PTH complex and use thereof

Abstract

The present invention relates to the field of biomedicine, and more specifically, to a complex containing a PTH protein or active fragment thereof and an antibody or fragment thereof that specifically binds to PTH, and the related use thereof in the treatment of a disease.

Description

Pth complex and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411378879.3, filed September 30, 2024, and Chinese Patent Application No. 202510126281.3, filed January 27, 2025, the entire contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, and more particularly, the present application relates to a complex of PTH protein or active fragment thereof and an antibody or fragment thereof specifically binding to PTH, and related uses for disease treatment. BACKGROUND

[0004] Parathyroid hormone (PTH) is a hormone secreted by the parathyroid gland, and its full-length sequence consists of 84 amino acids. It mainly regulates blood calcium and blood phosphorus through the kidney, bone, and intestine, and has the effects of increasing blood calcium, reducing blood phosphorus, and promoting bone turnover. The N-terminal polypeptide of PTH retains all the biological activities of full-length PTH, but due to its small molecular weight, it is easily cleared by the kidney or degraded by proteases, resulting in a very short half-life of PTH in the body, making it difficult to exert long-acting effects.

[0005] Hypoparathyroidism (HP) is a rare endocrine disorder characterized by insufficient parathyroid hormone (PTH) levels or impaired activity, disruption of calcium and phosphorus homeostasis, leading to low blood calcium and high blood phosphorus. Resection or injury of the parathyroid gland during anterior neck surgery is the most common cause of HP, accounting for 75% of the total incidence, and the remaining 25% of the causes include autoimmune diseases, genetic diseases, and rare infiltrative diseases. The incidence of temporary and permanent hypocalcemia after thyroidectomy is 19%-38% and 0%-3%, respectively. In recent years, with the increase in patients undergoing thyroid surgery, the number of patients with HP has also shown a growing trend. In the United States, Europe, Japan, and Korea, HP affects about 200,000 patients, and according to relevant literature and foreign epidemiological data, the current number of HP patients in China is about 200,000. The clinical manifestations of HP are diverse and almost involve all organ systems of the human body. Mainly including: muscle spasms, convulsions, cramps, tingling hands, foot numbness, fatigue, anxiety, depression, seizures, etc.

[0006] The routine treatment of supplementing active vitamin D analogues and calcium agents cannot completely control the disease and can have the risk of causing kidney disease and ectopic calcification. In addition, the treatment with calcium agents and vitamin D cannot solve the problem of reduced bone turnover due to PTH deficiency. The marketed short-acting parathyroid hormone Teriparatide and Natpara have not solved the bottleneck problem of short half-life and far from the purpose of maintaining the balance of calcium and phosphorus metabolism in the human body, so most patients using the drug still need to continue taking calcium and active vitamin D. The drug for treating hypoparathyroidism needs to keep its blood concentration stable at the normal physiological level, so the development of long-acting and slow-release parathyroid hormone drugs can truly realize the hormone replacement therapy of HP. Yorvipath is a slow-release long-acting prodrug of parathyroid hormone PTH(1-34); after Natpara is off the market, Yorvipath is the only hormone replacement therapy drug for treating hypoparathyroidism, which aims to restore PTH to the physiological level for 24 hours a day, but still needs to be injected once a day, which cannot improve the compliance of patients.

[0007] To prolong the retention time of the drug in the body, a common improvement strategy is to fuse with Fc or albumin, which increases the molecular weight and has the FcRn-mediated recycling mechanism, reduces kidney clearance, and improves the half-life of the target molecule in the body. Because PTH is easily degraded by proteases in serum, it is still difficult to achieve long-acting purposes, and parathyroid hormone needs to control calcium in the blood within a very strict range, and fusion with Fc or albumin cannot achieve the slow-release effect of the drug. SUMMARY

[0008] The present application provides a complex of an anti-PTH antibody and a PTH polypeptide, and a complex of an anti-PTH antibody and a PTH-VHH fusion protein, which can effectively reduce the kidney clearance of PTH and protect PTH from being degraded by proteases in serum, achieve the purpose of long-acting and slow-release of PTH, thereby can significantly prolong the retention time of PTH in the body, and based on the complex, further provides a drug and a method for preventing and / or treating bone-related diseases (such as osteoporosis) or diseases caused by hypoparathyroidism (such as hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria).

[0009] I. Complex

[0010] In one aspect, the present application provides a complex comprising a first protein and a second protein, the first protein specifically binds to the second protein, wherein the first protein comprises or consists of a parathyroid hormone (PTH) protein or an active fragment thereof; the second protein comprises or consists of an antibody or an antigen-binding fragment thereof targeting the PTH protein or the active fragment thereof.

[0011] In certain embodiments, the complex is a recombinant protein that is not naturally occurring. By "not naturally occurring" it is meant that the complex does not include an antigen-antibody complex naturally formed by a PTH protein or an active fragment thereof and an antibody or an antigen-binding fragment thereof targeting the PTH protein or the active fragment thereof in vivo in an animal or in an isolated animal cell. In certain embodiments, the complex is a recombinant protein comprising a polypeptide comprising at least one amino acid modification.

[0012] In certain embodiments, the PTH active fragment comprises at least the amino acid residues in PTH corresponding to positions 1-14 (or 1-16, 1-18, 1-20, 1-22, 1-25, 1-30, 1-34) of SEQ ID NO: 99.

[0013] In certain embodiments, the PTH active fragment comprises at least the amino acid residues in PTH corresponding to positions 1-34 of SEQ ID NO: 99.

[0014] In certain embodiments, the PTH active fragment consists of the amino acid residues in PTH corresponding to positions 1-34 of SEQ ID NO: 99.

[0015] In the context of the present application, when referring to amino acid positions of PTH, the reference is made to the sequence set forth in SEQ ID NO: 99. For example, the expression "amino acid residues in PTH corresponding to positions 1-34 of SEQ ID NO: 99" means the amino acid positions in the sequence of the PTH under comparison that are in equivalent positions to the amino acid residues at positions 1-34 of SEQ ID NO: 99, when the sequence of the PTH is optimally aligned with SEQ ID NO: 99, i.e. when the sequence of the PTH is aligned with SEQ ID NO: 99 to obtain the highest percentage identity.

[0016] Unless specifically indicated otherwise or clearly contradicted by context, the meaning of the remaining similar expressions herein is defined in a manner analogous to the above.

[0017] I-i. Complex: PTH and anti-PTH antibody

[0018] In certain embodiments, the first protein is a PTH protein or an active fragment thereof.

[0019] In certain embodiments, the PTH protein is a wild-type PTH protein.

[0020] In certain embodiments, the wild-type PTH protein comprises a sequence set forth in SEQ ID NO: 99.

[0021] In certain embodiments, the first protein is a PTH active fragment comprising at least the amino acid residues in PTH corresponding to positions 1-14 (or 1-16, 1-18, 1-20, 1-22, 1-25, 1-30, 1-34) of SEQ ID NO: 99.

[0022] In certain embodiments, the PTH active fragment comprises at least the amino acid residues in PTH corresponding to positions 1-34 of SEQ ID NO: 99.

[0023] In certain embodiments, the PTH active fragment consists of the amino acid residues in PTH corresponding to positions 1-14, or 1-16, or 1-18, or 1-20, or 1-22, or 1-25, or 1-30, or 1-34 of SEQ ID NO: 99.

[0024] In certain embodiments, the active fragment comprises a sequence set forth in SEQ ID NO: 25.

[0025] In certain embodiments, the PTH protein comprises a modification compared to a wild-type PTH protein or an active fragment thereof:

[0026] (1) comprises a mutation at one or more of the positions corresponding to positions 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99; and / or,

[0027] (2) comprises an aliphatic organic acid.

[0028] In certain embodiments, the mutation is an amino acid substitution.

[0029] In certain embodiments, the mutation is selected from one or more of the following:

[0030] (a) the amino acid at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 is substituted with a non-natural amino acid (e.g., alpha- aminoisobutyric acid (Aib));

[0031] (b) the PTH protein has a substitution at the position corresponding to position 16 of SEQ ID NO: 99 from asparagine (N) to, for example, alanine (A), serine (S);

[0032] (c) the PTH protein has a substitution at the position corresponding to position 17 of SEQ ID NO: 99 from serine (S) to, for example, glutamic acid (E);

[0033] (d) the PTH protein has a substitution at the position corresponding to position 34 of SEQ ID NO: 99 from phenylalanine (F) to, for example, lysine (K).

[0034] In certain embodiments, the PTH protein has a substitution at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 to a non-natural amino acid (e.g., alpha- aminoisobutyric acid (Aib)).

[0035] In certain embodiments, the PTH protein has a substitution at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 to a non-natural amino acid (e.g., alpha- aminoisobutyric acid (Aib)).

[0036] In certain embodiments, the PTH protein has a substitution at the position corresponding to position 16 of SEQ ID NO: 99 from asparagine (N) to, for example, alanine (A), serine (S);

[0037] In certain embodiments, the PTH protein has a substitution at the position corresponding to position 17 of SEQ ID NO: 99 from serine (S) to, for example, glutamic acid (E).

[0038] In certain embodiments, the PTH protein has a substitution at the position corresponding to position 34 of SEQ ID NO: 99 from phenylalanine (F) to, for example, lysine (K).

[0039] In certain embodiments, the PTH protein: (I) is substituted at an amino acid position corresponding to position 1, 25 of SEQ ID NO: 99 with an unnatural amino acid (e.g., a- amino isobutyric acid (Aib)); (II) has a substitution at the asparagine (N) corresponding to position 16 of SEQ ID NO: 99 (e.g., to alanine (A), serine (S)); and / or, (III) has a substitution at the serine (S) corresponding to position 17 of SEQ ID NO: 99 (e.g., to glutamic acid (E)).

[0040] In certain embodiments, the PTH protein: (I) is substituted at an amino acid position corresponding to position 1, 25 of SEQ ID NO: 99 with an unnatural amino acid (e.g., a- amino isobutyric acid (Aib)); (II) has a substitution at the asparagine (N) corresponding to position 16 of SEQ ID NO: 99 (e.g., to alanine (A), serine (S)); (III) has a substitution at the serine (S) corresponding to position 17 of SEQ ID NO: 99 (e.g., to glutamic acid (E)); and / or, (IV) comprises an aliphatic organic acid.

[0041] In certain embodiments, the first protein is a PTH active fragment consisting of amino acid residues corresponding to positions 1-14, or 1-16, or 1-18, or 1-20, or 1-22, or 1-25, or 1-30, or 1-34 of SEQ ID NO: 99 in the modified PTH protein.

[0042] In certain embodiments, the PTH active fragment comprises, compared to a wild-type PTH protein or an active fragment thereof:

[0043] (1) a mutation at one or more of positions corresponding to positions 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99; and / or,

[0044] (2) an aliphatic organic acid.

[0045] In certain embodiments, the mutation is an amino acid substitution.

[0046] In certain embodiments, the mutation is selected from one or more of:

[0047] (a) the PTH active fragment has an amino acid at one or more of positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 substituted with an unnatural amino acid (e.g., a- amino isobutyric acid (Aib));

[0048] (b) the PTH active fragment has a substitution at the position corresponding to position 16 of SEQ ID NO: 99, e.g., to alanine (A), serine (S);

[0049] (c) the PTH active fragment has a substitution at the position corresponding to position 17 of SEQ ID NO: 99, e.g., to glutamic acid (E);

[0050] (d) the PTH active fragment has a substitution at the position corresponding to position 34 of SEQ ID NO: 99, e.g., to lysine (K).

[0051] In certain embodiments, the PTH active fragment has a substitution at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 to a non-natural amino acid, such as alpha-aminobutyric acid (Aib).

[0052] In certain embodiments, the PTH active fragment has a substitution at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 to a non-natural amino acid, such as alpha-aminobutyric acid (Aib).

[0053] In certain embodiments, the PTH active fragment has a substitution at the position corresponding to position 16 of SEQ ID NO: 99, e.g., to alanine (A), serine (S);

[0054] In certain embodiments, the PTH active fragment has a substitution at the position corresponding to position 17 of SEQ ID NO: 99, e.g., to glutamic acid (E).

[0055] In certain embodiments, the PTH active fragment has a substitution at the position corresponding to position 34 of SEQ ID NO: 99, e.g., to lysine (K).

[0056] In certain embodiments, the PTH active fragment: (I) is substituted at the amino acid position corresponding to position 1, 25 of SEQ ID NO: 99 with an unnatural amino acid (such as alpha-aminoisobutyric acid (Aib)); (II) is substituted at the asparagine (N) corresponding to position 16 of SEQ ID NO: 99 (e.g., substituted with alanine (A), serine (S)); and / or, (III) is substituted at the serine (S) corresponding to position 17 of SEQ ID NO: 99 (e.g., substituted with glutamic acid (E)).

[0057] In certain embodiments, the PTH active fragment: (I) is substituted at the amino acid position corresponding to position 1, 25 of SEQ ID NO: 99 with an unnatural amino acid (such as alpha-aminoisobutyric acid (Aib)); (II) is substituted at the asparagine (N) corresponding to position 16 of SEQ ID NO: 99 (e.g., substituted with alanine (A), serine (S)); (III) is substituted at the serine (S) corresponding to position 17 of SEQ ID NO: 99 (e.g., substituted with glutamic acid (E)); and / or, (IV) comprises an aliphatic organic acid.

[0058] In certain embodiments, the PTH active fragment comprises a sequence as set forth in any one of SEQ ID NOs: 100-102, 73-76, 105, 106.

[0059] In certain embodiments, the aliphatic organic acid is optionally linked to:

[0060] (1) any amino acid in the PTH protein or active fragment thereof located between the amino acid at position 1 and the C-terminus;

[0061] (2) any amino acid in the PTH protein or active fragment thereof located between the amino acid at position 11 and the C-terminus;

[0062] (3) any amino acid in the PTH protein or active fragment thereof located between the amino acid at position 12 and the C-terminus;

[0063] (4) any amino acid in the PTH protein or active fragment thereof located between the amino acid at position 13 and the C-terminus;

[0064] (5) any amino acid in the PTH protein or active fragment thereof located between the amino acid at position 14 and the C-terminus;

[0065] (6) any amino acid in the PTH protein or active fragment thereof located between the amino acid at position 15 and the C-terminus;

[0066] (7) any amino acid in the PTH protein or active fragment thereof located from the 16th amino acid to the C-terminus;

[0067] (8) any amino acid in the PTH protein or active fragment thereof located from the 17th amino acid to the C-terminus;

[0068] (9) any amino acid in the PTH protein or active fragment thereof located from the 18th amino acid to the C-terminus;

[0069] (10) any amino acid in the PTH protein or active fragment thereof located from the 19th amino acid to the C-terminus;

[0070] (11) any amino acid in the PTH protein or active fragment thereof located from the 23rd amino acid to the C-terminus;

[0071] (12) any amino acid in the PTH protein or active fragment thereof located from the 26th amino acid to the C-terminus; or

[0072] (13) any amino acid in the PTH protein or active fragment thereof located from the 29th amino acid to the C-terminus;

[0073] (14) any amino acid in the PTH protein or active fragment thereof located from the 31st amino acid to the C-terminus;

[0074] (15) any amino acid in the PTH protein or active fragment thereof located from the 32nd amino acid to the C-terminus;

[0075] (16) any amino acid in the PTH protein or active fragment thereof located from the 33rd amino acid to the C-terminus;

[0076] (17) any amino acid in the PTH protein or active fragment thereof located from the 34th amino acid to the C-terminus;

[0077] wherein the positions are amino acid positions relative to SEQ ID NO: 99.

[0078] In certain embodiments, the aliphatic organic acid is optionally linked to the PTH protein or active fragment thereof at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, or 84th amino acid of the PTH protein or active fragment thereof.

[0079] In certain embodiments, the aliphatic organic acid is optionally linked to the C-terminal amino acid of the PTH protein or active fragment thereof via a spacer.

[0080] In certain embodiments, the aliphatic organic acid is optionally linked to the amino acid at the position corresponding to position 34 of SEQ ID NO: 99 of the PTH protein or active fragment thereof via a spacer.

[0081] In certain embodiments, the amino acid is selected from the group consisting of lysine, d-lysine, ornithine, cysteine, and homocysteine.

[0082] In certain embodiments, the PTH protein or active fragment thereof is substituted with a non-natural amino acid, such as alpha-aminoisobutyric acid (Aib) at an amino acid position selected from the group consisting of: (i) 1st, 25th; (ii) 1st; and, the aliphatic organic acid is optionally linked to the amino acid at the position corresponding to position 34 of SEQ ID NO: 99 of the PTH protein or active fragment thereof via a spacer.

[0083] In certain embodiments, the spacer comprises an acidic amino acid residue (e.g., gamma glutamic acid).

[0084] In certain embodiments, the spacer is selected from the group consisting of gamma glutamic acid-gamma glutamic acid dipeptide, *-[COCH2(OCH2CH2) k NH] q -gamma glutamic acid or gamma glutamic acid-[COCH2(OCH2CH2) k NH] q -gamma glutamic acid, wherein k is an integer selected from 1 to 20, q is an integer selected from 1 to 20, the *-end is attached to the side chain of an amino acid.

[0085] In certain embodiments, the k is 1 or 2.

[0086] In certain embodiments, the q is 1 or 2.

[0087] In certain embodiments, the aliphatic organic acid is attached to the PTH protein or active fragment thereof via a spacer to any amino acid from the 3rd amino acid to the C-terminus; the position is the amino acid position relative to SEQ ID NO: 99; the structural composition of the aliphatic organic acid and spacer is shown in formula (A):

[0088] III-(II)m-(I)n- Formula (A),

[0089] wherein,

[0090] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;

[0091] I is -C(=O)CH2(OCH2CH2)2NH-;

[0092] II is an acidic amino acid residue;

[0093] III is COOH-(CH2)r-C(=O)-, r is an integer selected from 12 to 30; and

[0094] III, II, and I are connected through an amide bond,

[0095] the carbonyl of I is connected to the amino group of the side chain of an amino acid in the PTH protein or active fragment thereof.

[0096] In certain embodiments, the carbonyl of I is connected to the amino group of the side chain of lysine.

[0097] In certain embodiments, the n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0098] In certain embodiments, the n is 1 or 2.

[0099] In certain embodiments, the II is selected from a gamma glutamic acid residue and an aspartic acid residue.

[0100] In certain embodiments, the II is a gamma glutamic acid residue having the structure shown below:

[0101] In certain embodiments, the m is 1.

[0102] In certain embodiments, the r is an integer selected from 16 to 22.

[0103] In certain embodiments, the III is COOH-(CH2) 16 -C(=0)-, COOH-(CH2) 17 -C(=0)-, or COOH-(CH2) 18 -C(=0)-.

[0104] In certain embodiments, the formula (A) has a structure selected from the group consisting of:

[0105] In certain embodiments, the PTH protein or active fragment thereof comprises a sequence as set forth in SEQ ID NO: 103 or 104.

[0106] In certain embodiments, the first protein comprises a sequence as set forth in any one of SEQ ID NOs: 73-76, 100-106, 25, 99.

[0107] In certain embodiments, the second protein is an antibody or antigen-binding fragment thereof that targets a PTH protein or active fragment thereof, which specifically binds to PTH(20-28).

[0108] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: 3 CDRs contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 53; and / or 3 CDRs contained in a light chain variable region (VL) as set forth in any one of SEQ ID NOs: 59, 63, 66, 69. In certain embodiments, the CDRs are defined by the Kabat, Chothia, or IMGT numbering system.

[0109] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 50; CDR-H2 of SEQ ID NO: 51; and CDR-H3 of SEQ ID NO: 52; and a light chain variable region comprising CDR-L1 of SEQ ID NO: 56, 62, 65, or 68; CDR-L2 of SEQ ID NO: 57; and CDR-L3 of SEQ ID NO: 58. In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0110] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising a sequence set forth in SEQ ID NO: 53 or a sequence at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical thereto; and a VL comprising a sequence set forth in any one of SEQ ID NO: 59, 63, 66, 69 or a sequence at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical thereto.

[0111] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4) and a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda).

[0112] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a variant of a human IgG (e.g., IgGl or IgG4) heavy chain constant region that possesses a mutation that reduces or abrogates effector function (e.g., ADCC, ADCP, and / or CDC activity) as compared to the wild-type sequence from which it is derived.

[0113] In certain embodiments, the variant of a human IgG heavy chain constant region is a variant of a human IgGl heavy chain constant region that possesses one or more of the following substitutions as compared to the wild-type sequence: L234A, L235A, and P329G (positions according to the EU numbering system).

[0114] In certain embodiments, the variant of a human IgGl heavy chain constant region comprises a sequence set forth in SEQ ID NO: 54 or 71.

[0115] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0116] (a) a heavy chain having the sequence set forth in SEQ ID NO: 55 and a light chain having the sequence set forth in SEQ ID NO: 61 ;

[0117] (b) a heavy chain having the sequence set forth in SEQ ID NO: 72 and a light chain having the sequence set forth in SEQ ID NO: 64;

[0118] (c) a heavy chain having the sequence set forth in SEQ ID NO: 72 and a light chain having the sequence set forth in SEQ ID NO: 67; or

[0119] (d) a heavy chain having the sequence set forth in SEQ ID NO: 72 and a light chain having the sequence set forth in SEQ ID NO: 70.

[0120] In certain embodiments, the first protein is as defined in Section I-i of the Invention ("Complex: PTH and Anti-PTH Antibody") and the second protein is as defined in Section I-i of the Invention ("Complex: PTH and Anti-PTH Antibody").

[0121] In certain embodiments, in the complex:

[0122] (1) the first protein is a PTH active fragment set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 55 and a light chain having the sequence set forth in SEQ ID NO: 61 ;

[0123] (2) the first protein is a PTH active fragment set forth in SEQ ID NO: 75 and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 55 and a light chain having the sequence set forth in SEQ ID NO: 61 ;

[0124] (3) the first protein is a PTH active fragment set forth in SEQ ID NO: 76 and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 55 and a light chain having the sequence set forth in SEQ ID NO: 61 ;

[0125] (4) the first protein is a PTH active fragment set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 72 and a light chain having the sequence set forth in SEQ ID NO: 67;

[0126] (5) the first protein is a PTH active fragment as set forth in SEQ ID NO: 75, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 72 and a light chain having the sequence as set forth in SEQ ID NO: 67;

[0127] (6) the first protein is a PTH active fragment as set forth in SEQ ID NO: 76, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 72 and a light chain having the sequence as set forth in SEQ ID NO: 67;

[0128] (7) the first protein is a PTH active fragment as set forth in SEQ ID NO: 101, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 72 and a light chain having the sequence as set forth in SEQ ID NO: 67;

[0129] (8) the first protein is a PTH active fragment as set forth in SEQ ID NO: 103, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 72 and a light chain having the sequence as set forth in SEQ ID NO: 67; or

[0130] (9) the first protein is a PTH active fragment as set forth in SEQ ID NO: 104, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 72 and a light chain having the sequence as set forth in SEQ ID NO: 67.

[0131] I-ii. Complex: PTH-VHH fusion protein and anti-PTH antibody

[0132] In certain embodiments, the first protein is a fusion protein comprising (i) a PTH protein or an active fragment thereof and (ii) a nanobody or an antigen binding fragment thereof that targets serum albumin.

[0133] In certain embodiments, the PTH protein is a wild-type PTH protein.

[0134] In certain embodiments, the wild-type PTH protein comprises the sequence set forth in SEQ ID NO: 99.

[0135] In certain embodiments, the fusion protein comprises a PTH active fragment that comprises at least the amino acid residues in PTH corresponding to positions 1-14 (or 1-16, 1-18, 1-20, 1-22, 1-25, 1-30, 1-34) of SEQ ID NO: 99.

[0136] In certain embodiments, the PTH active fragment comprises at least the amino acid residues at positions corresponding to positions 1-34 of SEQ ID NO: 99 in PTH.

[0137] In certain embodiments, the PTH active fragment consists of the amino acid residues at positions corresponding to positions 1-14, or 1-16, or 1-18, or 1-20, or 1-22, or 1-25, or 1-30, or 1-34 of SEQ ID NO: 99 in PTH.

[0138] In certain embodiments, the PTH active fragment comprises a sequence as set forth in SEQ ID NO: 25.

[0139] In certain embodiments, the PTH protein or active fragment thereof comprises a modification comprising a mutation at one or more of positions corresponding to positions 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99, as compared to a wild-type PTH protein or active fragment thereof.

[0140] In certain embodiments, the mutation is an amino acid substitution.

[0141] In certain embodiments, the mutation is selected from one or more of the following:

[0142] (a) the amino acid at one or more of positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 in the PTH protein or active fragment thereof is substituted with a non-natural amino acid (e.g., alpha-aminobutyric acid (Aib));

[0143] (b) the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99 in the PTH protein or active fragment thereof is substituted, e.g., substituted with alanine (A), serine (S);

[0144] (c) the serine (S) at the position corresponding to position 17 of SEQ ID NO: 99 in the PTH protein or active fragment thereof is substituted, e.g., substituted with glutamic acid (E);

[0145] (d) the phenylalanine (F) at the position corresponding to position 34 of SEQ ID NO: 99 in the PTH protein or active fragment thereof is substituted, e.g., substituted with lysine (K).

[0146] In certain embodiments, the PTH protein is a modified PTH protein in which at least one naturally occurring asparagine (N) is substituted, e.g., substituted with alanine (A) or serine (S), as compared to a wild-type PTH protein.

[0147] In certain embodiments, the modified PTH protein has a substitution of the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99, for example, to alanine (A) or serine (S).

[0148] In certain embodiments, the fusion protein comprises a PTH active fragment consisting of the amino acid residues at positions corresponding to positions 1-34 of SEQ ID NO: 99 in the modified PTH protein.

[0149] In certain embodiments, the PTH active fragment has a substitution of the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99, for example, to alanine (A) or serine (S).

[0150] In certain embodiments, the PTH active fragment comprises a sequence as set forth in SEQ ID NO: 93 or 94.

[0151] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises 3 CDRs contained in a heavy chain variable region (VHH) as set forth in any one of SEQ ID NOs: 7, 12, 18, 23, 96, 98. In certain embodiments, the CDRs are defined by the Kabat, Chothia, or IMGT numbering system.

[0152] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises:

[0153] (1) a CDR1 as set forth in SEQ ID NO: 4, a CDR2 as set forth in SEQ ID NO: 5 or 16, and a CDR3 as set forth in SEQ ID NO: 6, 95, or 97;

[0154] (2) a CDR1 as set forth in SEQ ID NO: 9, a CDR2 as set forth in SEQ ID NO: 10, and a CDR3 as set forth in SEQ ID NO: 11. In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0155] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (i) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 5, 6, respectively; (ii) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 6, respectively; (iii) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 95, respectively; or (iv) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 97, respectively. In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0156] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 7, 12, 18, 23, 96, 98 (e.g., SEQ ID NOs: 18, 23, 96, 98), or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto.

[0157] In certain embodiments, the PTH protein or active fragment thereof is optionally linked to the N-terminus or C-terminus of the nanobody or antigen-binding fragment thereof via a linker.

[0158] In certain embodiments, the PTH protein or active fragment thereof is directly linked to the N-terminus of the nanobody or antigen-binding fragment thereof.

[0159] In certain embodiments, the PTH protein or active fragment thereof is directly linked to the N-terminus of the nanobody or antigen-binding fragment thereof.

[0160] In certain embodiments, the linker is a peptide linker (e.g., a rigid peptide linker or a flexible peptide linker).

[0161] In certain embodiments, the linker is a peptide linker comprising one or more glycines and / or one or more serines.

[0162] In certain embodiments, the peptide linker is (GmS)n, m, n is independently an integer no less than 0, e.g., independently 1, 2, 3, or 4.

[0163] In certain embodiments, the peptide linker comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 26, 31-34.

[0164] In certain embodiments, the first protein comprises a fusion protein as set forth in any one of SEQ ID NOs: 27-30, 35-49.

[0165] In certain embodiments, the first protein comprises a fusion protein as set forth in any one of SEQ ID NOs: 29, 30, 45-49.

[0166] In certain embodiments, the second protein is an antibody or antigen-binding fragment thereof that specifically binds PTH(1-7) targeting a PTH protein or active fragment thereof.

[0167] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: 3 CDRs contained in a VH as set forth in SEQ ID NO: 80 or 88; and / or, 3 CDRs contained in a VL as set forth in SEQ ID NO: 85 or 91. In certain embodiments, the CDRs are defined by the Kabat, Chothia, or IMGT numbering system.

[0168] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising 3 CDRs: CDR-H1 of SEQ ID NO: 77; CDR-H2 of SEQ ID NO: 78 or 87; CDR-H3 of SEQ ID NO: 79; and, a VL comprising 3 CDRs: CDR-L1 of SEQ ID NO: 82 or 90; CDR-L2 of SEQ ID NO: 83; CDR-L3 of SEQ ID NO: 84. In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0169] In certain embodiments, (a) the VH comprises: CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 77, 78, 79, respectively; and the VL comprises: CDR-L1, CDR-L2, CDR-L3 of SEQ ID NOs: 82, 83, 84, respectively; or (b) the VH comprises: CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 77, 87, 79, respectively; and the VL comprises: CDR-L1, CDR-L2, CDR-L3 of SEQ ID NOs: 90, 83, 84, respectively. In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0170] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VHcomprising a sequence as set forth in SEQ ID NO: 80 or 88, or a sequence at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical thereto, and a VLcomprising a sequence as set forth in SEQ ID NO: 85 or 91, or a sequence at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical thereto.

[0171] In certain embodiments, (a) a VHcomprising a sequence as set forth in SEQ ID NO: 80 and a VLcomprising a sequence as set forth in SEQ ID NO: 85; or (b) a VHcomprising a sequence as set forth in SEQ ID NO: 88 and a VLcomprising a sequence as set forth in SEQ ID NO: 91.

[0172] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4) and a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda).

[0173] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a variant of a human IgG (e.g., IgGl or IgG4) heavy chain constant region that possesses a mutation that reduces or abolishes effector function (e.g., ADCC, ADCP, and / or CDC activity) as compared to the wild-type sequence from which it is derived.

[0174] In certain embodiments, the variant of a human IgG heavy chain constant region is a variant of a human IgGl heavy chain constant region that possesses one or more of the following substitutions: L234A, L235A, and P329G (positions according to the EU numbering system) as compared to the wild-type sequence.

[0175] In certain embodiments, the variant of a human IgGl heavy chain constant region comprises a sequence as set forth in SEQ ID NO: 54 or 71.

[0176] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0177] (i) a heavy chain having a sequence as set forth in SEQ ID NO: 81 and a light chain having a sequence as set forth in SEQ ID NO: 86; or

[0178] (ii) a heavy chain having the sequence set forth in SEQ ID NO: 89 and a light chain having the sequence set forth in SEQ ID NO: 92.

[0179] In certain embodiments, the first protein is as defined in Section I-ii of the present application ("Complex: PTH-VHH fusion protein with anti-PTH antibody"), and the second protein is as defined in Section I-ii of the present application ("Complex: PTH-VHH fusion protein with anti-PTH antibody").

[0180] In certain embodiments, in the complex:

[0181] (1) the first protein is a fusion protein set forth in SEQ ID NO: 29, and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 81 and a light chain having the sequence set forth in SEQ ID NO: 86;

[0182] (2) the first protein is a fusion protein set forth in SEQ ID NO: 30, and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 81 and a light chain having the sequence set forth in SEQ ID NO: 86;

[0183] (3) the first protein is a fusion protein set forth in SEQ ID NO: 45, and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 89 and a light chain having the sequence set forth in SEQ ID NO: 92;

[0184] (4) the first protein is a fusion protein set forth in SEQ ID NO: 46, and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 89 and a light chain having the sequence set forth in SEQ ID NO: 92;

[0185] (5) the first protein is a fusion protein set forth in SEQ ID NO: 30, and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 89 and a light chain having the sequence set forth in SEQ ID NO: 92;

[0186] (6) the first protein is a fusion protein set forth in SEQ ID NO: 48, and the second protein is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 89 and a light chain having the sequence set forth in SEQ ID NO: 92;

[0187] (7) the first protein is a fusion protein of SEQ ID NO: 49, and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 89 and a light chain having the sequence of SEQ ID NO: 92; or

[0188] (8) the first protein is a fusion protein of SEQ ID NO: 47, and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 89 and a light chain having the sequence of SEQ ID NO: 92.

[0189] I-iii. Complex: PTH-VHH fusion protein and anti-PTH antibody / PTH and anti-PTH antibody

[0190] In another aspect, the present application provides a complex comprising a first protein as defined in section I-i (“Complex: PTH and anti-PTH antibody”) or I-ii (“Complex: PTH-VHH fusion protein and anti-PTH antibody”) of the present application and a second protein as defined in section I-i (“Complex: PTH and anti-PTH antibody”) or I-ii (“Complex: PTH-VHH fusion protein and anti-PTH antibody”) of the present application.

[0191] In certain embodiments, the first protein is as defined in section I-i (“Complex: PTH and anti-PTH antibody”) of the present application, and the second protein is as defined in section I-i (“Complex: PTH and anti-PTH antibody”) of the present application.

[0192] In certain embodiments, the first protein is as defined in section I-ii (“Complex: PTH-VHH fusion protein and anti-PTH antibody”) of the present application, and the second protein is as defined in section I-ii (“Complex: PTH-VHH fusion protein and anti-PTH antibody”) of the present application.

[0193] II. Compositions

[0194] In another aspect, the present application provides a composition comprising:

[0195] (i) a first protein, or a nucleic acid molecule or a vector encoding said first protein, or a host cell comprising said nucleic acid molecule or vector, said first protein being a first protein comprised by a complex of the present application;

[0196] (ii) a second protein, or a nucleic acid molecule or a vector encoding said second protein, or a host cell comprising said nucleic acid molecule or vector, said second protein being a second protein comprised by a complex of the present application.

[0197] In certain embodiments, the composition comprises a feature selected from the group consisting of:

[0198] (1) the first protein is as defined in Section I-i of the Invention ("Complex: PTH and anti-PTH antibody"), and the second protein is as defined in Section I-i of the Invention ("Complex: PTH and anti-PTH antibody");

[0199] (2) the first protein is as defined in Section I-ii of the Invention ("Complex: PTH-VHH fusion protein and anti-PTH antibody"), and the second protein is as defined in Section I-ii of the Invention ("Complex: PTH-VHH fusion protein and anti-PTH antibody").

[0200] In certain embodiments, the composition comprises:

[0201] (i) the first protein comprised by the complex of Section I-i of the Invention, or a nucleic acid molecule or vector encoding the second protein, or a host cell comprising the nucleic acid molecule or vector; and

[0202] (ii) the second protein comprised by the complex of Section I-i of the Invention, or a nucleic acid molecule or vector encoding the second protein, or a host cell comprising the nucleic acid molecule or vector.

[0203] In certain embodiments, the composition comprises:

[0204] (i) the first protein comprised by the complex of Section I-ii of the Invention, or a nucleic acid molecule or vector encoding the first protein, or a host cell comprising the nucleic acid molecule or vector; and

[0205] (ii) the second protein comprised by the complex of Section I-ii of the Invention, or a nucleic acid molecule or vector encoding the second protein, or a host cell comprising the nucleic acid molecule or vector.

[0206] In another aspect, the present invention provides a method of preparing a complex of the present invention, comprising:

[0207] In another aspect, the present invention provides a method of preparing a complex of the present invention, comprising:

[0208] (1) providing a first protein and a second protein of a complex of the present invention;

[0209] (2) contacting (e.g., incubating) the first protein and the second protein under conditions suitable for complex formation, to obtain a complex of the application.

[0210] III. Uses

[0211] In another aspect, the application provides a pharmaceutical composition comprising a complex, composition of the application, and a pharmaceutically acceptable carrier and / or excipient.

[0212] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0213] In certain embodiments, the additional pharmaceutically active agent is selected from an agent that treats a bone-related disease, or an agent that treats a disease caused by hypoparathyroidism.

[0214] In certain embodiments, the bone-related disease is osteoporosis.

[0215] In certain embodiments, the disease caused by hypoparathyroidism is selected from hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria.

[0216] In another aspect, the application provides use of a complex, composition, or pharmaceutical composition of the application for the manufacture of a medicament for preventing and / or treating a bone-related disease or a disease caused by hypoparathyroidism in a subject.

[0217] In certain embodiments, the subject is a mammal, e.g., a human.

[0218] In certain embodiments, the complex, composition, or pharmaceutical composition is used alone or in combination with an additional pharmaceutically active agent.

[0219] In certain embodiments, the additional pharmaceutically active agent is selected from an agent that treats a bone-related disease, or an agent that treats a disease caused by hypoparathyroidism.

[0220] In certain embodiments, the bone-related disease is osteoporosis.

[0221] In certain embodiments, the disease caused by hypoparathyroidism is selected from hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria.

[0222] In another aspect, the application provides a method for preventing and / or treating a bone-related disease or a disease caused by hypoparathyroidism in a subject, comprising: administering to a subject in need thereof an effective amount of a complex, composition, or pharmaceutical composition of the application.

[0223] In certain embodiments, the subject is a mammal, e.g., a human.

[0224] In certain embodiments, the complex, composition, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent.

[0225] In certain embodiments, the additional pharmaceutically active agent is selected from an agent that treats a bone-related disease, or an agent that treats a disease caused by hypoparathyroidism.

[0226] In certain embodiments, the bone-related disease is osteoporosis.

[0227] In certain embodiments, the disease caused by hypoparathyroidism is selected from hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria.

[0228] The complex, composition, or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, e.g., tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The complex, composition, or pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the complex, composition, or pharmaceutical composition of the present application in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Furthermore, sterile injectable solutions can be prepared as sterile freeze-dried powders (e.g., by vacuum drying or freeze-drying) to allow reconstitution with a suitable solvent before use. Such sterile freeze-dried powders can be reconstituted into a suitable solvent, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), dextrose solution (e.g., 5% dextrose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH-buffered solution (e.g., phosphate-buffered saline), Ringer's solution, and any combination thereof, before use.

[0229] The complex, composition, or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrafoliar, intracellular reticulum, inguinal, intravesical, topical (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In certain embodiments, the complex, composition, or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0230] IV. Constituent molecules of the complex

[0231] PTH polypeptide or active fragment thereof

[0232] In another aspect, the present application provides a PTH polypeptide or active fragment thereof that, compared to a wild-type PTH polypeptide or active fragment thereof:

[0233] (1) comprises a mutation at one or more of the positions corresponding to positions 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99; and / or,

[0234] (2) comprises an aliphatic organic acid.

[0235] In certain embodiments, the mutation is an amino acid substitution.

[0236] In certain embodiments, the mutation is selected from one or more of the following:

[0237] (a) the amino acid at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted with a non-natural amino acid (e.g., alpha-aminobutyric acid (Aib));

[0238] (b) the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted, e.g., with alanine (A), serine (S);

[0239] (c) the serine (S) at the position corresponding to position 17 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted, e.g., with glutamic acid (E);

[0240] (d) the phenylalanine (F) at the position corresponding to position 34 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted, e.g., with lysine (K).

[0241] In certain embodiments, the PTH polypeptide or active fragment thereof comprises at least the amino acid residues in PTH corresponding to positions 1-14 (or 1-16, 1-18, 1-20, 1-22, 1-25, 1-30, 1-34) of SEQ ID NO: 99.

[0242] In certain embodiments, the PTH polypeptide or active fragment thereof comprises at least the amino acid residues in PTH corresponding to positions 1-34 of SEQ ID NO: 99, and is substituted at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 with a non-natural amino acid, such as alpha-aminobutyric acid (Aib).

[0243] In certain embodiments, the PTH polypeptide or active fragment thereof consists of the amino acid residues in PTH corresponding to positions 1-14, or 1-16, or 1-18, or 1-20, or 1-22, or 1-25, or 1-30, or 1-34 of SEQ ID NO: 99.

[0244] In certain embodiments, the PTH polypeptide or active fragment thereof is substituted with a non-natural amino acid, such as alpha-aminobutyric acid (Aib), at an amino acid position selected from the following relative to SEQ ID NO: 99: (i) positions 1, 3, 25; (ii) positions 3, 25; (iii) positions 1, 25; (iv) position 25; or (v) position 1.

[0245] In certain embodiments, the PTH polypeptide or active fragment thereof is substituted at the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99, for example, substituted with alanine (A), serine (S).

[0246] In certain embodiments, the PTH polypeptide or active fragment thereof is substituted at the serine (S) at the position corresponding to position 17 of SEQ ID NO: 99, for example, substituted with glutamic acid (E).

[0247] In certain embodiments, the PTH polypeptide or active fragment thereof is substituted at the phenylalanine (F) at the position corresponding to position 34 of SEQ ID NO: 99, for example, substituted with lysine (K).

[0248] In certain embodiments, the PTH polypeptide or active fragment thereof: (I) is substituted at the amino acid position corresponding to position 1, 25 of SEQ ID NO: 99 with a non-natural amino acid (such as alpha-aminobutyric acid (Aib)); (II) is substituted at the asparagine (N) corresponding to position 16 of SEQ ID NO: 99 (e.g., substituted with alanine (A), serine (S)); and / or, (III) is substituted at the serine (S) corresponding to position 17 of SEQ ID NO: 99 (e.g., substituted with glutamic acid (E)).

[0249] In certain embodiments, the PTH polypeptide or active fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 100-102, 73-76, 105, 106.

[0250] (1) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 1 to the C-terminus;

[0251] (2) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 11 to the C-terminus;

[0252] (3) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 12 to the C-terminus;

[0253] (4) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 13 to the C-terminus;

[0254] (5) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 14 to the C-terminus;

[0255] (6) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 15 to the C-terminus;

[0256] (7) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 16 to the C-terminus;

[0257] (8) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 17 to the C-terminus;

[0258] (9) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 18 to the C-terminus;

[0259] (10) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 19 to the C-terminus;

[0260] (11) any amino acid in the PTH polypeptide or active fragment thereof from the amino acid at position 23 to the C-terminus;

[0261] (12) any amino acid in the PTH polypeptide or active fragment thereof located from amino acid position 26 to the C-terminus; or

[0262] (13) any amino acid in the PTH polypeptide or active fragment thereof located from amino acid position 29 to the C-terminus;

[0263] (14) any amino acid in the PTH polypeptide or active fragment thereof located from amino acid position 31 to the C-terminus;

[0264] (15) any amino acid in the PTH polypeptide or active fragment thereof located from amino acid position 32 to the C-terminus;

[0265] (16) any amino acid in the PTH polypeptide or active fragment thereof located from amino acid position 33 to the C-terminus;

[0266] (17) any amino acid in the PTH polypeptide or active fragment thereof located from amino acid position 34 to the C-terminus;

[0267] wherein the positions are amino acid positions relative to SEQ ID NO: 99.

[0268] In certain embodiments, the aliphatic organic acid is optionally linked to amino acid position 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 of the PTH polypeptide or active fragment thereof via a spacer.

[0269] In certain embodiments, the aliphatic organic acid is optionally linked to the amino acid at the C-terminus of the PTH polypeptide or active fragment thereof via a spacer.

[0270] In certain embodiments, the aliphatic organic acid is optionally linked via a spacer to an amino acid at a position corresponding to position 34 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof.

[0271] In certain embodiments, the amino acid is selected from the group consisting of lysine, d-lysine, ornithine, cysteine, and homocysteine.

[0272] In certain embodiments, the PTH polypeptide or active fragment thereof is substituted with a non-natural amino acid, such as alpha-aminoisobutyric acid (Aib) at a position selected from the group consisting of: (i) position 1, position 25; (ii) position 1; and, the aliphatic organic acid is optionally linked via a spacer to an amino acid at a position corresponding to position 34 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof.

[0273] In certain embodiments, the spacer comprises an acidic amino acid residue (e.g., gamma glutamic acid).

[0274] In certain embodiments, the spacer is selected from the group consisting of gamma glutamic acid-gamma glutamic acid dipeptide, *-[COCH2(OCH2CH2) k NH] q -gamma glutamic acid or gamma glutamic acid-[COCH2(OCH2CH2) k NH] q -gamma glutamic acid, wherein k is an integer selected from 1 to 20, q is an integer selected from 1 to 20, the * end is attached to an amino acid side chain.

[0275] In certain embodiments, the k is 1 or 2.

[0276] In certain embodiments, the q is 1 or 2.

[0277] In certain embodiments, the aliphatic organic acid is linked via a spacer to any amino acid in the PTH polypeptide or active fragment thereof located between the amino acid at position 3 to the C-terminus; the positions are amino acid positions relative to SEQ ID NO: 99; the structural composition of the aliphatic organic acid and spacer is shown in formula (A):

[0278] III-(II)m-(I)n- Formula (A),

[0279] wherein,

[0280] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;

[0281] I is -C(=0)CH2(OCH2CH2)2NH-;

[0282] II is an acidic amino acid residue;

[0283] III is COOH-(CH2)r-C(=0)-, r is an integer selected from 12 to 30; and

[0284] III, II, and I are connected by an amide bond,

[0285] the carbonyl of I is connected to an amino group of an amino acid side chain in the PTH polypeptide or active fragment thereof.

[0286] In certain embodiments, the carbonyl of I is connected to an amino group of a lysine side chain.

[0287] In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0288] In certain embodiments, n is 1 or 2.

[0289] In certain embodiments, II is selected from a gamma glutamic acid residue and an aspartic acid residue.

[0290] In certain embodiments, II is a gamma glutamic acid residue having the structure shown below:

[0291] In certain embodiments, m is 1.

[0292] In certain embodiments, III is COOH-(CH2)r-C(=0)-, r is an integer selected from 16 to 22.

[0293] In certain embodiments, III is COOH-(CH2) 16 -C(=0)-, COOH-(CH2) 17 -C(=0)-, or COOH-(CH2) 18 -C(=0)-.

[0294] In certain embodiments, the formula (A) has a structure selected from the group consisting of:

[0295] In certain embodiments, the PTH polypeptide or active fragment thereof comprises a sequence as set forth in SEQ ID NO: 103 or 104.

[0296] In another aspect, the present application provides a PTH polypeptide or active fragment thereof comprising a sequence as set forth in any one of SEQ ID NOs: 73-76, 100-106.

[0297] Nanobody or antigen-binding fragment thereof

[0298] In another aspect, the present application provides a nanobody or antigen-binding fragment thereof that specifically binds serum albumin, comprising: 3 CDRs contained in a VHH as set forth in any one of SEQ ID NOs: 7, 12, 18, 23, 96, 98. In certain embodiments, the CDRs are defined by the Kabat, Chothia, or IMGT numbering system.

[0299] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises:

[0300] (1) a CDR1 as set forth in SEQ ID NO: 4, a CDR2 as set forth in SEQ ID NO: 5 or 16, and a CDR3 as set forth in SEQ ID NO: 6, 95, or 97; or

[0301] (2) a CDR1 as set forth in SEQ ID NO: 9, a CDR2 as set forth in SEQ ID NO: 10, and a CDR3 as set forth in SEQ ID NO: 11. In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0302] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises: (i) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 5, 6, respectively; (ii) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 6, respectively; (iii) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 95, respectively; or (iv) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 97, respectively. In certain embodiments, the CDRs are defined by the IMGT numbering system.

[0303] In certain embodiments, the nanobody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 7, 12, 18, 23, 96, 98, or a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical thereto.

[0304] Polypeptide construct

[0305] In another aspect, the present application provides a polypeptide construct specifically binding to serum albumin, comprising a Nanobody or antigen-binding fragment thereof of the present application, and an immunoglobulin Fc domain.

[0306] In certain embodiments, the immunoglobulin Fc domain is optionally linked to the N- and / or C-terminus (e.g. C-terminus) of the Nanobody or antigen-binding fragment thereof via a peptide linker.

[0307] In certain embodiments, the immunoglobulin Fc domain is an Fc domain of IgG (e.g. Fc domain of IgG1, IgG2, IgG3 or IgG4).

[0308] In certain embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 14.

[0309] In certain embodiments, the polypeptide construct comprises the sequence set forth in any one of SEQ ID NOs: 8, 13, 19, 24.

[0310] Fusion protein

[0311] In another aspect, the present application provides a fusion protein comprising a PTH polypeptide or active fragment thereof, and a Nanobody or antigen-binding fragment thereof of the present application; wherein the PTH polypeptide or active fragment thereof is a wild-type PTH polypeptide or active fragment thereof, or the PTH polypeptide or active fragment thereof comprises a modification comprising a mutation at one or more of the positions corresponding to position 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99, as compared to the wild-type PTH polypeptide or active fragment thereof.

[0312] In certain embodiments, the mutation is an amino acid substitution.

[0313] In certain embodiments, the mutation is selected from one or more of the following:

[0314] (a) the amino acid at one or more of the positions corresponding to position 1, 3, 25 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted with a non-natural amino acid (e.g. alpha-aminobutyric acid (Aib));

[0315] (b) the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted, e.g. with alanine (A), serine (S);

[0316] (c) the PTH polypeptide or active fragment thereof has a substitution at the position corresponding to position 17 of SEQ ID NO: 99, e.g., a substitution to glutamic acid (E), from serine (S);

[0317] (d) the PTH polypeptide or active fragment thereof has a substitution at the position corresponding to position 34 of SEQ ID NO: 99, e.g., a substitution to lysine (K), from phenylalanine (F).

[0318] In certain embodiments, the PTH polypeptide or active fragment thereof comprises at least the amino acid residues in PTH at positions corresponding to positions 1-14 (or 1-16, 1-18, 1-20, 1-22, 1-25, 1-30, 1-34) of SEQ ID NO: 99.

[0319] In certain embodiments, the PTH polypeptide or active fragment thereof consists of the amino acid residues in PTH at positions corresponding to positions 1-34 of SEQ ID NO: 99.

[0320] In certain embodiments, the PTH polypeptide or active fragment thereof has at least one naturally occurring asparagine (N) substituted, e.g., to alanine (A) or serine (S), as compared to wild-type PTH protein.

[0321] In certain embodiments, the PTH polypeptide or active fragment thereof has a substitution at the position corresponding to position 16 of SEQ ID NO: 99, e.g., a substitution to alanine (A) or serine (S), from asparagine (N).

[0322] In certain embodiments, the PTH polypeptide or active fragment thereof comprises a sequence as set forth in SEQ ID NO: 93 or 94.

[0323] In certain embodiments, the PTH polypeptide or active fragment thereof is optionally linked to the N- or C-terminus, e.g., the N-terminus, of the nanobody or antigen-binding fragment thereof via a linker.

[0324] In certain embodiments, the PTH polypeptide or active fragment thereof is directly linked to the N-terminus of the nanobody or antigen-binding fragment thereof.

[0325] In certain embodiments, the linker is a peptide linker (e.g., a rigid peptide linker or a flexible peptide linker).

[0326] In certain embodiments, the linker is a peptide linker comprising one or more glycines and / or one or more serines.

[0327] In certain embodiments, the peptide linker is (GmS)n, m, n is independently an integer no less than 0, for example, independently 1, 2, 3, or 4.

[0328] In certain embodiments, the peptide linker comprises or consists of the sequence set forth in any one of SEQ ID NOs: 26, 31-34.

[0329] In certain embodiments, the fusion protein comprises the sequence set forth in any one of SEQ ID NOs: 27-30, 35-49, for example, the sequence set forth in any one of SEQ ID NOs: 29, 30, 45-49.

[0330] In another aspect, the present application provides an isolated nucleic acid molecule encoding a PTH polypeptide or an active fragment thereof, a Nanobody or an antigen binding fragment thereof, a polypeptide construct, a fusion protein of the present application.

[0331] In another aspect, the present application provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present application.

[0332] In certain embodiments, the vector of the present application is, for example, a plasmid, a cosmid, a phage, and the like.

[0333] In another aspect, the present application provides a host cell comprising an isolated nucleic acid molecule or a vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells, human cells, and the like).

[0334] In another aspect, the present application provides a method of making a PTH polypeptide or an active fragment thereof, a Nanobody or an antigen binding fragment thereof, a polypeptide construct, or a fusion protein of the present application, comprising: culturing a host cell as described above under conditions that allow expression of the PTH polypeptide or an active fragment thereof, a Nanobody or an antigen binding fragment thereof, a polypeptide construct, or a fusion protein, and recovering the PTH polypeptide or an active fragment thereof, a Nanobody or an antigen binding fragment thereof, a polypeptide construct, or a fusion protein from the cultured host cell culture.

[0335] In another aspect, the present application provides a pharmaceutical composition containing a PTH polypeptide or an active fragment thereof, or a fusion protein of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0336] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0337] In certain embodiments, the additional pharmaceutically active agent is selected from an agent that treats a bone-related disease, or an agent that treats a disease caused by hypoparathyroidism.

[0338] In certain embodiments, the bone-related disease is osteoporosis.

[0339] In certain embodiments, the disease caused by hypoparathyroidism is selected from hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria.

[0340] In another aspect, the present application provides use of a PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition of the present application for the manufacture of a medicament for preventing and / or treating a bone-related disease or a disease caused by hypoparathyroidism in a subject.

[0341] In certain embodiments, the subject is a mammal, e.g., a human.

[0342] In certain embodiments, the PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition is used alone or in combination with an additional pharmaceutically active agent.

[0343] In certain embodiments, the additional pharmaceutically active agent is selected from an agent that treats a bone-related disease, or an agent that treats a disease caused by hypoparathyroidism.

[0344] In certain embodiments, the bone-related disease is osteoporosis.

[0345] In certain embodiments, the disease caused by hypoparathyroidism is selected from hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria.

[0346] In another aspect, the present application provides a method for preventing and / or treating a bone-related disease or a disease caused by hypoparathyroidism in a subject, comprising: administering to a subject in need thereof an effective amount of a PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition of the present application.

[0347] In certain embodiments, the subject is a mammal, e.g., a human.

[0348] In certain embodiments, the PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition is used alone or in combination with an additional pharmaceutically active agent.

[0349] In certain embodiments, the additional pharmaceutically active agent is selected from an agent that treats a bone-related disease, or an agent that treats a disease caused by hypoparathyroidism.

[0350] In certain embodiments, the bone-related disease is osteoporosis.

[0351] In certain embodiments, the disease caused by hypoparathyroidism is selected from hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria.

[0352] The PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition of the present application in the required amount in an appropriate solvent with one or more of the other ingredients, including, but not limited to, pH adjusting agents, surfactants, adjuvants, ion strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives, stabilizers, or any combination thereof, followed by filtered sterilization. In addition, sterile injectable solutions can be prepared as sterile, lyophilized powders (e.g., by vacuum drying or freeze-drying), for reconstitution with a suitable vehicle, e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), dextrose solution (e.g., 5% dextrose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered saline), Ringer's solution, and any combination thereof, prior to use.

[0353] The PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, endocytic reticulum, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or mode of administration will vary depending upon the desired goal. In certain embodiments, the PTH polypeptide or active fragment thereof, fusion protein, or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0354] Terminology Definitions

[0355] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Also, the virology, biochemistry, immunology laboratory procedures used herein are conventional procedures well established in the respective fields. Also, for better understanding of the present application, the definitions and explanations of the related terms are provided as follows.

[0356] When the terms "for example", "for instance", "such as", "including", "containing", or variations thereof are used herein, these terms are not to be interpreted in an exclusive sense, but are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components.

[0357] Unless otherwise indicated herein, or in the context of the specification, the terms "a" and "an" and the like are to be construed to cover both singular and plural references. Unless otherwise indicated herein, or in the context of the specification, the terms "comprising", "comprises" and the like are to be construed as not excluding the presence of other elements or steps.

[0358] As used herein, the term "parathyroid hormone (PTH)" is a basic, single-chain polypeptide hormone secreted by the chief cells of the parathyroid glands. PTH can regulate the metabolism of calcium and phosphorus in vertebrates, specifically can increase the concentration of calcium in the blood and decrease the concentration of phosphorus in the blood, the main target organs of action are bone and kidney, and can be used for the treatment of bone metabolic diseases. An exemplary amino acid sequence of PTH can be found in SEQ ID NO: 99, or obtained from public databases (e.g., GenBank database, UniProt database).

[0359] In this document, when referring to the amino acid sequence of PTH, the sequence set forth in SEQ ID NO: 99 is used for description. For example, the expression "the Xth amino acid residue of PTH" refers to the Xth amino acid residue of the polypeptide set forth in SEQ ID NO: 99. However, one skilled in the art understands that mutations or variations can be naturally occurring or artificially introduced in the amino acid sequence of PTH without affecting its biological function. Therefore, in the present application, the term "PTH" and its similar expressions shall include all such sequences, including, for example, the sequence set forth in SEQ ID NO: 99 and its natural or artificial variants (e.g., variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity). Also, when describing the residue positions or sequence fragments of PTH, they include not only the residue positions or sequence fragments of SEQ ID NO: 99, but also the corresponding residue positions or corresponding sequence fragments in its natural or artificial variants. For example, the expression "the Xth amino acid residue of PTH" includes the Xth amino acid residue of SEQ ID NO: 99 and the corresponding position in its variants (natural or artificial). According to the present application, the expression "corresponding position" or "corresponding fragment" refers to the residue or fragment at the equivalent position in the sequence being compared when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity. The optimal alignment of sequences for comparison can be performed by computerized implementation of known algorithms or by visual inspection. Existing sequence alignment and multiple sequence alignment algorithms include BLAST, ClustalW / ClustalW2 / Clustal Omega, etc.

[0360] As used herein, the term "PTH peptide" (also referred to as "PTH polypeptide", both of which are used interchangeably) has the meaning generally understood by one skilled in the art, which generally represents an active fragment of a full-length PTH protein (e.g., a wild-type PTH protein or a modified PTH protein), or, in certain embodiments, the PTH peptide can also be used to refer to the full-length PTH protein (e.g., a wild-type PTH protein or a modified PTH protein) itself. Generally, a PTH peptide typically possesses the biological activity (e.g., PTH1R binding activity and / or PTH1R activation activity) of the full-length PTH protein (e.g., a wild-type PTH protein or a modified PTH protein) from which it is derived. In the present application, non-limiting examples of PTH peptides include: PTH 3-16 , PTH 3-17 , PTH 3-27 , PTH 3-33 , PTH 3- 34 , PTH 3-35 , PTH 3-40 , PTH 3-50PTH 3-60 PTH 3-70 PTH 3-80 PTH 3-84 PTH 1-16 PTH 1- 17 PTH 1-27 PTH 1-33 PTH 1-34 PTH 1-35 PTH 1-40 PTH 1-50 PTH 1-60 PTH 1-70 PTH 1- 80 and PTH 1-84 The term "protein" as used herein refers to an organic macromolecule compound formed by one or more polypeptide chains formed by a plurality of amino acids connected by peptide bonds. In some embodiments, the amino acids have a general structure, e.g., R-CH(NH2)-COOH, with R being a side chain group. In some embodiments, the amino acids are naturally occurring amino acids. In some embodiments, the amino acids are non-natural amino acids. In some embodiments, the amino acids are D-amino acids; in some embodiments, the amino acids are L-amino acids. A "standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than a standard amino acid, whether prepared synthetically or obtained from a natural source. In the present disclosure, the terms "polypeptide", "protein", and "peptide" have the same meaning and are used interchangeably.

[0361] As used herein, the term "active fragment of a PTH protein" refers to a fragment having a function of a PTH protein, which can be a part of a PTH protein or a fragment obtained after modification (e.g., deletion, addition, or substitution of amino acids) of a PTH protein; for example, the active fragment is a fragment comprising a part of a PTH protein that binds to a ligand or a receptor, or a fragment that retains a function of a PTH protein after deletion, addition, or substitution of amino acids.

[0362] As used herein, the term "acidic amino acid" refers to an amino acid containing two carboxyl groups, such as glutamic acid and aspartic acid.

[0363] As used herein, the term "amino acid residue" refers to the remaining structure after the amino group of an amino acid loses one hydrogen and / or the carboxyl group loses one hydroxyl. An amino acid residue (e.g., in the context of a polypeptide sequence) as used herein can be represented by its full name, its one-letter code, and / or its three-letter code. These three ways are fully equivalent and are used interchangeably.

[0364] The nomenclature of the twenty standard amino acids referred to herein follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Also in the present application, amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0365] As used herein, the term "aliphatic organic acid" refers to a saturated or unsaturated organic acid having an aliphatic hydrocarbon chain (straight chain or branched). The "aliphatic organic acid" includes fatty acids and fatty diacids. The term "fatty acid" refers to a carboxylic acid having an aliphatic hydrocarbon chain (straight chain or branched), e.g., containing 10 to 30 carbon atoms, and one of the carbon atoms forms a carboxyl group. Examples of fatty acids include, but are not limited to, myristic acid, palmitic acid, stearic acid, and arachidic acid. The term "fatty diacid" refers to a dicarboxylic acid having an aliphatic hydrocarbon chain (straight chain or branched), e.g., containing 10 to 30 carbon atoms, and two of the carbon atoms form carboxyl groups. Examples of fatty diacids include, but are not limited to, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, eicosanedioic acid, docosanedioic acid, tetracosanedioic acid.

[0366] As used herein, the term "linker" or "spacer" is a bond, molecule, or molecular group that binds two separate entities to one another. The linker can provide optimal spacing of the two entities, or can otherwise provide a labile linkage that separates the two entities from one another. Labile bonds include photocleavable groups, acid-labile moieties, base-labile moieties, and enzyme-cleavable groups.

[0367] As used herein, the term "AEEA-AEEA-GAMMA-GLU-C18DIACID" is also referred to as "AEEA-AEEA-γGlu-C18DIACID" or "OEG-OEG-γE-C18 diacid". Wherein GAMMA-GLU is used interchangeably with γGlu, γ-Glu, and γE. The structure of AEEA-AEEA-GAMMA-GLU-C18DIACID is: wherein the carbon marked with "*" can be in the racemic form, or in the R or S configuration form. Preferably, the structure is: When it modifies a particular amino acid and / or amino acid residue in a sequence, one skilled in the art will understand that AEEA-AEEA-GAMMA-GLU-C18DIACID, in the absence of further specification, means the structural fragment of the compound above after reaction with the amino acid and / or amino acid residue: Preferably, the structural fragment is:

[0368] The term "antibody", as used herein, refers to a molecule derived from an immunoglobulin that is capable of specifically binding to a target antigen through at least one antigen-combining site located in its variable region. An "intact antibody" typically comprises two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibodies, as IgM, IgD, IgG, IgA, and IgE, respectively. Within light chains and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains exhibit less inter- species variability than the variable domains, and are not directly involved in binding of the antibody to an antigen. However, they exhibit various effector functions, such as mediating immunoglobulin- mediated and host tissue or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains (VH and VL, respectively) form the antigen binding site of antibodies. Assignment of amino acids to each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)); or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883; or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0369] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in a Nanobody, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g., as defined by the numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or IMGT (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In the present application, the CDRs contained by the antibodies of the application or antigen-binding fragments thereof can be determined according to various numbering systems known in the art, e.g., by the Kabat, Chothia, IMGT, or AbM numbering system.

[0370] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0371] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide that comprises a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". Antigen-binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, single-chain antibodies (e.g., scFv), diabodies, multispecific antibodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.

[0372] As used herein, the term "full length antibody" means an antibody composed of two "full length heavy chains" and two "full length light chains". Among them, the "full length heavy chain" refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain in the direction from N-terminus to C-terminus; and, when the full length antibody is of IgE isotype, optionally further comprising a heavy chain constant region CH4 domain. Preferably, the "full length heavy chain" is a polypeptide chain composed of VH, CH1, HR, CH2 and CH3 in the direction from N-terminus to C-terminus. The "full length light chain" is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full length antibody chains are connected together by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full length heavy chains. The full length antibody of the present application can be from a single species, for example, human; it can also be a chimeric antibody or a humanized antibody. The full length antibody of the present application comprises two antigen binding sites formed by a pair of VH and VL, respectively, which specifically recognize / bind the same antigen.

[0373] As used herein, the term "Fd fragment" means an antibody fragment composed of VH and CH1 domains; the term "Fab fragment" means an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bond connecting the two heavy chain fragments in the F(ab')2 fragment, composed of one complete light chain and a Fd fragment of heavy chain (composed of VH and CH1 domains); the term "Fv fragment" means an antibody fragment composed of VL and VH domains of a single arm of an antibody.

[0374] As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. In some cases, a disulfide bond can also exist between the VH and VL of the scFv.

[0375] As used herein, the term "diabody" means that the VH and VL domains thereof are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and generating two antigen binding sites.

[0376] Each of the above antibody fragments maintains the ability to specifically bind to the same antigen bound by the full-length antibody from which the fragment was derived, and / or competes with the full-length antibody for specific binding to the antigen.

[0377] Antigen-binding fragments of antibodies (e.g., the above antibody fragments) can be obtained using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) from a given antibody (e.g., an antibody provided herein), and screened for specificity in the same manner as used for the intact antibody.

[0378] In the present context, the term "antibody" when used in reference to a term "antigen-binding fragment thereof, includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context specifically indicates otherwise.

[0379] As used herein, the term "nanobody" has the meaning commonly understood by those of skill in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Nanobodies can also be referred to as "single-domain antibodies" (sdAbs), and the two terms are used interchangeably. Typically, a nanobody consists of 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, so long as antigen binding and specificity are substantially maintained.

[0380] In the present context, the term "nanobody" when used in reference to a term "antigen-binding fragment thereof, includes not only intact nanobodies, but also antigen-binding fragments of nanobodies. As used herein, the term "antigen-binding fragment of a nanobody" refers to a polypeptide comprising a fragment of a nanobody that maintains the ability to specifically bind to the same antigen bound by the nanobody from which the fragment was derived, and / or competes with the nanobody for specific binding to the antigen. In some embodiments, the "antigen-binding fragment of a nanobody" can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, as compared to the full-length nanobody, so long as antigen binding and specificity are substantially maintained.

[0381] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0382] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (kaor kon) and the "dissociation rate constant" (kdisor koff) can be calculated from the concentrations and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 : 186-187). The ratio kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon, and kdis values can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore. In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0383] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, and the vector's genetic material elements are then expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain a replication origin.

[0384] As used herein, the term "host cell" refers to a cell that can be used for the introduction of a vector, including but not limited to, a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as fibroblast cell, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK 293 cell, or human cell.

[0385] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, physiologically and / or pharmacologically, with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives, stabilizers. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Adjuvants include, but are not limited to, inorganic adjuvants (such as aluminum hydroxide, vanadate), biological adjuvants (such as Mycobacterium tuberculosis, BCG, Corynebacterium parvum, Bordetella pertussis, Gram-negative bacterial endotoxins, B subunit of cholera toxin, muramyl dipeptide, cytokines), synthetic adjuvants (such as double-stranded polyribonucleotides, uridine acid), oil agents (such as Freund's complete adjuvant, peanut oil emulsion), and nano-adjuvants, etc. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial agents and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carriers or excipients include sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solution, and any combination thereof.

[0386] As used herein, the term "prevention" refers to a method undertaken in order to stop or delay the occurrence of a disease or disorder or symptoms thereof in a subject. As used herein, the term "treatment" refers to a method undertaken in order to obtain a beneficial or desired clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether or not detectable to the patient. Further, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0387] As used herein, the term "subject" refers to a mammal, for example, a human. In certain embodiments, the subject (e.g., human) has a bone-related disease (e.g., osteoporosis) or a disease caused by hypoparathyroidism (e.g., hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria), or is at risk of having such a disease.

[0388] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a bone-related disease or a disease caused by hypoparathyroidism) refers to an amount that is sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of the drug, and other therapies that the patient is undergoing, etc.

[0389] Advantages of the Invention

[0390] The present application provides an anti-PTH antibody and PTH polypeptide complex, which reduces kidney clearance through antibody binding, prolongs half-life through FcRn recycling mechanism, and protects PTH from protease degradation in serum after binding of the anti-PTH antibody to PTH. The present application also provides an anti-PTH antibody and PTH-VHH fusion protein complex, which can reversibly bind to serum albumin after entering the body, has complete serum albumin binding activity after the PTH-VHH fusion protein is separated from the anti-PTH antibody, provides protection to PTH, and prolongs the half-life, and finally the free PTH-VHH binds to PTH1R on the cell membrane to exert biological function. The present application also provides an anti-PTH antibody and PTH polypeptide complex modified by aliphatic organic acid chains, which protects the complex from protease degradation in serum through antibody binding, prolongs the half-life of PTH, and on the other hand, the aliphatic organic acid on the side chain of the PTH polypeptide can reversibly bind to serum albumin, providing protection to PTH and prolonging the half-life. The complex of the present application can effectively prolong the retention time of PTH in the body while ensuring the activity of PTH, which has important clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0391] Figure 1: Affinity detection results of SZ0396 and HSA, CSA, RSA, MSA. Among them, at 120s, the four curves in the figure from top to bottom represent the affinity detection results of SZ0396 and CSA, the affinity detection results of SZ0396 and HSA, the affinity detection results of SZ0396 and MSA, and the affinity detection results of SZ0396 and RSA.

[0392] Figure 2: Affinity detection results of GS002-KT06 and HSA, CSA, RSA, MSA. Among them, at 120s, the four curves in the figure from top to bottom represent the affinity detection results of GS002-KT06 and MSA, the affinity detection results of GS002-KT06 and CSA, the affinity detection results of GS002-KT06 and RSA, and the affinity detection results of GS002-KT06 and HSA.

[0393] Figure 3: Affinity detection results of SZ0630 and HSA, CSA, RSA, MSA. Among them, at 120s, the four curves in the figure from top to bottom represent the affinity detection results of SZ0630 and HSA, the affinity detection results of SZ0630 and CSA, the affinity detection results of SZ0630 and RSA, and the affinity detection results of SZ0630 and MSA.

[0394] Figure 4: The affinity detection results of SZ0695 and HSA, CSA, RSA, MSA. In the figure, at 120s, the four curves from top to bottom represent the affinity detection results of SZ0695 and CSA, the affinity detection results of SZ0695 and MSA, the affinity detection results of SZ0695 and HSA, and the affinity detection results of SZ0695 and RSA, respectively.

[0395] Figure 5: The schematic diagram of the molecular mechanism of PTH-VHH fusion protein.

[0396] Figure 6: The blood calcium concentration changes of rats after single administration of PTH-VHH (P011, P020).

[0397] Figure 7: The blood drug concentration changes of rats after single administration of PTH-VHH (P011, P020).

[0398] Figure 8: The blood calcium concentration changes of rats after single administration of humanized PTH-VHH (P065).

[0399] Figure 9: The blood calcium concentration changes of rats after single administration of humanized PTH-VHH (P090).

[0400] Figure 10: The blood calcium concentration changes of rats after single administration of PTH-VHH with shortened Linker (P092, P094, P095).

[0401] Figure 11: The schematic diagram of the molecular mechanism of the first generation complex (anti-PTH antibody / PTH complex).

[0402] Figure 12: The protein SEC purification profile of anti-PTH antibody / PTH complex.

[0403] Figure 13: The blood calcium concentration changes of rats after single administration of SZ01590160 / PEP025.

[0404] Figure 14: The blood calcium concentration changes of rats after single administration of SZ08830849 / PEP067.

[0405] Figure 15: The blood calcium concentration changes of rats after multiple administrations of SZ08830849 / PEP067.

[0406] Figure 16: The blood phosphorus concentration changes of rats after multiple administrations of SZ08830849 / PEP067.

[0407] Figure 17: The schematic diagram of the molecular mechanism of the second generation complex (anti-PTH antibody / PTH-VHH complex).

[0408] Figure 18: The protein SEC purification profile of anti-PTH antibody / PTH-VHH complex.

[0409] Figure 19: Changes in blood calcium concentration in rats after single administration of SZ07240725 / P065, SZ07240725 / P090.

[0410] Figure 20: Changes in blood calcium concentration in rats after single administration of SZ07240725 / P065, SZ07240725 / P090, SZ08850854 / P090, SZ08850854 / P126, SZ08850854 / P132.

[0411] Figure 21: Changes in blood calcium concentration in rats after single administration of SZ08850854 / P146, SZ08850854 / P149, SZ08850854 / P142.

[0412] Figure 22: Changes in blood calcium concentration in rats after multiple administration of SZ08850854 / P146, SZ08850854 / P149, SZ08850854 / P142.

[0413] Figure 23: Changes in blood phosphorus concentration in rats after multiple administration of SZ08850854 / P146, SZ08850854 / P149, SZ08850854 / P142.

[0414] Figure 24: Schematic diagram of the molecular mechanism of action of the third generation complex (anti-PTH antibody / PTH-FA (fatty acid chain modified PTH) complex).

[0415] Figure 25: Protein SEC purification profile of the anti-PTH antibody / PTH-FA complex.

[0416] Figure 26: Changes in blood calcium concentration in rats after single administration of SZ08830849 / PTH-FA complex.

[0417] Figure 27: Changes in blood phosphorus concentration in rats after single administration of SZ08830849 / PTH-FA complex.

[0418] Figure 28: Changes in blood calcium concentration in rats after multiple administration of SZ08830849 / PTH-FA complex.

[0419] Figure 29: Changes in blood phosphorus concentration in rats after multiple administration of SZ08830849 / PTH-FA complex.

[0420] Sequence information

[0421] The information of the sequences involved in the present application is described in the following table:

[0422] Table 1: Sequence description

[0423] Wherein, all CDRs in Table 1 are defined by IMGT numbering system; (Aib) represents alpha- aminoisobutyric acid; {K(AEEA-AEEA-GAMMA-GLU-C18DIACID)} represents lysine modified by aliphatic organic acid chain (AEEA-AEEA-GAMMA-GLU-C18DIACID). DETAILED DESCRIPTION

[0424] The present application will now be described in the following non-limiting examples.

[0425] Those skilled in the art will appreciate that the examples describe the present application by way of example only, and are not intended to limit the scope of the application as claimed. Experimental methods in the examples are conventional unless otherwise specified. Where specific conditions are not specified in the examples, conventional conditions or manufacturer's recommended conditions are used. Where manufacturers of reagents or instruments are not specified, conventional products available commercially are used.

[0426] Example 1: Preparation of anti-albumin VHH

[0427] 1.1 VHH screening

[0428] Goat and camel (goat and camel sources: Shenzhen Kangti Life and Ningxia Baishi) were immunized with human serum albumin (HSA), cynomolgus serum albumin (CSA) and rattus serum albumin (RSA) mixed in equal mass proportions, and peripheral blood was taken after 4 immunizations for constructing goat and camel immune libraries.

[0429] The sources of albumin of each species are as follows:

[0430] HSA (SEQ ID NO: 1), CSA (SEQ ID NO: 2) and RSA sequences (SEQ ID NO: 3) were downloaded from https: / / www.uniprot.org / , and mammalian cell expression was performed. After affinity purification, HSA, CSA and RSA proteins available for immunization were obtained.

[0431] Fresh peripheral blood 50ml of the immunization reached the titer was extracted, PBMC was separated, total RNA was extracted, and total cDNA was synthesized by reverse transcription. The reverse transcription product was used as a template for two rounds of PCR: the first round of PCR designed primers in the vector signal peptide and antibody heavy chain constant region, and two bands of about 1000bp and 750bp were amplified. The 750bp band was recovered and used as a template for the second round of PCR. The second round of PCR was designed in the antibody heavy chain FR1 and FR4 region and introduced a restriction site in the phagemid vector. The second round of PCR product was digested and recovered by agarose gel, and then loaded into the digested phagemid vector, and TG1 E. coli was electrotransformed to complete the construction of the immune library.

[0432] The immune library was packaged into a phage library, and biotinylated HSA (HSA-biotin) or RSA (RSA-biotin) was used as an antigen for liquid phase panning. The bacteria solution after the first and second rounds of panning was diluted and plated, and cultured overnight. Single colonies were picked from the plate, packaged with helper phage M13KO7, and the phage supernatant was subjected to ELISA screening. The positive clones were sequenced. The anti-HSA VHH screened from the immune library was designed for fusion expression with Human IgG1 Fc, and the amino acid sequence composition is shown in Table 2.

[0433] Table 2: Sequence composition of anti-serum albumin fusion protein

[0434] 1.2 Affinity identification

[0435] ForteBio Octet HTX was used to detect the affinity of VHH-Fc fusion protein to HSA, CSA, RSA, and mouse serum albumin (MSA): AHC (brand: Sartorius; item number: 18-5060) probes were used to capture the anti-HSA fusion protein (SZ0396, GS002-KT06) antibodies obtained above at 200s, and then HSA / CSA / RSA / MSA was used as the analyte, with a binding reaction of 120s and a dissociation reaction of 180s. The affinity of anti-HSA fusion protein to HSA, CSA, RSA, and MSA was analyzed.

[0436] The sources of albumin from various species are as follows:

[0437] The CSA sequence was downloaded from https: / / www.uniprot.org / , and the monkey serum albumin obtained after affinity purification was used for mammalian cell expression. The sequence is as follows (SEQ ID NO: 2).

[0438] HSA (brand: sigma; item number: SLCL8079)

[0439] RSA (brand: Equitech-Bio; part number: RTSA62-0100)

[0440] MSA (brand: abcam; part number: ab183228)

[0441] The experimental results are shown in Figures 1, 2 and Table 3, and the results show that the two screened anti-serum albumin fusion proteins have similar affinities for the serum albumins of the four species tested, and the affinities are all high.

[0442] Table 3: Affinity determination of anti-serum albumin fusion proteins to HSA, CSA, RSA, MSA

[0443] 1.3 Humanization of anti-HSA fusion proteins

[0444] The variable region sequences of the two screened anti-serum albumin fusion proteins (SZ0396, GS002-KT06) described above were designed for humanization, using the CDR grafting strategy, and partial back-mutation was performed. The designed variable region was constructed into a fusion protein with a human Fc region sequence, expressed in mammalian cells, and affinity purified. The fusion protein obtained after humanization of SZ0396 is called SZ0630, and the fusion protein obtained after humanization of GS002-KT06 is called SZ0695. The amino acid sequence composition of the humanized fusion proteins is shown in Table 4 below.

[0445] Table 4: Amino acid sequence composition of humanized fusion proteins

[0446] 1.4 Affinity identification after humanization

[0447] The affinities of the humanized anti-serum albumin VHH-Fc fusion proteins (SZ0630, SZ0695) to serum albumins of various species were detected using ForteBio Octet HTX. The AHC (brand: Sartorius; part number: 18-5060) probe was used to capture Anti-HSA antibody 200s, and then serum albumin was used as the analyte. The binding reaction was 120 s, and the dissociation reaction was 180 s. The affinities of anti-HSA to HSA, CSA, RSA, and MSA were analyzed.

[0448] The experimental results are shown in Figures 3, 4 and Table 5, and the results show that the two humanized anti-serum albumin fusion proteins have high affinities for the serum albumins of the four species tested.

[0449] Table 5: Affinity determination of humanized anti-serum albumin fusion proteins to HSA, CSA, RSA, MSA

[0450] Example 2: PTH-VHH fusion proteins

[0451] In order to improve the retention time of PTH in vivo, PTH is fused with anti-serum albumin nanobody (PTH-VHH), which prolongs the half-life by binding of nanobody to albumin, and the steric hindrance formed after the fusion protein binds to albumin protects the PTH polypeptide from proteases in serum; and after binding to serum albumin, it can partially block the function of PTH, reduce receptor-mediated clearance, and has complete activity when serum albumin is free. The molecular mechanism is shown in Figure 5.

[0452] 2.1 PTH-VHH fusion proteins

[0453] 2.1.1 Preparation of PTH-VHH fusion proteins

[0454] The PTH (1-34) polypeptide was constructed into a fusion protein with anti-HSA VHH, and the fusion proteins obtained by constructing the PTH (1-34) polypeptide with SZ0396 VHH and GS002-KT06 VHH were named P011 and P020, respectively, and the end of the fusion protein was mutated with a PP suffix. The amino acid sequence composition of fusion proteins P011 and P020 is as follows in Table 6.

[0455] Table 6: Amino acid sequence composition of fusion proteins P011 and P020

[0456] 2.1.2 cAMP activity detection of PTH-VHH fusion proteins

[0457] Preparation of serum incubation samples: The samples to be tested (P011 and P020) were mixed with human serum (manufacturer: ORiCELLS, item number: FSU-MIX-100ml) at a volume ratio of 1:2 for cAMP activity detection. The sample dilution and transfer equipment used in this experiment was a Thermo electric multi-channel pipettor (manufacturer: Thermo Scientific, item number: 4672060BT / 4672010BT).

[0458] cAMP activity detection method: according to the cAMP Gs Dynamic kit kit (brand: Cisbio; item number: 62AM4PEC) instructions to prepare two kinds of buffers, assay buffer 1 (0.5% BSA in DMEM) and assay buffer 2 (0.5mM IBMX in assay buffer 1). Add 5uL assay buffer 1 diluted sample of each concentration to the 384-well plate, add 5uL assay buffer 2 diluted Saos-2 cell suspension to the 384-well plate, and incubate in the cell incubator for 30 minutes. Add 5uL 1X cAMP-d2 and 5uL 1X Anti-cAMP-Cryptate in turn, incubate at room temperature for 1 hour, read data on PerkinElmer EnVision Multilabel 2103 (665nm and 620nm), calculate HTRF Ratio = Signal 665nm / Signal 620nm x 10 (665nm and 620nm) on PerkinElmer EnVision Multilabel 2103 (665nm and 620nm), calculate HTRF Ratio = Signal 665nm / Signal 620nm x 10 4 , cAMP% = [1-(HTRF Ratio sample -HTRF Ratioagonist max) / (HTRF Ratio cell only -HTRF Ratioagonist max)]x100%, wherein sample is the sample well, cell only is the cell only well, and agonist max is the PTH(1-34) well. Data analysis was performed by GraphPad Prism 10.2.3 software.

[0459] The results are shown in Table 7. P011 and P020 have activity in PBS comparable to PTH(1-34). After binding to serum albumin in serum, P011 and P020 partially block the activity of PTH, which may have a slow-release effect, consistent with the original design.

[0460] Table 7: cAMP activity detection

[0461] 2.1.3 Human serum stability of PTH-VHH fusion proteins

[0462] Preparation of serum incubation samples: mix the samples to be tested (P011 and P020) with human serum (manufacturer: ORiCELLS, item number: FSU-MIX-100ml) at a ratio of 1:2 by volume, and incubate in a 37°C incubator. After the incubation time, remove and store in a -20°C refrigerator. After collecting all time point samples, perform cAMP activity detection. The cAMP activity detection method is the same as that in Example 2.1.2.

[0463] The experimental results are shown in Table 8. From the results, the human serum stability of P011 and P020 is significantly improved compared with PTH (1-34), indicating that the anti-albumin VHH of P011 and P020 can well protect the PTH polypeptide from being degraded by proteases in serum after binding to serum albumin.

[0464] Table 8: Human serum stability

[0465] 2.2 Humanized PTH-VHH fusion protein

[0466] 2.2.1 Preparation of humanized PTH-VHH fusion protein

[0467] The PTH (1-34) polypeptide and the humanized anti-HSA VHH are constructed into a fusion protein. The fusion proteins constructed by the PTH (1-34) polypeptide and the SZ0630 VHH and the SZ0695 VHH are named P065 and P090, respectively. The fusion protein has two amino acids PP- at the end, i.e., the humanized PTH-VHH P011 corresponds to the humanized PTH-VHH P065, and the humanized PTH-VHH P020 corresponds to the humanized PTH-VHH P090. The amino acid sequence composition of the fusion proteins P065 and P090 is shown in Table 9.

[0468] Table 9: Amino acid sequence composition of fusion proteins P065 and P090

[0469] 2.2.2 cAMP activity detection of humanized PTH-VHH fusion protein

[0470] Preparation of serum incubation samples: The samples to be tested (P065 and P090) are mixed with human serum (manufacturer: ORiCELLS, product number: FSU-MIX-100ml) at a volume ratio of 1:2, and then subjected to cAMP activity detection. The cAMP activity detection method is the same as that in Example 2.1.2.

[0471] The experimental results are shown in Table 10. P065 and P090 have the effect of blocking PTH activity in serum, and there is a possibility of slow release.

[0472] Table 10: cAMP activity detection

[0473] 2.2.3 Serum stability of PTH-VHH fusion protein

[0474] Preparation of serum incubation samples: The samples to be tested (P065 and P090) were mixed with human serum (manufacturer: ORiCELLS, product number: FSU-MIX-100ml) and rat serum (rat serum was obtained by sterile operation in the company) at a ratio of 1:2 by volume, and then incubated in a 37°C incubator. After the incubation time, the samples were temporarily stored in a -20°C refrigerator. After all the samples were collected at the time points, the cAMP activity was detected. The cAMP activity detection method was the same as in Example 2.1.2, except that the sample dilution and transfer equipment used in this experiment was NAYON 96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYON 96-ACC2-F003-C1).

[0475] The experimental results are shown in Table 11. The results show that the stability of the humanized PTH-VHH fusion protein in serum does not change significantly. After the anti-albumin VHH binds to serum albumin, the PTH polypeptide can be well protected from degradation by proteases in serum.

[0476] Table 11: Stability of human and rat serum

[0477] 2.3 PTH-VHH fusion protein after linker shortening

[0478] 2.3.1 Molecular design

[0479] P065 and P090 were selected for amino acid reduction of the linker to find molecules that maintain activity without binding to albumin and have more activity reduction after binding to albumin. The original molecules and the number and design of the truncated linkers are shown in Table 12.

[0480] Table 12: Number and composition of original PTH-VHH and truncated linker PTH-VHH

[0481] 2.3.2 cAMP activity detection of PTH-VHH fusion protein after linker shortening

[0482] Preparation of serum incubation samples: The samples to be tested (P065, P085, P086, P087, P088, P089, P090, P091, P092, P093, P094, P095) were mixed with human serum (manufacturer: ORiCELLS, product number: FSU-MIX-100ml) and rat serum (rat serum was obtained by sterile operation in the company) at a ratio of 1:2 by volume, and then incubated in a 37°C incubator. After the incubation time, the samples were temporarily stored in a -20°C refrigerator. After all the samples were collected at the time points, the cAMP activity was detected. The cAMP activity detection method was the same as in Example 2.1.2, except that the sample dilution and transfer equipment used in this experiment was NAYON 96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYON 96-ACC2-F003-C1).

[0483] The experimental results are shown in Table 13, and the results show that the blocking of the activity of the PTH-VHH fusion protein by albumin in serum is significantly stronger as the linker is shortened, especially in human serum.

[0484] Table 13: cAMP activity detection

[0485] 2.3.3 Serum stability of PTH-VHH fusion proteins after linker shortening

[0486] The samples to be tested (P065, P085, P086, P087, P088, P089, P090, P091, P092, P093, P094, P095) were mixed with human serum (manufacturer: ORiCELLS, product number: FSU-MIX-100ml) and rat serum (rat serum obtained by sterile operation in the company) at a ratio of 1:2 by volume, and then incubated in a 37°C incubator. After the incubation time, they were temporarily stored in a -20°C refrigerator. After all the samples at different time points were collected, cAMP activity detection was performed, and the cAMP activity detection method was the same as in Example 2.1.2, except that the sample dilution and transfer equipment used in this experiment was NAYO N96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYO N96-ACC2-F003-C1).

[0487] The experimental results are shown in Tables 14 and 15, and compared with PTH(1-34), the serum stability of the PTH-VHH fusion proteins with shortened linkers is significantly improved.

[0488] Table 14: Human and rat serum stability of P090-P095

[0489] Table 15: Human and rat serum stability of P065, P085-P089 Note: NA represents not detected.

[0490] 2.4 In vivo efficacy in rats

[0491] 2.4.1 PTH-VHH in vivo wild-type rat experiment

[0492] The positive control PC-3 (Eneboparatide analogue, reference DOI: 10.1002 / jbm4.10367) was synthesized according to the literature report.

[0493] The purpose of this study is to test the PTH-VHH molecules in vivo regulation of wild-type rats blood calcium. This experiment uses 7-week-old male SD rats (n = 4 / group, experimental animals from Bai Ao Sai Tu), using a single subcutaneous administration of the same dose (25 nmol / kg, solvent: 1*PBS) of different PTH-VHH molecules (P011 and P020), before administration (0 hours), 3 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours after administration Time points for blood sampling, and using a biochemical instrument (model: sysmex BX4000) and reagent kit (brand: Maccura; item number: CH0101251 and CH0101257) to detect the concentration of inorganic calcium in the serum of the animals.

[0494] Conclusion: The experimental results are shown in Figure 6. The sCa of wild-type rats in each test group before administration was consistent. After administration of PTH-VHH, the sCa of rats in the administration group increased significantly compared with the PBS solvent control group, and reached a peak at 24 hours after administration, and fell to the sCa level of the PBS solvent group at 72 hours after administration. The duration of sCa regulation in rats by PTH-VHH molecules was comparable to that of the control drug PC-3, and had a long-acting effect.

[0495] This blood drug concentration (PK) study aims to test the metabolism of PTH-VHH molecules in vivo and their relevance to blood calcium. Serum samples were taken before administration (0h), 3h, 10h, 24h, 48h, 72h, 96h, 120h, 144h, 168h after administration, and the blood drug concentration (PK) was detected.

[0496] Conclusion: The experimental results are shown in Figure 7 and Table 16. The blood calcium concentration and the blood drug concentration of the fusion protein showed good correlation, and the T1 / 2 of the fusion protein was significantly longer than that of the PTH(1-34) polypeptide.

[0497] Table 16: PTH-VHH pharmacokinetic parameter determination

[0498] 2.4.2 Humanized PTH-VHH in vivo wild-type rat experiment

[0499] (1) The purpose of this study is to test the regulation of sCa in wild type rats in vivo after humanization of PTH-VHH molecule (P065). 7-week-old male SD rats (n=4 / group, experimental animals from Bao Saigu) were used in this experiment. A single subcutaneous administration of 25 nmol / kg of PTH-VHH molecule was used. Blood was collected at time points of pre-administration (0 hours), 3 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, and 168 hours after administration. The concentration of inorganic calcium in the serum of the animals was detected using a biochemical instrument (model: sysmex BX4000) and a kit (brand: Maccura; item number: CH0101251 and CH0101257).

[0500] Conclusion: The experimental results are shown in Figure 8. The sCa of wild type rats in each test group before administration was consistent. After administration of P065, the sCa of rats in the administration group increased significantly to varying degrees compared with the PBS solvent control group, and reached a peak at 24 hours after administration. The sCa level fell to the PBS solvent group level at 72 hours after administration. The duration of sCa regulation in vivo of humanized PTH-VHH (P065) molecule was equivalent to that of the control drug PC-3, i.e., equivalent to that of the PTH-VHH molecule before humanization.

[0501] (2) The purpose of this study is to test the regulation of sCa in PTX (parathyroidectomy) model rats in vivo after a single administration of humanized PTH-VHH (P090) molecule. 8-week-old male SD rats (n=4 / group) were used in this experiment. A single subcutaneous administration was used. Blood was collected at time points of pre-administration (D0), 24 hours (D1), 48 hours (D2), 96 hours (D4), D5, and D6 after administration. The concentration of inorganic calcium in the serum of the animals was detected using a biochemical instrument (model: sysmex BX4000) and a kit (brand: Maccura; item number: CH0101251 and CH0101257).

[0502] Conclusion: The experimental results are shown in Figure 9. The sCa of PTx model rats before administration was consistent, and was significantly lower than that of Sham (sham operation group) rats. After administration of P090, the sCa of rats in the administration group increased significantly and exceeded the sCa level of the Sham group, and reached a peak at 48 hours after the first administration, and then gradually fell until the fourth day after administration, still close to the Sham group level. The duration of sCa regulation in vivo of humanized PTH-VHH molecule P090 was equivalent to that of the PTH-VHH molecule before humanization.

[0503] 2.4.3 In vivo PTx model rat experiment of PTH-VHH after linker shortening

[0504] The purpose of this study is to test the regulation of blood calcium in PTX model rats by single administration of PTH-VHH molecules with shortened linkers, including duration and dose dependence. In this experiment, 8-week-old male SD rats (n = 4 / group, experimental animals from Baiuo Saigu) were administered different doses of different PTH-VHH molecules (P092, P094, P095) (vehicle: 1*PBS) by single subcutaneous administration, and blood was collected at the time points of before administration (D0), 24 hours after administration (D1), 48 hours after administration (D2), 96 hours after administration (D4), D5, and D6, and the concentration of inorganic calcium in the serum of the animals was detected using a biochemical instrument (model: sysmex BX4000) and a kit (brand: Maccura; item number: CH0101251 and CH0101257).

[0505] Conclusion: The experimental results are shown in Figure 10. The sCa of each PTX model group of rats before administration was consistent, and was significantly lower than that of the Sham (sham operation group) rats. After administration of PTH-VHH molecules, the sCa of the administration group rats increased significantly and exceeded the sCa level of the Sham group, and reached a peak at 48 hours after the first administration, and then gradually fell until the 5th day after administration, still close to the level of the Sham group. The duration of sCa regulation in rats by PTH-VHH molecules with different lengths of linkers was comparable.

[0506] 2.5 PTM risk site modification

[0507] 2.5.1 CDR PTM modification

[0508] Since P065 has PTM risk sites in the VHH CDR region, point mutation is used for improvement. After mutation, the PTH-VHH names are P126 and P132, respectively, and the sequences are shown in SEQ ID NO: 45 and SEQ ID NO: 46. Among them, for P126, the fourth amino acid in VHH CDR3 (SEQ ID NO: 17) of P065 is mutated from glycine (G) to alanine (A). For P132, the fourth amino acid in VHH CDR3 (SEQ ID NO: 17) of P065 is mutated from glycine (G) to tyrosine (Y).

[0509] 2.5.2 Serum albumin binding

[0510] ForteBio Octet HTX was used to detect the affinity of P065, P126, P132 fusion proteins to serum albumin of various species to detect the change in affinity before and after PTM modification. AHC (brand: Sartorius; item number: 18-5060) probe was used to capture anti-PTH (SZ07240725) for 200 s, and then P065, P126, P132 were activated for 200 s, respectively, with Albumin as the analyte, binding reaction for 120 s, dissociation reaction for 180 s.

[0511] The experimental results are shown in Table 17, and the results show that after the anti-albumin VHH CDR in P065 is removed from the PTM risk site, the affinity to serum albumin of various species is still high, with only slight changes.

[0512] Table 17: Affinity determination of fusion proteins to serum albumin of various species

[0513] 2.5.3 PTH N16 mutation

[0514] PTH-VHH fusion protein P090 and modified P126, P132 have deamination risk at N16 at the PTH end, so the site is mutated, and the mutated numbers are P142, P146, P149, respectively, and the sequences are shown in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49. Among them, for P142, by mutating the 16th amino acid of PTH (1-34) (SEQ ID NO: 25) from asparagine (N) to alanine (A) in P090. For P146, by mutating the 16th amino acid of PTH (1-34) (SEQ ID NO: 25) from asparagine (N) to serine (S) in P126. For P149, by mutating the 16th amino acid of PTH (1-34) (SEQ ID NO: 25) from asparagine (N) to serine (S) in P132.

[0515] 2.5.4 Serum albumin affinity

[0516] In this experiment, Biacore molecular interaction analyzer (cytiva, Biacore 8K+) was used, and SA chip was used to capture biotin-HSA, RSA, CSA, MSA, and P090, P126, P132 and their N16 mutation (P142, P146, P149) were used as analytes to flow through the chip, binding for 180 s, dissociation for 420 s, and kinetic analysis was performed according to the collected data by Biacore Insight Evaluation Software 3.0.12 software.

[0517] The results are shown in Table 18, which show that after mutation of the N16 site of PTH, P142, P146, P149 have little change in affinity to serum albumin of various species compared with P090, P126, P132.

[0518] Table 18: Affinity determination of fusion proteins to serum albumin of various species

[0519] 2.5.5 cAMP activity

[0520] The samples to be tested (P090, P126, P132 and their N16 mutation (P142, P146, P149), PTH (1-34) as control) were mixed with human plasma (manufacturer: Saili, product number: SLB-HWB-70A) and rat plasma (obtained by sterile operation in the company) in a ratio of 1:2 by volume, and then cAMP activity detection was performed. The cAMP activity detection method was the same as in Example 2.1.2, except that the sample dilution and transfer equipment used in this experiment was NAYO N96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYO N96-ACC2-F003-C1).

[0521] The results are shown in Table 19, which show that P142 has improved activity in PBS compared with before N16 mutation, but the blocking activity in plasma is weaker. P146 and P149 have no significant change in activity in PBS, human plasma and rat plasma compared with before N16 mutation.

[0522] Table 19 cAMP activity

[0523] Example 3: First generation complex

[0524] In order to enable PTH to be retained in the body for a long time, the strategy of using anti-PTH antibody to form a complex with PTH polypeptide is adopted. This strategy reduces kidney clearance through antibody binding and prolongs half-life through FcRn recycling mechanism. In addition, after the combination of anti-PTH antibody and PTH, PTH can be protected from protease degradation in serum. The schematic diagram of the molecular action is shown in Figure 11.

[0525] 3.1 Anti-PTH antibody

[0526] 3.1.1 Anti-PTH (20-28) antibody

[0527] According to the description of antibody mab183 in patent (US_7318925_B2), the antibody epitope is located at PTH(20-28) and can inhibit PTH-induced downstream signals. The variable region sequence of mab183 was constructed into a complete antibody SZ01590160, and the constant region was humanized IgG1(L234A / L235A / P329G) / K. The sequence composition of SZ01590160 is shown in Table 20 and Table 21.

[0528] Table 20: Sequence composition of heavy chain of SZ01590160

[0529] Table 21: Sequence composition of light chain of SZ01590160

[0530] 3.1.2 Affinity characterization

[0531] Using a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), a protein A chip (brand: cytiva, catalog number: 29127566) was used to capture SZ01590160 molecules, and PTH(1-34) was used as an analyte to flow through the chip, with a binding time of 180 s and a dissociation time of 420 s. Kinetic analysis was performed according to the collected data by Biacore Insight Evaluation Software 3.0.12 software, and the results are shown in Table 22. SZ01590160 has good binding affinity to PTH(1-34).

[0532] Table 22: Affinity determination

[0533] 3.1.3 Post-translational modification (PTM) site engineering

[0534] Since there is a risk of isomerization in the DG motif in the light chain CDR1 of antibody SZ01590160, an attempt was made to mutate this motif to reduce the risk of isomerization while maintaining affinity to PTH. The 7th amino acid of the light chain CDR1 of SZ01590160 was replaced with glycine (G) to construct full-length antibody SZ08830816 using human IgG1 (L234A / L235A) / K constant region; the 8th amino acid of the light chain CDR1 of SZ01590160 was replaced with glutamic acid (E) to construct full-length antibody SZ08830849 using human IgG1 (L234A / L235A) / K constant region; and the 7th amino acid of the light chain CDR1 of SZ01590160 was replaced with serine (S) to construct full-length antibody SZ08830850 using human IgG1 (L234A / L235A) / K constant region. The sequence composition of SZ08830816, SZ08830849, and SZ08830850 is shown in Table 23 and Table 24.

[0535] Table 23: Heavy chain sequence composition of SZ08830816, SZ08830849, and SZ08830850

[0536] Table 24: Light chain sequence composition of SZ08830816, SZ08830849, and SZ08830850

[0537] 3.1.4 Affinity after PTM site modification

[0538] Using a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), the Anti-human antibody was coupled to a CM-5 chip (brand: cytiva, catalog number: BR100530) according to the instructions of Human Antibody Capture kit, type 2 (brand: cytiva, catalog number: 29234600), and then the chip was used to capture molecules such as SZ01590160, and PTH (1-34) was used as the analyte to flow through the chip, with a binding time of 180 s and a dissociation time of 420 s. Kinetic analysis was performed using Biacore Insight Evaluation Software 3.0.12 software based on the collected data, and the results are shown in Table 25. After PTM site mutation, the affinity of anti-PTH antibody SZ01590160 did not decrease significantly.

[0539] Table 25: Affinity determination

[0540] 3.2 PTH (1-34) mutants

[0541] 3.2.1 Polypeptide synthesis

[0542] PTH(1-34) polypeptide is easily degraded by proteases in serum, and attempts were made to replace protease-sensitive sites with unnatural amino acids and to synthesize them by a supplier (Nanjing Kingsway). The designed mutant sequences are shown in Table 26.

[0543] Table 26: PTH(1-34) and its mutant sequences

[0544] PEP005 is the original human PTH(1-34) sequence, the sequence of which is shown in SEQ ID NO: 25. PEP023 is obtained by replacing the 1st, 3rd and 25th amino acids of PEP005 with Aib, the sequence of which is shown in SEQ ID NO: 73; PEP024 is obtained by replacing the 3rd and 25th amino acids of PEP005 with Aib, the sequence of which is shown in SEQ ID NO: 74; PEP025 is obtained by replacing the 1st and 25th amino acids of PEP005 with Aib, the sequence of which is shown in SEQ ID NO: 75; and PEP026 is obtained by replacing the 25th amino acid of PEP005 with Aib, the sequence of which is shown in SEQ ID NO: 76.

[0545] 3.2.2 cAMP activity

[0546] PEP005, PEP023, PEP024, PEP025, and PEP026 were subjected to cAMP activity detection, and the cAMP activity detection method was the same as in Example 2.1.2, except that the detection was performed on Saos-2 cells and UMR106 cells, respectively. The results are shown in Table 27, which show that the cAMP activity of the mutated polypeptides in human Saos-2 cells did not change significantly, and only PEP025 activity was slightly enhanced. In rat-derived UMR106 cells, PEP023 and PEP025 remained unchanged, and the activity of PEP024 and PEP026 decreased.

[0547] Table 27: cAMP activity detection of PTH(1-34) and its variants (PEP023, PEP024, PEP025, PEP026)

[0548] 3.2.3 Serum stability

[0549] The sample to be tested (PEP005, PEP023, PEP024, PEP025, PEP026) was mixed with human serum (manufacturer: ORiCELLS, product number: FSU-MIX-100ml) at a ratio of 1:2 by volume and placed in a 37°C incubator. After the incubation time, the sample was temporarily stored in a -20°C refrigerator. After all the samples were collected at the specified time points, the cAMP activity was detected using the method described in Example 2.1.2.

[0550] As shown in Table 28, the stability of the mutant polypeptide in human serum was significantly improved, and the activity did not decrease significantly after incubation in human serum for 7 days.

[0551] Table 28: Human serum stability test

[0552] 3.2.4 Anti-PTH antibody affinity

[0553] Using a Biacore molecular interaction analyzer (cytiva, Biacore 8K+), a protein A chip (brand: cytiva, product number: 29127566) was used to capture the SZ01590160 molecule, and PTH(1-34), PEP023, PEP024, PEP025, PEP026 were used as analytes to flow through the chip, with a binding time of 180s and a dissociation time of 420s. Kinetic analysis was performed using Biacore Insight Evaluation Software 3.0.12 software based on the collected data, and the results are shown in Table 29. The results show that the mutant polypeptide still has a high level of affinity with the anti-PTH antibody SZ01590160.

[0554] Table 29: Antibody affinity test

[0555] 3.3 PEP025 PTM risk site modification

[0556] 3.3.1 PEP025 N16 mutation

[0557] PEP025 N16 has a risk of deamination, so the site was mutated. The mutant numbers are PEP066, PEP067 and PEP068, respectively. The mutant sequences were synthesized by a supplier (Nanjing Kingsway) and are shown in Table 30.

[0558] Table 30: PEP025 and its mutant sequences

[0559] Among them, PEP066 is obtained by mutating the 16th amino acid of PEP025 from asparagine (N) to alanine (A), as shown in SEQ ID NO:100; PEP067 is obtained by mutating the 16th amino acid of PEP025 from asparagine (N) to serine (S), as shown in SEQ ID NO:101; PEP068 is obtained by mutating the 17th amino acid of PEP025 from serine (S) to glutamic acid (E), as shown in SEQ ID NO:102.

[0560] 3.3.2 cAMP activity and plasma stability

[0561] The test samples (PEP025, PEP066, PEP067, PEP068) were mixed with human plasma (manufacturer: Saili, catalog number: SLB-HWB-70A) and rat plasma (rat plasma obtained aseptically in-house) at a volume ratio of 1:2 and incubated at 37°C. After the incubation time, the samples were removed and temporarily stored at -20°C. After all time points of sample collection were completed, cAMP activity was detected. The cAMP activity detection method was the same as in Example 2.1.2, except that the human plasma-incubated samples were detected on Saos-2 cells, and the rat plasma-incubated samples were detected on UMR106 cells. The sample dilution and transfer equipment used in this experiment was a NAYO N96 fully automated pipetting workstation (manufacturer: Naiyou Biotechnology, model: NAYO N96-ACC2-F003-C1).

[0562] The results are shown in Table 31 below. Compared with the unmutated N16 (PEP025), the activities of the mutant peptides (PEP066, PEP067, PEP068) after incubation in human and rat plasma for 7 days were not significantly different.

[0563] Table 31: Detection of cAMP activity and plasma stability of PEP025 and its mutant peptides

[0564] 3.3.3 Affinity of anti-PTH antibody

[0565] Using Biacore molecular interaction analyzer (cytiva, Biacore 8K+), using protein A chip (brand: cytiva, part number: 29127566), capture SZ08830849 molecules, PTH(1-34), PEP025, PEP066, PEP067, PEP068 as analytes respectively flow through the chip, bind for 180s, dissociate for 420s, according to the collected data by Biacore Insight Evaluation Software 3.0.12 software for kinetic analysis, the results are shown in Table 32 below, the mutant polypeptide still has a high level of affinity with anti-PTH antibody SZ08830849.

[0566] Table 32: Affinity detection of PEP025 and its mutant polypeptide with antibody SZ08830849

[0567] 3.4 Anti-PTH antibody / PTH complex

[0568] 3.4.1 Preparation of antibody / PTH complex

[0569] After mixing the antibody and polypeptide at a molar ratio (anti-PTH:peptide = 1:8), incubate at room temperature for 3h, separate and purify by SEC (HiLoad 26 / 600 Superdex 200pg), collect the main peak of anti-PTH / PTH complex 1:2 binding, and the SEC purification profile of anti-PTH / PTH complex protein is shown in Figure 12.

[0570] PEP025, PEP067 were mixed with antibody SZ01590160 and antibody SZ08830849 after removal of PTM risk sites to prepare complexes, the names and compositions of the complexes are shown in Table 33 below.

[0571] Table 33: Composition of anti-PTH antibody / PTH complex

[0572] PEP005 is the original PTH(1-34).

[0573] 3.4.2 cAMP activity and plasma stability

[0574] The samples to be tested (SZ01590160 / PEP025, SZ08830849 / PEP025) were mixed with human plasma (manufacturer: Saili, product number: SLB-HWB-70A) and rat plasma (rat plasma was obtained by sterile operation in the company) in a ratio of 1:2 by volume, and then incubated in a 37°C incubator. After the incubation time, the samples were temporarily stored in a -20°C refrigerator. After all the samples at different time points were collected, cAMP activity detection was performed. The cAMP activity detection method was the same as in Example 2.1.2, except that the sample dilution and transfer equipment used in this experiment was NAYO N96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYO N96-ACC2-F003-C1).

[0575] The results are shown in Table 34. The results show that the cAMP activity of the complex has no obvious change, and the activity has no obvious change after 14 days of treatment in plasma. Compared with PTH(1-34), the plasma stability of the complex is obviously improved.

[0576] Table 34: cAMP activity and plasma stability detection of complex

[0577] 3.4.3 PTM modification of SZ08830849 / PEP067 complex cAMP activity and plasma stability

[0578] The samples to be tested (SZ08830849 / PEP067) were mixed with human plasma (manufacturer: Saili, product number: SLB-HWB-70A), rat plasma (rat plasma was obtained by sterile operation in the company) and cyno plasma (cynomolgus monkey plasma was obtained by sterile operation in Huazhen Biological) in a ratio of 1:2 by volume, and then incubated in a 37°C incubator. After the incubation time, the samples were temporarily stored in a -20°C refrigerator. After all the samples at different time points were collected, cAMP activity detection was performed. The cAMP activity detection method was the same as in Example 2.1.2, except that the human plasma and cyno plasma incubated samples were detected on Saos-2 cells, and the rat plasma incubated samples were detected on UMR106 cells. The sample dilution and transfer equipment used in this experiment was NAYO N96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYO N96-ACC2-F003-C1).

[0579] The results are shown in Table 35. The activity of the complex has no obvious change after 14 days of treatment in plasma. Compared with PTH(1-34), the plasma stability of the complex is obviously improved.

[0580] Table 35: cAMP activity and plasma stability assay of the complex

[0581] 3.5 In vivo efficacy

[0582] 3.5.1 Single dose of Anti-PTH / PTH complex

[0583] Positive control PC-3 was synthesized according to the literature report, reference DOI: 10.1002 / jbm4.10367.

[0584] The purpose of this study is to test the Anti-PTH / PTH complex molecule (SZ01590160 / PEP025) in vivo for the regulation of blood calcium in wild-type rats. 6-8-week-old male SD rats (n = 4 / group, experimental animals from Baiosaitu) were used in this experiment. A single subcutaneous dose of 7.3 nmol / kg (vehicle: 1*PBS) of Anti-PTH / PTH complex molecule (SZ01590160 / PEP025) was administered, and blood was collected at time points before administration, 3 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, and 168 hours after administration. The inorganic calcium concentration in the serum of the animals was detected using a biochemical instrument (model: sysmex BX4000) and reagent kit (brand: Maccura; catalog number: CH0101251 and CH0101257).

[0585] Conclusion: The results are shown in Figure 13. The sCa of wild-type rats in each test group was consistent before administration. After administration of Anti-PTH / PTH complex molecule (SZ01590160 / PEP025), the sCa of rats in the administration group increased to varying degrees compared with the PBS vehicle control group, and all reached a peak at 24 hours after administration, and fell to the sCa level of the PBS vehicle group at 96 hours after administration. The duration of Anti-PTH / PTH complex molecule for regulating sCa in rats in vivo was comparable to that of the control drug PC-3, showing obvious long-acting effect in vivo.

[0586] 3.5.2 Single dose of Anti-PTH / PTH complex after PTM modification

[0587] PC-1 (Transcon PTH analogue, literature DOI number: 10.1002 / jbmr.3824) was used as a control drug.

[0588] The purpose of this study is to test the Anti-PTH / PTH complex molecules after PTM modification in vivo for the regulation of sCa in PTx model rats. This experiment uses 9-10 week old male SD rats (n=5 / group, experimental animals from Vivotec Laboratories). Single subcutaneous administration of different doses (solvent: 1*PBS) of different Anti-PTH / PTH complex molecules (SZ08830849 / PEP067) is given, and blood is collected at pre-dose (Day 0), Day 1, Day 2, Day 4, Day 5, Day 6, Day 7 time points, and the inorganic calcium concentration in the serum of the animals is detected by using biochemical instrument (model: sysmex BX4000) and kit (brand: Maccura; item number: CH0101251 and CH0101257).

[0589] Conclusion: As shown in Figure 14, the sCa of each test group of rats before administration is consistent, after administration of different doses of Anti-PTH / PTH complex molecules (SZ08830849 / PEP067), the sCa of the administration group of rats is increased to different degrees compared with the PBS solvent control group, and reaches the peak at 24 hours after administration, and falls to the sCa level of the PBS solvent group at 3 or 4 days after administration. The duration of Anti-PTH / PTH complex molecules (SZ08830849 / PEP067) for regulating sCa in rats is better than that of the control drug PC-1, showing obvious long-acting effect in vivo.

[0590] 3.5.3 Anti-PTH / PTH complex multiple administration after PTM modification

[0591] PC-1 is used as a control drug.

[0592] The purpose of this study is to test the Anti-PTH / PTH complex molecules after PTM modification in vivo for the regulation of sCa in PTx model rats. This experiment uses 9-10 week old male SD rats (n=5 / group, experimental animals from Vivotec Laboratories). Single subcutaneous administration of different doses (solvent: 1*PBS) of different Anti-PTH / PTH complex molecules (SZ08830849 / PEP067) is given, and blood is collected at pre-dose (Day 0), Day 1, Day 2, Day 4, Day 5, Day 6, Day 7 time points, and the inorganic calcium concentration in the serum of the animals is detected by using biochemical instrument (model: sysmex BX4000) and kit (brand: Maccura; item number: CH0101251 and CH0101257).

[0593] Conclusion: As shown in Figure 15 and Figure 16, the sCa of each PTx model group of rats was consistent before administration, and was significantly lower than that of the Sham (sham operation group) rats. After administration of the Anti-PTH / PTH complex molecule (SZ08830849 / PEP067), the sCa of the administration group of rats increased significantly and exceeded the sCa level of the Sham group, and reached a peak at 24 hours after the first administration, and then gradually fell until the 3rd day after administration, still close to the level of the Sham group. Within 30 days of multiple administrations, blood calcium and blood phosphorus can be maintained within the normal physiological range.

[0594] Example 4: Second-generation complex

[0595] Both PTH-VHH fusion protein and antibody / PTH complex significantly prolonged the duration of PTH efficacy in vivo, so it is considered that if the two designs are superimposed, the duration of in vivo efficacy can be further prolonged. The target molecule of the design adopts the mode of PTH-VHH fusion protein forming a complex with anti-PTH antibody. After entering the body, it can reversibly bind to serum albumin, and after the PTH-VHH fusion protein is released from the anti-PTH antibody, it can also bind to serum albumin, providing protection for PTH and prolonging the half-life. Finally, the free PTH-VHH binds to PTH1R on the cell membrane to exert biological function. The specific molecular mechanism is shown in Figure 17.

[0596] 4.1 Anti-PTH antibody

[0597] 4.1.1 Antibody before modification

[0598] According to the description of antibody sc275 in the patent (US_7318925_B2), the epitope of the antibody is located in PTH(1-7). The heavy and light chain variable regions of sc275 are constructed into full-length antibody SZ07240725, using human IgG1(L234A / L235 / P329G) / K constant region. The sequence composition of SZ07240725 is shown in Table 36 and Table 37.

[0599] Table 36: Sequence composition of heavy chain of SZ07240725

[0600] Table 37: Sequence composition of light chain of SZ07240725

[0601] 4.1.2 Affinity

[0602] Anti-human antibody was coupled on CM-5 chip (brand: cytiva, part number: BR100530) according to the operation instruction of Human Antibody Capture kit, type 2 (brand: cytiva, part number: 29234600), then the chip was used to capture SZ07240725 molecule, PTH(1-34), P065, P090 were used as analyte to flow through the chip, binding for 180s, dissociation for 420s, kinetic analysis was performed according to the collected data by Biacore Insight Evaluation Software 3.0.12 software, the results are shown in Table 38, the results show that SZ07240725 has high affinity with PTH(1-34), P065 and P090.

[0603] Table 38: Affinity determination

[0604] 4.1.3 PTM site modification

[0605] Since the heavy chain CDR2 of anti-PTH antibody SZ07240725 has a DG motif, there is a risk of isomerization, and there is a risk of NG deamination in the light chain CDR1, so it is considered to remove these two PTM risk sites, change DG in the heavy chain CDR2 to DA, and mutate NG in the light chain CDR1 to SG. The mutated heavy and light chain variable regions were constructed into full-length antibody SZ08850854, using human IgG1(L234A / L235A) / K constant region. The sequence composition of SZ08850854 is shown in Table 39 and Table 40.

[0606] Table 39: Heavy chain sequence composition of SZ08850854

[0607] Table 40: Light chain sequence composition of SZ08850854

[0608] 4.1.4 Affinity identification after modification

[0609] Anti-human antibody was coupled on CM-5 chip (brand: cytiva, part number: BR100530) using Biacore molecule interaction analyzer (cytiva, Biacore 8K+) according to the operation instruction of Human Antibody Capture kit, type 2 (brand: cytiva, part number: 29234600), then the chip was used to capture SZ08850854 molecule, PTH(1-34) and PTH-VHH fusion protein were used as analyte to flow through the chip, binding 180s, dissociation 420s, kinetic analysis was performed according to the collected data by Biacore Insight Evaluation Software 3.0.12 software, the results are shown in Table 41. The results show that the affinity of anti-PTH antibody SZ08850854 after PTM modification to PTH and PTH-VHH is maintained at a high level.

[0610] Table 41: Affinity determination

[0611] 4.2 Anti-PTH antibody / PTH-VHH complex

[0612] 4.2.1 Preparation of antibody / PTH-VHH complex

[0613] Anti-PTH antibody (SZ07240725, SZ08850854) and PTH-VHH fusion protein were expressed in mammalian cells respectively, after one-step affinity purification, mixed and incubated according to the molar ratio of antibody: fusion protein greater than 1:3, then purified by SEC (HiLoad 26 / 600 Superdex 200pg) to remove excess PTH-VHH fusion protein, and collect the complex with a molar ratio of antibody: fusion protein of 1:2. The SEC purification profile of anti-PTH / PTH-VHH complex protein is shown in Figure 18.

[0614] The PTH-VHH fusion protein was mixed with the antibody to prepare the complex, and the names and compositions of the complexes are shown in Table 42.

[0615] Table 42: Composition of anti-PTH antibody / PTH-VHH complex

[0616] 4.2.2 cAMP activity of SZ07240725 / PTH-VHH complex

[0617] The samples to be tested (SZ07240725 / P065, SZ07240725 / P090) were mixed with human plasma (manufacturer: Saili, product number: SLB-HWB-70A) and rat plasma (rat plasma was obtained by sterile operation in the company) at a ratio of 1:2 by volume, and then cAMP activity detection was performed. The cAMP activity detection method was the same as in Example 2.1.2, except that the sample dilution and transfer equipment used in this experiment was NAYO N96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYO N96-ACC2-F003-C1).

[0618] Conclusion: As shown in Table 43, the cAMP activity of SZ07240725 / PTH-VHH complex in human or rat plasma was significantly lower than that in PBS, suggesting the possibility of sustained release, which met the design expectation.

[0619] Table 43: cAMP activity detection of SZ07240725 / PTH-VHH complex

[0620] 4.2.3 cAMP activity of SZ08850854 / PTH-VHH complex

[0621] The samples to be tested (SZ08850854 / P126, SZ08850854 / P132, SZ08850854 / P090) were mixed with human plasma (manufacturer: Saili, product number: SLB-HWB-70A) and rat plasma (rat plasma was obtained by sterile operation in the company) at a ratio of 1:2 by volume, and then cAMP activity detection was performed. The cAMP activity detection method was the same as in Example 2.1.2, except that the sample dilution and transfer equipment used in this experiment was NAYO N96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYO N96-ACC2-F003-C1).

[0622] Conclusion: As shown in Table 44, the activity of SZ08850854 / P126, SZ08850854 / P132 and SZ08850854 / P090 complex molecules after PTM modification, and the fold decrease in cAMP activity after the addition of human and rat plasma, showed no significant difference compared with before modification.

[0623] Table 44: cAMP activity detection of SZ08850854 / PTH-VHH complex

[0624] 4.2.4 cAMP activity of PTM modified SZ08850854 / PTH-VHH complex

[0625] The samples to be tested (SZ08850854 / P142, SZ08850854 / P090, SZ08850854 / P146, SZ08850854 / P126, SZ08850854 / P149, SZ08850854 / P132) were mixed with human plasma (manufacturer: Saili, product number: SLB-HWB-70A) and rat plasma (rat plasma was obtained by sterile operation in the company) in a ratio of 1:2 by volume, and then cAMP activity detection was performed. The cAMP activity detection method was the same as in Example 2.1.2, except that the human plasma mixed sample was detected on Saos-2 cells, and the rat plasma mixed sample was detected on UMR106 cells. The sample dilution and transfer equipment used in this experiment was NAYO N96 fully automatic pipetting workstation (manufacturer: Naiyou Biological, model: NAYO N96-ACC2-F003-C1).

[0626] Conclusion: The results are shown in Table 45. The activity of the SZ08850854 / P142, SZ08850854 / P146, and SZ08850854 / P149 complex molecules did not show significant differences compared with before modification. The fold decrease in cAMP activity of SZ08850854 / P146 and SZ08850854 / P149 after adding human and rat plasma did not show significant differences compared with before modification. The fold decrease in cAMP activity of SZ08850854 / P142 after adding human and rat plasma was less than before modification, indicating that its activity was improved.

[0627] Table 45: cAMP activity detection of PTM modified SZ08850854 / PTH-VHH complex

[0628] 4.2.5 Plasma stability of PTM modified SZ08850854 / PTH-VHH complex

[0629] The samples to be tested (SZ08850854 / P146, SZ08850854 / P149, SZ08850854 / P142) were mixed with human plasma (manufacturer: Saili, catalog number: SLB-HWB-70A), rat plasma (rat plasma obtained by sterile operation in the company), and cyno plasma (macaque plasma obtained by sterile operation in Huazhen Biotech) at a ratio of 1:2 by volume, and then cAMP activity detection was performed. The cAMP activity detection method was the same as in Example 2.1.2, except that the human plasma and cyno plasma incubated samples were detected on Saos-2 cells, and the rat plasma incubated samples were detected on UMR106 cells. The sample dilution and transfer equipment used in this experiment was NAYO N96 fully automatic pipetting workstation (manufacturer: Naiyou Biotech, model: NAYO N96-ACC2-F003-C1).

[0630] Conclusion: As shown in Tables 46-48, SZ08850854 / P142, SZ08850854 / P146, and SZ08850854 / P149 showed no significant decrease in activity after incubation in human, rat, and monkey plasma for 0, 7, and 14 days, indicating that the complex molecule is stable in serum and significantly better than PTH(1-34).

[0631] Table 46: PTM-modified SZ08850854 / PTH-VHH complex human plasma stability test

[0632] Table 47: PTM-modified SZ08850854 / PTH-VHH complex rat plasma stability test

[0633] Table 48: PTM-modified SZ08850854 / PTH-VHH complex monkey plasma stability test

[0634] 4.3 In vivo efficacy

[0635] 4.3.1 Single dose of SZ07240725 / PTH-VHH complex

[0636] PC-2 (PTH peptide analog, sequence see patent US2023 / 0285578A1 SEQ ID NO: 87) and PC-1 were used as control drugs.

[0637] The purpose of this study is to test the regulation of Anti-PTH / PTH-VHH complex molecules on blood calcium in PTx model rats after single administration. In this experiment, 9-10-week-old male SD rats (n=5 / group, experimental animals from Vantianhua) were used. Single subcutaneous administration was used to attempt to administer different doses of Anti-PTH / PTH-VHH complex molecules (SZ07240725 / P065, SZ07240725 / P090) (vehicle: 1*PBS). Blood was collected at 0 hours before administration, 10 hours, 24 hours, 48 hours, 96 hours, 120 hours, 144 hours, and 168 hours after administration. Biochemical instrument (model: sysmex BX4000) and reagent kit (brand: Maccura; catalog number: CH0101251 and CH0101257) were used to detect the concentration of inorganic calcium in the serum of the animals.

[0638] Conclusion: As shown in Figure 19, the sCa of each PTx model group was consistent before administration, and was significantly lower than that of the Sham (sham operation group) rats. After administration of Anti-PTH / PTH-VHH complex molecules, the sCa of the administration group rats increased significantly and exceeded the sCa level of the Sham group. It reached a peak at 96 hours after the first administration, and then gradually fell until the 6th day after administration, which was close to the level of the Sham group and remained within the normal physiological range of blood calcium. Both SZ07240725 / P065 and SZ07240725 / P090 complex molecules significantly maintained stable blood calcium for a long time, and the duration of the experiment was significantly longer than that of the control drugs PC-1 and PC-2.

[0639] 4.3.2 SZ08850854 / PTH-VHH complex single administration

[0640] The purpose of this study is to test the Anti-PTH antibody and Anti-PTH / PTH-VHH complex molecules after PTM modification of VHH part, the regulation effect on blood calcium of PTx model rats after single administration. In this experiment, 9-10 week old male SD rats (n=6 / group, experimental animals from Weitong Lihua) were used, and different doses of different Anti-PTH / PTH-VHH complex molecules (SZ08850854 / P065, SZ08850854 / P126, SZ08850854 / P132, SZ08850854 / P090) were administered by single subcutaneous administration (solvent: 1*PBS), and blood was collected at the time points of before administration (0 hours), 24 hours, 48 hours, 96 hours, D5, D6, D7, D9 after administration, and the concentration of inorganic calcium in the serum of the animals was detected by biochemical instrument (model: sysmex BX4000) and kit (brand: Maccura; item number: CH0101251 and CH0101257).

[0641] Conclusion: As shown in Figure 20, the sCa of each PTx model rat before administration was consistent, and was significantly lower than that of the Sham (sham operation group) rats. After administration of Anti-PTH / PTH-VHH complex molecules, the sCa of the administration group rats increased significantly and exceeded the sCa level of the Sham group. By comparing SZ07240725 / P090 and SZ08850854 / P090 complex molecules, it was found that the PTM modification of Anti-PTH antibody did not have a significant effect on the regulation of blood calcium by complex molecules. Similarly, by comparing SZ07240725 / P065, SZ08850854 / P126, and SZ08850854 / P132 groups, it was found that the PTM modification of Anti-PTH antibody and the PTM modification of VHH did not have a significant effect on the regulation of blood calcium by complex molecules. In addition, by comparing the control drugs PC-1 and PC-2, the Anti-PTH / PTH-VHH complex molecules after PTM modification can maintain blood calcium stable for a longer period of time.

[0642] 4.3.3 PTM modification of SZ08850854 / PTH-VHH complex single administration

[0643] The purpose of this study is to test the Anti-PTH / PTH-VHH complex molecule after the modification of PTH part PTM site for the regulation of blood calcium in PTX model rats after single administration. In this experiment, 9-10 week old male SD rats (n=6 / group, experimental animals from Weitong Lihua) were used, and different doses of different Anti-PTH / PTH-VHH complex molecules (SZ08850854 / P146, SZ08850854 / P149, SZ08850854 / P142) were administered by single subcutaneous administration (solvent: 1*PBS), and blood was collected at the time points of before administration, 24h (D1), 48h (D2), 96h (D4), 120h (D5), 144h (D6), 168 (D7), D9 after administration, and the concentration of inorganic calcium in the serum of the animals was detected by biochemical instrument (model: sysmex BX4000) and kit (brand: Maccura; Catalog number: CH0101251 and CH0101257).

[0644] Conclusion: As shown in Figure 21, the sCa of each PTX model group of rats before administration was consistent, and was significantly lower than that of the Sham (sham operation group) rats. After administration of Anti-PTH / PTH-VHH complex molecules, the sCa of the administration group rats increased significantly and exceeded the sCa level of the Sham group, reaching a peak at 48 hours after administration, and then gradually falling until the 6th day after administration, still close to the level of the Sham group, and remained within the normal physiological range of blood calcium, and at the same time showed a good dose-effect relationship. The results show that after removing the PTM risk site in PTH, the in vivo efficacy of the complex molecule is similar to that before modification, and also has the effect of stabilizing blood calcium for a long time, and the duration is significantly longer than that of the control drug and PC-1 and PC-2.

[0645] 4.3.4 PTM modification SZ08850854 / PTH-VHH complex multiple administration

[0646] The purpose of this study is to test the regulation of Anti-PTH / PTH-VHH complex molecules on blood calcium and phosphorus in PTX model rats. In this experiment, 9-10 week old male SD rats (n=6 / group, experimental animals from Vivotec Laboratories) were given different doses of different Anti-PTH / PTH-VHH complex molecules (SZ08850854 / P146, SZ08850854 / P149, SZ08850854 / P142) (vehicle: 1*PBS) by single subcutaneous administration. Blood was collected at the time points of pre-administration (D0), 24h after administration (D1), 48h after administration (D2), 96h after administration (D4), 120h after administration (D5), 144h after administration (D6), 192h after administration (D8), D9, D10, D12, and the concentration of inorganic calcium and inorganic phosphorus in the serum of the animals was detected by biochemical instrument (model: sysmex BX4000) and kit (brand: Maccura; catalog number: CH0101251 and CH0101257).

[0647] Conclusion: As shown in Figures 22 and 23, the sCa of each PTX model group was consistent before administration, and was significantly lower than that of the Sham (sham operation group) rats. After administration of Anti-PTH / PTH-VHH complex molecules, the sCa of the administration group rats increased significantly and exceeded the sCa level of the Sham group. After multiple administrations, blood calcium and phosphorus were stable and remained within the normal physiological range of blood calcium and phosphorus, showing a good dose-effect relationship.

[0648] Example 5: Third-generation complex

[0649] The target molecule is designed to form a complex of PTH molecules modified with stacked fatty acid chains and non-natural amino acids with anti-PTH antibodies. After entering the body, it can be reversibly combined with serum albumin through the fatty acid chains, and after the PTH molecules modified with stacked fatty acid chains and non-natural amino acids are released from the anti-PTH antibodies, they can also bind to serum albumin, providing protection for PTH and prolonging the half-life. The final free PTH molecules bind to PTH1R on the cell membrane to exert biological functions. The specific molecular mechanism is shown in Figure 24.

[0650] 5.1 PEP067 fatty acid (FA) modified mutant

[0651] 5.1.1 PTH-FA polypeptide synthesis

[0652] To further improve the serum stability of PEP067 polypeptide, fatty acid chains were used to modify PEP067, and the sequence was designed for synthesis by the supplier (Nanjing Kingsway). The mutant sequence is shown in Table 49.

[0653] Table 49: PEP067 and its fatty acid chain modified sequences

[0654] In the above, the 34th amino acid of PEP067 is mutated from phenylalanine (F) to lysine (K) (the sequence is shown as SEQ ID NO: 105), and the mutated lysine side chain is connected with a fatty diacid chain (AEEA-AEEA-GAMMA-GLU-C18DIACID) to obtain PEP071, the sequence of PEP071 is shown as SEQ ID NO: 103; the 25th amino acid of PEP067 is mutated from Aib to arginine (R), and the 34th amino acid is mutated from phenylalanine (F) to lysine (K) (the sequence is shown as SEQ ID NO: 106), and the mutated lysine side chain is connected with a fatty acid chain (AEEA-AEEA-GAMMA-GLU-C18DIACID) to obtain PEP072, the sequence of PEP072 is shown as SEQ ID NO: 104.

[0655] 5.1.2 cAMP activity and plasma stability of PTH-FA polypeptides

[0656] The cAMP activity of PEP071 and PEP072 (stock solution) was detected, and the detection method was as follows:

[0657] According to the instructions of cAMP Gs Dynamic kit (brand: Cisbio; product number: 62AM4PEC), two buffers were prepared, namely assay buffer 1 (8.3% human plasma or rat plasma in DMEM) and assay buffer 2 (0.5mM IBMX in assay buffer 1). 5μL of each concentration of sample (PEP071, PEP072, PEP025, PTH(1-34)) diluted with assay buffer 1 was added to a 384-well plate, and 5μL of Saos-2 cell (Beina Biotech, BNCC338485) or UMR106 cell (Beina Biotech, BNCC100334) cell suspension diluted with assay buffer 2 was added to the 384-well plate, and incubated in a cell incubator for 30 minutes. 5μL of 1X cAMP-d2 and 5μL of 1X Anti-cAMP-Cryptate were added in turn, and incubated at room temperature for 1 hour. Data was read on PerkinElmer EnVision Multilabel 2103 (665nm and 620nm), and HTRF Ratio=Signal 665nm / Signal 620nmx10 was calculated (665nm and 620nm) above.4 cAMP % = [1 - (HTRF Ratio sample - HTRF Ratio cell only) / (HTRF Ratio agonist max - HTRF Ratio cell only)] x 100%, wherein, sample is the sample well, cell only is the cell only well, agonist max is the PTH(1-34) well. Data analysis was performed by GraphPad Prism 10.2.3 software. The sample dilution and transfer equipment used in this experiment was NAYO N96 fully automated pipetting workstation (manufacturer: Naiyou Biological, model: NAYO N96-ACC2-F003-C1). sample cell only Ratio agonist max

[0658] The plasma stability of PEP071 and PEP072 was detected, and the plasma stability detection method was as follows:

[0659] The samples to be tested (PEP071, PEP072, PEP025, PTH(1-34)) were mixed with human plasma (obtained by sterile operation of Saisai Biological) and rat plasma (obtained by sterile operation of the company) at a ratio of 1:2 by volume, and then incubated in a 37°C incubator. After the incubation time, it was temporarily stored in a -20°C refrigerator. After all time point samples (samples not incubated after mixing with plasma (D0), samples incubated for 1 day after mixing with plasma (D1), samples incubated for 3 days after mixing with plasma (D3), samples incubated for 7 days after mixing with plasma (D7)) were collected, cAMP activity detection was performed, and the cAMP activity detection method was the same as above.

[0660] The results are shown in Table 50. The cAMP activities of PEP071 and PEP072 on Saos-2 cells and UMR106 cells were weaker than those of PTH(1-34), but the cAMP activities of PEP071 and PEP072 were still very high, with EC50 in the nM level, meeting the subsequent in vivo drug requirements. The stabilities of PEP071 and PEP072 modified by fatty diacid chains in human and rat plasma were better than those of PEP025 and PTH(1-34).

[0661] Table 50: Plasma stability detection of PEP025, PEP071, PEP072

[0662] 5.1.3 Anti-PTH antibody affinity

[0663] ​​​Using Biacore molecule interaction analyzer (cytiva, Biacore 8K+), using anti-Human capture chip (brand: cytiva, part number: 29234600), capture SZ08830849 molecule, PEP025, PEP071, PEP072 as analyte flow through the chip respectively, binding 180s, dissociation 420s, according to the collected data by Biacore Insight Evaluation Software 3.0.12 software for kinetic analysis, the results are shown in Table 51, PEP071 and PEP072 have nM level affinity with anti-PTH antibody SZ08830849, meet the requirements of complex preparation.

[0664] Table 51: antibody affinity detection

[0665] 5.2 Anti-PTH antibody / PTH-FA complex

[0666] 5.2.1 Preparation of anti-PTH antibody / PTH-FA complex

[0667] The anti-PTH antibody (SZ08830849) and PTH-FA polypeptide (PEP071, PEP072) were mixed at a molar ratio of 1:4 (anti-PTH:peptide), incubated at room temperature for 3h, and then separated and purified by SEC (HiLoad 26 / 600 Superdex 200pg). The main peak of the anti-PTH / PTH-FA complex 1:2 binding was collected, and the SEC purification profile of the anti-PTH / PTH-FA complex protein is shown in Figure 25.

[0668] The complex name and composition are shown in Table 52.

[0669] Table 52: Composition of anti-PTH antibody / PTH-FA complex

[0670] 5.2.2 Plasma stability of SZ08830849 / PEP072 complex

[0671] The sample to be tested (SZ08830849 / PEP072) was mixed with human plasma (obtained by sterile operation of human blood plasma by SAIL BIO), rat plasma (obtained by sterile operation of rat blood plasma by the company), and cyno plasma (obtained by sterile operation of cynomolgus monkey blood plasma by Huazhen Biotech) in a ratio of 1:2 by volume, and then incubated in a 37°C incubator. After the incubation time, the samples were temporarily stored in a -20°C refrigerator. After all the samples were collected at the time points, the cAMP activity was detected, and the cAMP activity detection method was the same as that in Example 5.1.2.

[0672] The results are shown in Tables 53-55. After 14 days of incubation of SZ08830849 / PEP072 in human, rat, and cynomolgus monkey plasma, the activity did not change substantially. Compared with PTH (1-34), the plasma stability of the complex was significantly improved, indicating that SZ08830849 / PEP072 has a significant long-acting effect in vitro.

[0673] Table 53: Stability of SZ08830849 / PEP072 complex in human plasma

[0674] Table 54: Stability of SZ08830849 / PEP072 complex in cynomolgus monkey plasma

[0675] Table 55: Stability of SZ08830849 / PEP072 complex in rat plasma

[0676] 5.3 In vivo efficacy

[0677] 5.3.1 Single administration of SZ08830849 / PTH-FA complex

[0678] The purpose of this study is to test the effect of Anti-PTH / PTH-FA complex molecules on the regulation of serum calcium and phosphorus in PTX model rats. In this experiment, 9-10 week old male SD rats (n=4 / group, experimental animals from Vivotec Laboratories) were given a single subcutaneous dose of different Anti-PTH / PTH-FA complex molecules (1 nmol / kg SZ08830849 / PEP071, 1 nmol / kg SZ08830849 / PEP072) (vehicle: 1*PBS) and control drugs (40 nmol / kg PC-2). Blood samples were collected before and after administration at Day 1, Day 2, Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, and Day 12, and the concentrations of inorganic calcium and inorganic phosphorus in the serum of the animals were detected using a biochemical analyzer (model: sysmex BX4000) and reagent kits (brand: Maccura; catalog number: CH0101251 and CH0101257).

[0679] Conclusion: As shown in Figures 26-27, the sCa and sPi of the rats in each test group were consistent before administration. After administration of 1 nmol / kg SZ08830849 / PEP071 and SZ08830849 / PEP072 and 40 nmol / kg PC-2, the sCa of the rats in the administration groups increased to varying degrees compared to the PBS vehicle control group (PTX model rats given PBS vehicle), and was close to the normal physiological level of the Sham group (rats not subjected to PTX modeling). The sPi of the rats in the administration groups decreased to varying degrees compared to the PBS vehicle control group, and was close to the normal physiological level of the Sham group. The sCa of the 40 nmol / kg PC-2 group decreased to below the normal physiological level 4 to 5 days after administration, close to the PBS vehicle group level. The SZ08830849 / PEP071 group achieved the above effect at 1 nmol / kg, while the 1 nmol / kg SZ08830849 / PEP072 group could still maintain sCa and sPi at normal physiological levels 9 days after a single administration. Both SZ08830849 / PEP071 and SZ08830849 / PEP072 showed significant long-term effects in vivo.

[0680] 5.3.2 SZ08830849 / PTH-FA complex multiple administration

[0681] The purpose of this study is to test the regulation of Anti-PTH / PTH-FA complex molecule multiple doses on the blood calcium and phosphorus of PTX model rats in vivo. In this experiment, 9-10 week old male SRG rats (n=6 / group, experimental animals from Vinton Li Hua) were given different doses of SZ08830849 / PEP072 complex molecules and control drugs PC-2 and PC-1 by subcutaneous administration. The administration methods of each group are as follows:

[0682] Before administration, at day 1, day 4, day 7, day 8, day 11, day 14, day 15, day 18, day 21, day 22, day 25, day 28, day 29, day 32, day 35, day 36, day 39, day 42, day 43, day 46, day 49, day 50, day 53, day 56, day 57, day 60, day 63, day 54, day 67, day 70, day 71, day 74, day 77, day 78, day 81, day 84, day, day 85, day 88, day 91, day 92, day 95, day 98, etc. Time points, blood was collected and the concentration of inorganic calcium and inorganic phosphorus in the serum of the animals was detected by using a biochemical instrument (model: sysmex BX4000) and a kit (brand: Maccura; item number: CH0101251 and CH0101257).

[0683] Conclusion: The results are shown in Figures 28-29. Before administration, the sCa and sPi of the rats in each test group were consistent. After administration of different doses of SZ08830849 / PEP072, the sCa of the rats in the administration group increased to varying degrees compared with the PBS solvent control group (PTX model rats given PBS solvent), and was close to the normal physiological level of the Sham group (rats not subjected to PTX modeling). The sPi of the rats in the administration group decreased to varying degrees compared with the PBS solvent control group, and was close to the normal physiological level of the Sham group. SZ08830849 / PEP072 administered once a week can stably maintain the blood calcium and phosphorus levels within the normal physiological range, and has a lower administration frequency compared with the daily administration of control drugs, showing obvious long-acting effect in vivo.

[0684] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in accordance with all the teachings of the disclosure, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

A complex comprising a first protein and a second protein, the first protein specifically binding to the second protein, wherein, The first protein comprises or consists of a parathyroid hormone (PTH) protein or an active fragment thereof; and the second protein comprises or consists of an antibody or an antigen-binding fragment thereof that targets the PTH protein or the active fragment thereof. The complex of claim 1, wherein, The first protein is a PTH protein or an active fragment thereof; Preferably, the PTH protein is a wild-type PTH protein. Preferably, the wild-type PTH protein comprises the sequence set forth in SEQ ID NO:

99. Preferably, the PTH protein active fragment comprises at least the amino acid residues in PTH corresponding to positions 1-14 (or 1-16, 1-18, 1-20, 1-22, 1-25, 1-30, 1-34) of SEQ ID NO:

99. Preferably, the PTH protein active fragment comprises at least the amino acid residues in PTH corresponding to positions 1-34 of SEQ ID NO: 99; more preferably, the PTH protein active fragment consists of the amino acid residues in PTH corresponding to positions 1-34 of SEQ ID NO:

99. The complex of any one of claims 1 or 2, wherein, The PTH protein or the active fragment thereof comprises a modification compared to a wild-type PTH protein or the active fragment thereof: (1) comprises a mutation at one or more of the positions corresponding to positions 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99; and / or, (2) comprises an aliphatic organic acid. The complex of claim 3, wherein, The mutation is an amino acid substitution. Preferably, the mutation is selected from one or more of the following: (a) the amino acid at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 is substituted to a non-natural amino acid (e.g., alpha-aminoisobutyric acid (Aib)) in the PTH protein or the active fragment thereof; (b) the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99 is substituted, e.g., to alanine (A), serine (S), in the PTH protein or the active fragment thereof; (c) the serine (S) at the position corresponding to position 17 of SEQ ID NO: 99 is substituted, e.g., to glutamic acid (E), in the PTH protein or the active fragment thereof; (d) the phenylalanine (F) at the position corresponding to position 34 of SEQ ID NO: 99 is substituted, e.g., to lysine (K), in the PTH protein or the active fragment thereof; Preferably, the PTH protein or the active fragment thereof is substituted to a non-natural amino acid, such as alpha-aminoisobutyric acid (Aib), at an amino acid position selected from the following relative to SEQ ID NO: 99: (i) position 1, 3, 25; (ii) position 3, 25; (iii) position 1, 25; (iv) position 25; or (v) position 1. Preferably, the PTH protein or active fragment thereof: (I) is substituted with a non-natural amino acid (such as alpha-aminoisobutyric acid (Aib)) at the amino acid position corresponding to position 1, 25 of SEQ ID NO: 99; (II) is substituted at the asparagine (N) corresponding to position 16 of SEQ ID NO: 99 (e.g., substituted with alanine (A), serine (S)); and / or, (III) is substituted at the serine (S) corresponding to position 17 of SEQ ID NO: 99 (e.g., substituted with glutamic acid (E)). The complex of claim 3 or 4, wherein, The aliphatic organic acid is optionally linked to any amino acid in the PTH protein or active fragment thereof from the amino acid at position 1 to the C-terminus; the positions are amino acid positions relative to SEQ ID NO: 99; Preferably, the aliphatic organic acid is optionally linked to the amino acid at the C-terminus of the PTH protein or active fragment thereof; Preferably, the aliphatic organic acid is optionally linked to the amino acid at the C-terminus of the PTH protein or active fragment thereof; Preferably, the amino acid is selected from lysine, d-lysine, ornithine, cysteine, or homocysteine; Preferably, the PTH protein or active fragment thereof is substituted with a non-natural amino acid, such as alpha-aminoisobutyric acid (Aib), at an amino acid position selected from the following relative to SEQ ID NO: 99: (i) position 1, 25; (ii) position 1; and, the aliphatic organic acid is optionally linked to the amino acid at the C-terminus of the PTH protein or active fragment thereof. The complex of claim 5, wherein, The spacer comprises an acidic amino acid residue (e.g., gamma glutamic acid); Preferably, said spacer is selected from the group consisting of a gamma glutamic acid- gamma glutamic acid dipeptide, *-[COCH2(OCH2CH2) k NH] q - gamma glutamic acid or gamma glutamic acid -[COCH2(OCH2CH2) k NH] q - gamma glutamic acid, wherein k is an integer selected from 1 to 20, q is an integer selected from 1 to 20, the * end is attached to the amino acid side chain; Preferably, the k is 1 or 2; Preferably, the q is 1 or 2. The complex of claim 5 or 6, wherein, The aliphatic organic acid is optionally linked to any amino acid in the PTH protein or active fragment thereof from the amino acid at position 1 to the C-terminus; the positions are amino acid positions relative to SEQ ID NO: 99; the structural composition of the aliphatic organic acid and spacer is shown as Formula (A): III-(II)m-(I)n- Formula (A), wherein, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; I is -C(=O)CH2(OCH2CH2)2NH-; II is an acidic amino acid residue; III is COOH-(CH2)r-C(=O)-, r is an integer selected from 12 to 30; and III, II, and I are connected by an amide bond between them, the carbonyl of I is connected to an amino group of an amino acid side chain in the PTH protein or active fragment thereof; Preferably, the carbonyl of I is connected to an amino group of a lysine side chain; Preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Preferably, n is 1 or 2; Preferably, II is selected from a gamma glutamic acid residue and an aspartic acid residue; Preferably, said II is a gamma glutamic acid residue having the structure shown below: Preferably, m is 1; Preferably, r is an integer selected from 16 to 22; Preferably, said III is COOH-(CH2) 16 -C(=O)-, COOH-(CH2) 17 -C(=O)-, or COOH-(CH2) 18 -C(=O)-; Preferably, said formula (A) has a structure selected from the following: The complex of any one of claims 1-7, wherein, The first protein comprises a sequence as set forth in any one of SEQ ID NOs: 73-76, 100-106, 25, 99. The complex of any one of claims 1-8, wherein, The second protein is an antibody or antigen-binding fragment thereof that specifically binds to PTH(20-28) targeting a PTH protein or an active fragment thereof. The complex of claims 1-9, wherein, The antibody or antigen-binding fragment thereof comprises: 3 CDRs contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 53; and / or, 3 CDRs contained in a light chain variable region (VL) as set forth in any one of SEQ ID NOs: 59, 63, 66, 69; Preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising 3 CDRs: CDR-H1 of SEQ ID NO: 50; CDR-H2 of SEQ ID NO: 51; and CDR-H3 of SEQ ID NO: 52; and, a light chain variable region comprising 3 CDRs: CDR-L1 of SEQ ID NO: 56, 62, 65, or 68; CDR-L2 of SEQ ID NO: 57; and CDR-L3 of SEQ ID NO:

58. Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising a sequence as set forth in SEQ ID NO: 53 or a sequence with at least 80% identity thereto; and, a VL comprising a sequence as set forth in any one of SEQ ID NOs: 59, 63, 66, 69 or a sequence with at least 80% identity thereto. The complex of claim 9 or 10, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4) and a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda). The complex of claim 11, wherein, The antibody or antigen-binding fragment thereof comprises a variant of a human IgG (e.g., IgGl or IgG4) heavy chain constant region, which variant possesses a mutation that reduces or abolishes effector function (e.g., ADCC, ADCP, and / or CDC activity) as compared to the wild-type sequence from which it is derived; Preferably, the variant of a human IgG heavy chain constant region is a variant of a human IgGl heavy chain constant region, which variant possesses one or more of the following substitutions as compared to the wild-type sequence: L234A, L235A, and P329G (positions according to the EU numbering system); Preferably, the variant of a human IgGl heavy chain constant region comprises a sequence as set forth in SEQ ID NO: 54 or 71. The complex of any one of claims 9-12, wherein, The antibody or antigen-binding fragment thereof comprises: (a) a heavy chain having a sequence as set forth in SEQ ID NO: 55 and a light chain having a sequence as set forth in SEQ ID NO: 61; (b) a heavy chain having the sequence of SEQ ID NO: 72 and a light chain having the sequence of SEQ ID NO: 64; (c) a heavy chain having the sequence of SEQ ID NO: 72 and a light chain having the sequence of SEQ ID NO: 67; or (d) a heavy chain having the sequence of SEQ ID NO: 72 and a light chain having the sequence of SEQ ID NO:

70. The complex of any one of claims 1-13, wherein, the first protein is as defined in any one of claims 1 to 8 and the second protein is as defined in any one of claims 9 to 13; Preferably, in the complex: (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (1) the first protein is a PTH active fragment as set forth in SEQ ID NO: 25 and the second protein is an antibody comprising a heavy chain having the sequence of SEQ ID NO: 55 and a light chain having the sequence of SEQ ID NO: 61 ; (9) the first protein is a PTH active fragment as set forth in SEQ ID NO: 104, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 72 and a light chain having the sequence as set forth in SEQ ID NO:

67. The complex of claim 1 or 2, wherein, The first protein is a fusion protein comprising (i) a PTH protein or an active fragment thereof and (ii) a nanobody or an antigen-binding fragment thereof targeting serum albumin. The complex of claim 15, wherein, The PTH protein or the active fragment thereof comprises a modification comprising a mutation at one or more of positions corresponding to positions 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99, as compared to a wild-type PTH protein or the active fragment thereof. The complex of claim 16, wherein, The mutation is an amino acid substitution; Preferably, the mutation is selected from one or more of the following: (a) an amino acid at one or more of positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 of the PTH protein or the active fragment thereof is substituted with a non-natural amino acid (e.g., a-aminobutyric acid (Aib)); (b) an asparagine (N) at a position corresponding to position 16 of SEQ ID NO: 99 of the PTH protein or the active fragment thereof is substituted, e.g., with alanine (A), serine (S); (c) a serine (S) at a position corresponding to position 17 of SEQ ID NO: 99 of the PTH protein or the active fragment thereof is substituted, e.g., with glutamic acid (E); (d) a phenylalanine (F) at a position corresponding to position 34 of SEQ ID NO: 99 of the PTH protein or the active fragment thereof is substituted, e.g., with lysine (K); Preferably, the PTH protein or the active fragment thereof comprises a sequence as set forth in SEQ ID NO: 93 or 94. The complex of any one of claims 15-17, wherein, The nanobody or the antigen-binding fragment thereof comprises 3 CDRs contained in a heavy chain variable region (VHH) as set forth in any one of SEQ ID NOs: 7, 12, 18, 23, 96, 98; Preferably, the nanobody or the antigen-binding fragment thereof comprises: (1) CDR1 as set forth in SEQ ID NO: 4, CDR2 as set forth in SEQ ID NO: 5 or 16, and CDR3 as set forth in SEQ ID NO: 6, 95 or 97; (2) CDR1 as set forth in SEQ ID NO: 9, CDR2 as set forth in SEQ ID NO: 10, and CDR3 as set forth in SEQ ID NO: 11; Preferably, the Nanobody or antigen-binding fragment thereof comprises: (i) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 5, 6, respectively; (ii) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 6, respectively; (iii) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 95, respectively; or (iv) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 97, respectively; Preferably, the Nanobody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 7, 12, 18, 23, 96, 98 (e.g., SEQ ID NOs: 18, 23, 96, 98) or a sequence having at least 80% identity thereto. The complex of claims 15-18, wherein, The PTH protein or active fragment thereof is optionally linked to the N- or C-terminus of the Nanobody or antigen-binding fragment thereof via a linker; Preferably, the PTH protein or active fragment thereof is optionally linked to the N-terminus of the Nanobody or antigen-binding fragment thereof via a linker; Preferably, the linker is a peptide linker (e.g., a rigid peptide linker or a flexible peptide linker); Preferably, the linker is a peptide linker comprising one or more glycines and / or one or more serines; Preferably, the peptide linker is (GmS)n, m, n are independently integers not less than 0, e.g., independently 1, 2, 3, or 4; Preferably, the peptide linker comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 26, 31-34. The complex of any one of claims 15-19, wherein, The first protein comprises a fusion protein as set forth in any one of SEQ ID NOs: 27-30, 35-49; Preferably, the first protein comprises a fusion protein as set forth in any one of SEQ ID NOs: 29, 30, 45-49. The complex of any one of claims 15-20, wherein, The second protein is an antibody or antigen-binding fragment thereof targeting a PTH protein or active fragment thereof, which specifically binds to PTH(1-7). The complex of any one of claims 15-21, wherein, The antibody or antigen-binding fragment thereof comprises: 3 CDRs contained in a VH as set forth in SEQ ID NO: 80 or 88; and / or, 3 CDRs contained in a VL as set forth in SEQ ID NO: 85 or 91; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising 3 CDRs: CDR-H1 of SEQ ID NO: 77; CDR-H2 of SEQ ID NO: 78 or 87; CDR-H3 of SEQ ID NO: 79; and, a VL comprising 3 CDRs: CDR-L1 of SEQ ID NO: 82 or 90; CDR-L2 of SEQ ID NO: 83; CDR-L3 of SEQ ID NO: 84; Preferably, (a) the VH comprises: CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 77, 78, 79, respectively; and the VL comprises: CDR-L1, CDR-L2, CDR-L3 of SEQ ID NOs: 82, 83, 84, respectively; or (b) the VH comprises: CDR-H1, CDR-H2, CDR-H3 of SEQ ID NOs: 77, 87, 79, respectively; and the VL comprises: CDR-L1, CDR-L2, CDR-L3 of SEQ ID NOs: 90, 83, 84, respectively. Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising a sequence as set forth in SEQ ID NO: 80 or 88, or a sequence that is at least 80% identical thereto; and a VL comprising a sequence as set forth in SEQ ID NO: 85 or 91, or a sequence that is at least 80% identical thereto. Preferably, the antibody or antigen-binding fragment thereof comprises: (a) a VH comprising a sequence as set forth in SEQ ID NO: 80; and a VL comprising a sequence as set forth in SEQ ID NO: 85; or (b) a VH comprising a sequence as set forth in SEQ ID NO: 88; and a VL comprising a sequence as set forth in SEQ ID NO:

91. The complex of claim 21 or 22, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4) and a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda). The complex of claim 23, wherein, The antibody or antigen-binding fragment thereof comprises a variant of a human IgG (e.g., IgGl or IgG4) heavy chain constant region that possesses a mutation that reduces or abolishes effector function (e.g., ADCC, ADCP, and / or CDC activity) as compared to the wild-type sequence from which it is derived; Preferably, the variant of a human IgG heavy chain constant region is a variant of a human IgGl heavy chain constant region that possesses one or more of the following substitutions: L234A, L235A, and P329G (positions according to the EU numbering system) as compared to the wild-type sequence; Preferably, the variant of a human IgGl heavy chain constant region comprises a sequence as set forth in SEQ ID NO: 54 or 71. The complex of any one of claims 21-24, wherein, The antibody or antigen-binding fragment thereof comprises: (i) a heavy chain having a sequence as set forth in SEQ ID NO: 81 and a light chain having a sequence as set forth in SEQ ID NO: 86; or (ii) a heavy chain having a sequence as set forth in SEQ ID NO: 89 and a light chain having a sequence as set forth in SEQ ID NO:

92. The complex of any one of claims 21-25, wherein the first protein is as defined in any one of claims 15-20 and the second protein is as defined in any one of claims 21-25; Preferably, in the complex: (1) the first protein is a fusion protein as set forth in SEQ ID NO: 29, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 81 and a light chain having the sequence as set forth in SEQ ID NO: 86; (2) the first protein is a fusion protein as set forth in SEQ ID NO: 30, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 81 and a light chain having the sequence as set forth in SEQ ID NO: 86; (3) the first protein is a fusion protein as set forth in SEQ ID NO: 45, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 89 and a light chain having the sequence as set forth in SEQ ID NO: 92; (4) the first protein is a fusion protein as set forth in SEQ ID NO: 46, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 89 and a light chain having the sequence as set forth in SEQ ID NO: 92; (5) the first protein is a fusion protein as set forth in SEQ ID NO: 30, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 89 and a light chain having the sequence as set forth in SEQ ID NO: 92; (6) the first protein is a fusion protein as set forth in SEQ ID NO: 48, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 89 and a light chain having the sequence as set forth in SEQ ID NO: 92; (7) the first protein is a fusion protein as set forth in SEQ ID NO: 49, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 89 and a light chain having the sequence as set forth in SEQ ID NO: 92; or (8) the first protein is a fusion protein as set forth in SEQ ID NO: 47, and the second protein is an antibody comprising a heavy chain having the sequence as set forth in SEQ ID NO: 89 and a light chain having the sequence as set forth in SEQ ID NO:

92. a complex comprising a first protein as defined in any one of claims 1-8, 15-20 and a second protein as defined in any one of claims 9-13, 21-25; Preferably, the first protein is as defined in any one of claims 1-8, and the second protein is as defined in any one of claims 9-13. Preferably, the first protein is as defined in any one of claims 15-20, and the second protein is as defined in any one of claims 21-25. a composition comprising: (i) a first protein, or a nucleic acid molecule or a vector encoding the first protein, or a host cell comprising the nucleic acid molecule or the vector, the first protein being a first protein comprised by a complex according to any one of claims 1-27; (ii) a second protein, or a nucleic acid molecule or vector encoding the second protein, or a host cell comprising the nucleic acid molecule or vector, the second protein being a second protein comprised by the complex of any one of claims 1-27. The composition of claim 28, comprising: (i) a first protein comprised by the complex of any one of claims 3-14, or a nucleic acid molecule or vector encoding the first protein, or a host cell comprising the nucleic acid molecule or vector; and (ii) a second protein comprised by the complex of any one of claims 3-14, or a nucleic acid molecule or vector encoding the second protein, or a host cell comprising the nucleic acid molecule or vector. The composition of claim 28, comprising: (i) a first protein comprised by the complex of any one of claims 15-27, or a nucleic acid molecule or vector encoding the first protein, or a host cell comprising the nucleic acid molecule or vector; and (ii) a second protein comprised by the complex of any one of claims 15-27, or a nucleic acid molecule or vector encoding the second protein, or a host cell comprising the nucleic acid molecule or vector. A pharmaceutical composition comprising the complex of any one of claims 1-27, the composition of any one of claims 28-30, and a pharmaceutically acceptable carrier and / or excipient. Use of the complex of any one of claims 1-27, the composition of any one of claims 28-30, or the pharmaceutical composition of claim 31 for the manufacture of a medicament for preventing and / or treating a bone-related disease (such as osteoporosis) or a disease caused by hypoparathyroidism (such as hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria) in a subject; Preferably, the subject is a mammal, such as a human; Preferably, the complex, composition, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent. A PTH polypeptide or active fragment thereof, which, as compared to a wild-type PTH polypeptide or active fragment thereof: (1) comprises a mutation at one or more of the positions corresponding to positions 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99; and / or, (2) comprises an aliphatic organic acid. The PTH polypeptide or active fragment thereof of claim 33, wherein, The mutation is an amino acid substitution; Preferably, the mutation is selected from one or more of the following: (a) the amino acid at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted with a non-natural amino acid (e.g., alpha-aminobutyric acid (Aib)); (b) the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted, e.g., with alanine (A), serine (S); (c) the PTH polypeptide or active fragment thereof has a substitution at the position corresponding to Serine (S) at position 17 of SEQ ID NO: 99, for example to Glutamic acid (E); (d) the PTH polypeptide or active fragment thereof has a substitution at the position corresponding to Phenylalanine (F) at position 34 of SEQ ID NO: 99, for example to Lysine (K); Preferably, the PTH polypeptide or active fragment thereof has a substitution to a non-natural amino acid, such as alpha-aminoisobutyric acid (Aib), at an amino acid position selected from the following relative to SEQ ID NO: 99: (i) position 1, position 3, position 25; (ii) position 3, position 25; (iii) position 1, position 25; (iv) position 25; or (v) position 1; Preferably, the PTH polypeptide or active fragment thereof: (I) has a substitution to a non-natural amino acid (such as alpha-aminoisobutyric acid (Aib)) at an amino acid position corresponding to position 1, position 25 of SEQ ID NO: 99; (II) has a substitution at the position corresponding to Asparagine (N) at position 16 of SEQ ID NO: 99 (for example to Alanine (A), Serine (S)); and / or, (III) has a substitution at the position corresponding to Serine (S) at position 17 of SEQ ID NO: 99 (for example to Glutamic acid (E)). The PTH polypeptide or active fragment thereof of claim 33 or 34, wherein, Preferably, the aliphatic organic acid is optionally linked via a spacer to an amino acid of the PTH polypeptide or active fragment thereof at a position corresponding to position 34 of SEQ ID NO: 99; Preferably, the aliphatic organic acid is optionally linked via a spacer to an amino acid of the PTH polypeptide or active fragment thereof at a position corresponding to position 34 of SEQ ID NO: 99; Preferably, the aliphatic organic acid is optionally linked via a spacer to an amino acid of the PTH polypeptide or active fragment thereof at a position corresponding to position 34 of SEQ ID NO: 99; Preferably, the amino acid is selected from lysine, d-lysine, ornithine, cysteine or homocysteine; Preferably, the PTH polypeptide or active fragment thereof has a substitution to a non-natural amino acid, such as alpha-aminoisobutyric acid (Aib), at an amino acid position selected from the following relative to SEQ ID NO: 99: (i) position 1, position 25; (ii) position 1; and, the aliphatic organic acid is optionally linked via a spacer to an amino acid of the PTH protein or active fragment thereof at a position corresponding to position 34 of SEQ ID NO:

99. The PTH polypeptide or active fragment thereof of claim 35, wherein, Preferably, the spacer comprises an acidic amino acid residue (such as gamma glutamic acid); Preferably, said spacer is selected from the group consisting of a gamma glutamic acid- gamma glutamic acid dipeptide, *-[COCH2(OCH2CH2) k NH] q - gamma glutamic acid or gamma glutamic acid - [COCH2(OCH2CH2) k NH] q - gamma glutamic acid, wherein k is an integer selected from 1 to 20, q is an integer selected from 1 to 20, the * end is attached to an amino acid side chain; Preferably, the k is 1 or 2; Preferably, the q is 1 or 2. The PTH polypeptide or active fragment thereof of claim 35 or 36, wherein, Preferably, the aliphatic organic acid is optionally linked via a spacer to an amino acid of the PTH polypeptide or active fragment thereof at a position corresponding to position 34 of SEQ ID NO: 99; Preferably, the aliphatic organic acid is optionally linked via a spacer to an amino acid of the PTH polypeptide or active fragment thereof at a position corresponding to position 34 of SEQ ID NO: 99; III-(II)m-(I)n- Formula (A), wherein, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; I is -C(=O)CH2(OCH2CH2)2NH-; II is an acidic amino acid residue; III is COOH-(CH2)r-C(=O)-, r is an integer selected from 12 to 30; and III, II, and I are connected via amide bonds, the carbonyl of said I is connected to an amino group of an amino acid side chain in said PTH protein or active fragment thereof; preferably, the carbonyl of said I is connected to an amino group of a lysine side chain; preferably, said n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, said n is 1 or 2; preferably, said II is selected from a gamma glutamic acid residue and an aspartic acid residue; Preferably, said II is a gamma glutamic acid residue having the structure shown below: preferably, said m is 1; preferably, said III is COOH-(CH2)r-C(=O)-, r is an integer selected from 16 to 22; Preferably, said III is COOH-(CH2) 16 -C(=O)-, COOH-(CH2) 17 -C(=O)-, or COOH-(CH2) 18 -C(=O)-; Preferably, said formula (A) has a structure selected from the following: a PTH polypeptide or active fragment thereof comprising a sequence as set forth in any one of SEQ ID NOs: 73-76, 100-106. a Nanobody or antigen-binding fragment thereof that specifically binds serum albumin, comprising: 3 CDRs contained in a VHH as set forth in any one of SEQ ID NOs: 7, 12, 18, 23, 96, 98; preferably, said Nanobody or antigen-binding fragment thereof comprises: (1) a CDR1 as set forth in SEQ ID NO: 4, a CDR2 as set forth in SEQ ID NO: 5 or 16, and a CDR3 as set forth in SEQ ID NO: 6, 95 or 97; or (2) a CDR1 as set forth in SEQ ID NO: 9, a CDR2 as set forth in SEQ ID NO: 10, and a CDR3 as set forth in SEQ ID NO: 11; preferably, said Nanobody or antigen-binding fragment thereof comprises: (i) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 5, 6, respectively; (ii) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 6, respectively; (iii) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 95, respectively; or (iv) CDR1, CDR2, CDR3 as set forth in SEQ ID NOs: 4, 16, 97, respectively; preferably, said Nanobody or antigen-binding fragment thereof comprises a sequence as set forth in any one of SEQ ID NOs: 7, 12, 18, 23, 96, 98 or a sequence having at least 80% identity thereto. a polypeptide construct that specifically binds serum albumin, comprising a Nanobody or antigen-binding fragment thereof of claim 39, and an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is an Fc domain of IgG (e.g., an Fc domain of IgG1, IgG2, IgG3, or IgG4); Preferably, the immunoglobulin Fc domain is an Fc domain of IgG (e.g., an Fc domain of IgG1, IgG2, IgG3, or IgG4); Preferably, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 14; Preferably, the polypeptide construct comprises the sequence set forth in any one of SEQ ID NOs: 8, 13, 19, 24. A fusion protein comprising a PTH polypeptide or active fragment thereof, and a Nanobody of claim 39 or antigen binding fragment thereof; wherein, The PTH polypeptide is a wild-type PTH polypeptide, or the PTH polypeptide or active fragment thereof comprises a modification, which comprises a mutation at one or more of the positions corresponding to positions 1, 3, 16, 17, 25, 34 of SEQ ID NO: 99, as compared to a wild-type PTH polypeptide or active fragment thereof; Preferably, the mutation is an amino acid substitution; Preferably, the mutation is selected from one or more of the following: (a) the amino acid at one or more of the positions corresponding to positions 1, 3, 25 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted with a non-natural amino acid (e.g., alpha-aminobutyric acid (Aib)); (b) the asparagine (N) at the position corresponding to position 16 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted, e.g., substituted with alanine (A), serine (S); (c) the serine (S) at the position corresponding to position 17 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted, e.g., substituted with glutamic acid (E); (d) the phenylalanine (F) at the position corresponding to position 34 of SEQ ID NO: 99 of the PTH polypeptide or active fragment thereof is substituted, e.g., substituted with lysine (K); Preferably, the PTH polypeptide or active fragment thereof comprises the sequence set forth in SEQ ID NO: 93 or 94; Preferably, the PTH polypeptide or active fragment thereof is optionally linked to the N-terminus or C-terminus, e.g., N-terminus, of the nanobody or antigen-binding fragment thereof via a linker; Preferably, the linker is a peptide linker (e.g., a rigid peptide linker or a flexible peptide linker); Preferably, the peptide linker is a peptide linker comprising one or more glycines and / or one or more serines; Preferably, the peptide linker is (GmS)n, m, n are independently integers no less than 0, e.g., independently 1, 2, 3, or 4; Preferably, the peptide linker comprises or consists of the sequence set forth in any one of SEQ ID NOs: 26, 31-34; Preferably, the fusion protein comprises the sequence set forth in any one of SEQ ID NOs: 27-30, 35-49, e.g., the sequence set forth in any one of SEQ ID NOs: 29, 30, 45-49. An isolated nucleic acid molecule encoding a PTH polypeptide or an active fragment thereof according to any one of claims 33-38, a Nanobody or an antigen-binding fragment thereof according to claim 39, a polypeptide construct according to claim 40, a fusion protein according to claim 41. A vector comprising the isolated nucleic acid molecule according to claim 42. A host cell comprising the isolated nucleic acid molecule according to claim 42 or the vector according to claim 43. A pharmaceutical composition comprising a PTH polypeptide or an active fragment thereof according to any one of claims 33-38, or a fusion protein according to claim 41, and a pharmaceutically acceptable carrier and / or excipient. Use of a PTH polypeptide or an active fragment thereof according to any one of claims 33-38, a fusion protein according to claim 41, or a pharmaceutical composition according to claim 45, for the manufacture of a medicament for the prevention and / or treatment of a bone-related disease (such as osteoporosis) or a disease caused by hypoparathyroidism (such as hypoparathyroidism, hypocalcemia, hyperphosphatemia, or hypercalciuria) in a subject; Preferably, the subject is a mammal, such as a human; Preferably, the PTH polypeptide, fusion protein, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent.

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