPTH comprises the amino acid sequence shown in SEQ ID NO: 6. 〔Aspect 24〕 The polypeptide according to Aspect 23, wherein the amino acid sequence shown in SEQ ID NO: 6 is at the N-terminus of the sPTH. 〔Aspect 25〕 The polypeptide according to Aspect 19, wherein the sPTH comprises an amino acid sequence having at least 55% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 102-106 and SEQ ID NOs: 132-155. 〔Aspect 26〕 The polypeptide according to Aspect 19, wherein the sPTH comprises an amino acid sequence having 75 to 97% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 102-106 and SEQ ID NOs: 132-155. 〔Aspect 27〕 The polypeptide according to Aspect 1, wherein the sPTH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 102-106 and SEQ ID NOs: 132-155. 〔Aspect 28〕 The polypeptide according to Aspect 1, wherein the sPTH comprises the amino acid sequence of SEQ ID NO: 107. 〔Aspect 29〕 The polypeptide according to any one of Aspects 6, 12, and 21, wherein the at least one amino acid substitution is a conservative substitution. 〔Aspect 30〕 The polypeptide according to embodiment 29, wherein the at least one amino acid substitution is a highly conserved substitution. [Embodiment 31] The polypeptide according to any one of embodiments 2, 3, 7, 8, 13, 14, and 22, wherein the amino acid substitution is a conservative substitution. [Embodiment 32] The polypeptide according to embodiment 31, wherein the amino acid substitution is a highly conserved substitution. [Embodiment 33] The polypeptide according to any one of embodiments 1 to 32, wherein the sPTH binds to PTHR with an affinity that is at least about 5% higher than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. [Embodiment 34] The polypeptide according to any one of embodiments 1 to 32, wherein the sPTH binds to PTHR with an affinity that is at least about 5% lower than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. [Embodiment 35] The polypeptide according to any one of embodiments 1 to 32, wherein the sPTH binds to PTHR with an affinity that is about 5% or less lower than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. [Embodiment 36] The polypeptide according to any one of embodiments 1 to 35, wherein the sPTH is an agonist of PTHR. [Embodiment 37] The polypeptide according to embodiment 36, wherein the sPTH is a biased agonist of PTHR. [Embodiment 38] The polypeptide according to any one of embodiments 1 to 35, wherein the sPTH is an inverse agonist of PTHR. [Embodiment 39] The polypeptide according to any one of embodiments 1 to 35, wherein the sPTH is an antagonist of PTHR. [Embodiment 40] The polypeptide according to any one of embodiments 1 to 39, wherein the polypeptide is conjugated to a heterologous moiety. [Embodiment 41] The polypeptide according to embodiment 40, wherein the heterologous moiety is selected from the group consisting of polyethylene glycol (PEG), hexadecanoic acid, hydrogel, lipid nanoparticles, polymer nanoparticles, and heterologous polypeptide sequences, or combinations thereof. [Embodiment 42] The polypeptide according to embodiment 41, wherein the polymer nanoparticles comprise poly(lactic-co-glycolic) acid (PLGA). [Embodiment 43] The polypeptide according to embodiment 40, wherein the heterologous polypeptide sequence comprises a carrier polypeptide. [Embodiment 44] The polypeptide according to aspect 43, wherein the carrier polypeptide is albumin or an Fc polypeptide. 〔Aspect 45〕 A fusion protein comprising the polypeptide according to any one of aspects 1 to 44. 〔Aspect 46〕 A polynucleotide comprising a sequence encoding the polypeptide according to any one of aspects 1 to 44 or the fusion protein according to aspect 45. 〔Aspect 47〕 An expression vector comprising the polynucleotide according to aspect 46. 〔Aspect 48〕 A host cell comprising the polynucleotide according to aspect 46 or the expression vector according to aspect 47. 〔Aspect 49〕 A composition comprising the polypeptide according to any one of aspects 1 to 44 or the fusion protein according to aspect 45. 〔Aspect 50〕 The composition according to aspect 49, further comprising one or more pharmaceutical excipients, diluents, or carriers. 〔Aspect 51〕 A method for regulating PTHR signaling in the mammalian cell, comprising contacting the mammalian cell with an effective amount of the composition according to aspect 49 or 50. 〔Aspect 52〕 A method for treating a subject in need thereof, comprising administering to the subject an effective amount of the composition according to aspect 49 or 50. 〔Aspect 53〕 A method for regulating PTHR signaling in a subject in need thereof, comprising administering to the subject an effective amount of the composition according to aspect 49 or 50. 〔Aspect 54〕 The method according to aspect 52 or 53, wherein the subject is a mammal. 〔Aspect 55〕 The method according to aspect 54, wherein the subject is a human. 〔Aspect 56〕 The method according to any one of aspects 52 to 55, wherein the subject is an adult. 〔Aspect 57〕 The method according to any one of aspects 52 to 56, wherein the subject is at least 50 years old. 〔Aspect 58〕 The method according to any one of aspects 52 to 57, wherein the subject is a female. 〔Aspect 59〕 The method according to aspect 58, wherein the subject is in the perimenopausal, postmenopausal, or postmenopausal period. 〔Aspect 60〕 The method according to any one of aspects 52 to 59, wherein the subject has or is at risk of developing dysregulated calcium homeostasis. 〔Aspect 61〕 The method according to any one of Aspects 52 to 59, wherein the subject has a disease selected from osteoporosis, Blomstrand chondrodysplasia, familial primary failure of tooth eruption, Eiken syndrome, Ollier disease, hypercalcemia, hyperparathyroidism, Jansen metaphyseal chondrodysplasia, hypercalcemia, hypercalciuria, nephrocalcinosis, and combinations thereof, or has a risk of developing the same. [Aspect 62] The method according to Aspect 61, wherein the sPTH is an agonist of PTHR. [Aspect 63] The method according to Aspect 62, wherein the sPTH is a biased agonist of PTHR. [Aspect 64] The method according to any one of Aspects 52 to 63, wherein the subject has low bone density or has a risk of developing the same. [Aspect 65] The method according to any one of Aspects 52 to 59, wherein the subject has a disease selected from Blomstrand lethal chondrodysplasia, Ollier disease, familial primary failure of tooth eruption, Eiken syndrome, brachydactyly type E, hypoparathyroidism, osteoporosis, and combinations thereof, or has a risk of developing the same. [Aspect 66] The method according to any one of Aspects 52 to 59, wherein the subject has osteoporosis or has a risk of developing the same. [Aspect 67] The method according to any one of Aspects 52 to 66, wherein the amount is effective for promoting calcium homeostasis, increasing, maintaining or decreasing the reduction of bone mineral content, reducing the risk of fracture, promoting fracture healing, or a combination of the foregoing. [Aspect 68] The method according to any one of Aspects 52 to 67, wherein the effective amount does not induce catabolic bone resorption. [Aspect 69] The method according to any one of Aspects 52 to 59, wherein the subject has a disease selected from hypercalcemia, hyperparathyroidism, parathyroid cancer, metastatic bone disease, and combinations thereof, or has a high risk of developing the same. [Aspect 70] The method according to Aspect 69, wherein the hyperparathyroidism occurs as a complication of parathyroid cancer. [Aspect 71] The method according to Aspect 69 or 70, wherein the metastatic bone disease occurs with associated hypercalcemia. [Aspect 72] The method according to any one of aspects 52 to 59, wherein the subject has a disease selected from Jansen type metaphyseal chondrodysplasia, hyperparathyroidism, hypercalcemia, hypercalciuria, nephrocalcinosis, chronic kidney disease, and combinations thereof, or has a high risk of developing the disease. [[Aspect 73]] The method according to aspect 72, wherein the chronic kidney disease is associated with or results from hypercalcemia, hypercalciuria, nephrocalcinosis, or combinations thereof.

Claims

1. A polypeptide that specifically binds to the parathyroid hormone receptor (PTHR), wherein the polypeptide comprises synthetic parathyroid hormone (sPTH), and the sPTH comprises an amino acid sequence selected from SEQ ID NOs: 103, 132, 134, 136-138, 140-143, 145, 147-152, 154, and 155.

2. The polypeptide according to claim 1, wherein the sPTH binds to PTHR with an affinity that is at least 5% higher than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

5.

3. The polypeptide according to claim 1, wherein the sPTH binds to PTHR with an affinity that is 5% or less lower than that of a polypeptide having an amino acid sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

5.

4. A fusion protein comprising the polypeptide according to any one of claims 1 to 3.

5. A polynucleotide comprising a sequence encoding the polypeptide according to any one of claims 1 to 3 or the fusion protein according to claim 4.

6. An expression vector comprising the polynucleotide according to claim 5.

7. A host cell comprising the polynucleotide according to claim 5 or the expression vector according to claim 6.

8. A composition comprising the polypeptide according to any one of claims 1 to 3 or the fusion protein according to claim 4.

9. The composition according to claim 8, further comprising one or more pharmaceutical excipients, diluents, or carriers.

10. A PTHR signaling regulator comprising an effective amount of the composition according to claim 8 or 9 for administration to a subject.

11. The regulator according to claim 10, wherein the subject is a mammal.

12. The regulator according to claim 10, wherein the subject is a human.

13. The regulator according to any one of claims 10 to 12, wherein the subject has or is at risk of developing dysregulated calcium homeostasis.

14. The modulator according to any one of claims 10 to 12, wherein the subject has a disease selected from osteoporosis, Blomstrand chondrodysplasia, familial primary failure of tooth eruption, Eiken syndrome, Ollier disease, hypercalcemia, hyperparathyroidism, Jansen metaphyseal chondrodysplasia, hypercalcemia, hypercalciuria, nephrocalcinosis, and combinations thereof, or has a risk of developing the same.

15. The modulator according to claim 14, wherein the sPTH is an agonist of the PTHR.

16. The modulator according to claim 15, wherein the sPTH is a biased agonist of the PTHR.

17. The modulator according to any one of claims 10 to 16, wherein the subject has low bone density or has a risk of developing the same.

18. The modulator according to any one of claims 10 to 12, wherein the subject has a disease selected from Blomstrand lethal chondrodysplasia, Ollier disease, familial primary failure of tooth eruption, Eiken syndrome, brachydactyly type E, hypoparathyroidism, osteoporosis, and combinations thereof, or has a risk of developing the same.

19. The modulator according to any one of claims 10 to 12, wherein the subject has osteoporosis or has a risk of developing the same.

20. The modulator according to any one of claims 10 to 19, wherein the amount is effective for promoting calcium homeostasis, increasing, maintaining or decreasing the reduction of bone mineral content, reducing the risk of fracture, promoting fracture healing, or a combination of the foregoing.

21. The modulator according to any one of claims 10 to 20, wherein the effective amount does not induce catabolic bone resorption.

22. The modulator according to any one of claims 10 to 12, wherein the subject has a disease selected from hypercalcemia, hyperparathyroidism, parathyroid cancer, metastatic bone disease, and combinations thereof, or has a high risk of developing the same.

23. The modulator according to claim 22, wherein the hyperparathyroidism occurs as a complication of parathyroid cancer.

24. The modulator according to claim 22 or 23, wherein the metastatic bone disease occurs with associated hypercalcemia.

25. The modulator according to any one of claims 10 to 12, wherein the subject has a disease selected from Jansen type metaphyseal chondrodysplasia, hyperparathyroidism, hypercalcemia, hypercalciuria, nephrocalcinosis, chronic kidney disease, and combinations thereof, or has a high risk of developing the disease.

26. The modulator according to claim 25, wherein the chronic kidney disease is associated with or results from hypercalcemia, hypercalciuria, nephrocalcinosis, or combinations thereof.

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