阅读说明：本技术 甲状旁腺激素融合多肽 (Parathyroid hormone fusion polypeptides ) 是由 伊恩·威尔金森 理查德·罗斯 于 2018-04-27 设计创作，主要内容包括：本公开涉及了包含受体多肽的长效甲状旁腺激素或甲状旁腺激素样融合多肽,和其在治疗甲状旁腺功能减退和骨质疏松症中的用途。(The present disclosure relates to long-acting parathyroid hormone or parathyroid hormone-like fusion polypeptides comprising a receptor polypeptide, and their use in the treatment of hypoparathyroidism and osteoporosis.)

1. A fusion polypeptide comprising

A polypeptide comprising the amino acid sequence of parathyroid hormone or a biologically active fragment or analogue thereof,

A polypeptide comprising the amino acid sequence of a receptor polypeptide or a fragment or analog thereof, wherein said parathyroid hormone or a biologically active fragment or analog thereof is linked directly or indirectly to said receptor polypeptide as a translational fusion.

2. The fusion polypeptide of claim 1, wherein the fusion polypeptide comprises a parathyroid hormone comprising or consisting of the amino acid sequence set forth in SEQ ID No. 10.

3. The fusion polypeptide of claim 1 or 2, wherein the fusion polypeptide comprises a fragment of SEQ ID NO 10 comprising amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35.

4. The fusion polypeptide of claim 1 or 2, wherein the fusion polypeptide comprises parathyroid hormone comprising the sequence shown in SEQ ID NO 9.

5. The fusion polypeptide of claim 4, wherein the fusion polypeptide comprises a fragment of SEQ ID NO 9 comprising amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35.

6. The fusion polypeptide of claim 5, wherein the fusion polypeptide comprises a parathyroid hormone comprising or consisting of the amino acid sequence shown in SEQ ID NO 54.

7. the fusion polypeptide of claim 1 or 2, wherein the fusion polypeptide comprises a parathyroid hormone analogue or fragment thereof, which parathyroid hormone analogue comprises or consists of: amino acid sequence of formula (I)

Xaa01-Val-Xaa03-Glu-Ile-Gln-Leu-Xaa08-His-Xaa10-Xaa11-Xa a12-Xaa13-Xaa14- Leu-Xaa16-Xaa17-Xaa18-Arg-Arg-Arg-Xaa22-Ph e-Leu-Xaa25-Xaa26-Leu-Ile-Ala-Glu- Ile-His-Thr-Ala-Glu-Ile(SEQ ID NO:8)

(II) wherein Xaa01 is Ser or Ala; xaa03 is Ser or Ala; xaa08 is Met or Leu; xaa10 is Asn, Ala, Val, Asp, Glu or Gln; xaa11 is Leu, Ala, Val, Met, Lys, Arg, or Trp; xaa12 is Gly, Ala, His, or Arg; xaa13 is Lys, Ala, Leu, Gln, Arg, His, or Trp; xaa14 is His, Leu, Arg, Phe, Trp, or Ser; xaa16 is Gln or Asn; xaa17 is Asp or Ser; xaa18 is Ala, Leu, Met, Glu, Ser, or Phe; xaa22 is Ala, Phe, Glu, Ser, Leu, Asn, Trp, or Lys; xaa25 is His, Arg, Leu, Trp, or Lys; and Xaa26 is Lys, His, Ala, Ser, Asn, or Arg, or a fragment thereof comprising amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35 of formula (I), as long as Xaa18 is not Leu or Met, Xaa22 is not Phe, and Xaa26 is not His.

8. the fusion polypeptide of claim 1 or 2, wherein the fusion polypeptide comprises parathyroid hormone and parathyroid hormone analogue amino acid sequences comprising or consisting of the amino acid sequence shown in SEQ ID No. 37.

9. the fusion polypeptide of any one of claims 1 to 3, wherein the fusion polypeptide comprises a parathyroid hormone-related protein comprising the amino acid sequence shown in SEQ ID NO 40, or a biologically active fragment or analog thereof.

10. The fusion polypeptide of any one of claims 1-9, wherein the fusion polypeptide comprises a parathyroid hormone receptor extracellular domain-containing receptor polypeptide.

11. The fusion polypeptide of claim 10, wherein the fusion polypeptide comprises a parathyroid hormone receptor extracellular domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 3.

12. The fusion polypeptide of claim 10, wherein the fusion polypeptide comprises a parathyroid hormone receptor extracellular domain or fragment thereof comprising an amino acid sequence from 10% to 99% identical to the full-length amino acid sequence set forth in SEQ ID No. 3, and wherein the domain or fragment binds parathyroid hormone, a fragment or analog thereof.

13. The fusion polypeptide of claim 12, wherein the fusion polypeptide comprises a parathyroid hormone receptor extracellular domain or fragment thereof comprising an amino acid sequence 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 3, and wherein said domain binds to parathyroid hormone, a fragment or analog thereof.

14. The fusion polypeptide of any one of claims 11-13, wherein the fusion polypeptide comprises a modified amino acid sequence encoding the extracellular domain of parathyroid hormone receptor, wherein the modification is one or more amino acid substitutions selected from the group consisting of: I107K, D109A, P104L or L159A shown in SEQ ID NO 3.

15. The fusion polypeptide of claim 14, wherein the fusion polypeptide comprises a parathyroid hormone receptor extracellular domain comprising the I07K substitution set forth in SEQ ID No. 3.

16. the fusion polypeptide of any one of claims 10 to 15, wherein the fusion polypeptide comprises a modified parathyroid hormone receptor extracellular domain, wherein the modification is by addition, deletion or substitution of at least one amino acid residue, wherein the modified polypeptide substantially lacks parathyroid hormone binding activity or has reduced parathyroid hormone binding activity.

17. The fusion polypeptide of any one of claims 1-16, wherein the fusion polypeptide comprising a receptor polypeptide alternatively or additionally comprises a growth hormone binding domain polypeptide of a growth hormone receptor.

18. the fusion polypeptide of claim 17, wherein the fusion polypeptide comprises an extracellular growth hormone binding domain polypeptide of a human growth hormone receptor.

19. The fusion polypeptide of claim 18, wherein the fusion polypeptide comprises an extracellular growth hormone binding domain polypeptide comprising the amino acid sequence set forth in SEQ ID NO 5.

20. The fusion polypeptide of claim 19, wherein the fusion polypeptide comprises a modified extracellular growth hormone binding domain polypeptide, wherein the modification is by addition, deletion or substitution of at least one amino acid residue, wherein the modified polypeptide substantially lacks growth hormone binding activity or has reduced growth hormone binding activity.

21. The fusion polypeptide of claim 20, wherein the fusion polypeptide comprises one or more modifications of an amino acid residue selected from the group consisting of: w169, R43, E44, I103, W104, I105, P106, I164 and D165 as shown in SEQ ID NO 5.

22. The fusion polypeptide of claim 21, wherein the fusion polypeptide comprises a deletion of the amino acid residue tryptophan 104 of the amino acid sequence set forth in SEQ ID NO. 5.

23. The fusion polypeptide of claim 22, wherein the fusion polypeptide comprises a substitution of tryptophan 104 of the amino acid sequence set forth as SEQ ID No. 5.

24. The fusion polypeptide of claim 23, wherein the fusion polypeptide comprises an alanine to tryptophan 104 substitution as set forth in SEQ ID NO 7.

25. The fusion polypeptide of any one of claims 1 to 24, wherein the fusion polypeptide comprises or consists of a parathyroid hormone fragment comprising or consisting of amino acid residues 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35 of SEQ ID No. 8 or amino acid residues 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35 of SEQ ID No. 8.

26. The fusion polypeptide of claim 25, wherein the fusion polypeptide comprises or consists of the amino acid sequence shown as SEQ ID No. 54, wherein the parathyroid hormone amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue, and wherein the modified fusion polypeptide retains parathyroid hormone activity.

27. The fusion polypeptide of any one of claims 1-26, where the fusion polypeptide comprises a parathyroid receptor domain polypeptide, where the parathyroid receptor domain polypeptide is located at the carboxy terminus of the fusion polypeptide.

28. The fusion polypeptide of any one of claims 1-26, where the fusion polypeptide comprises a parathyroid receptor domain polypeptide, where the parathyroid receptor domain polypeptide is located at the amino-terminus of the fusion polypeptide.

29. The fusion polypeptide of any one of claims 1-26 wherein the fusion polypeptide comprises a growth hormone binding domain polypeptide, wherein the growth hormone binding domain polypeptide is located at the carboxy terminus of the fusion polypeptide.

30. The fusion polypeptide of any one of claims 1-26 wherein the fusion polypeptide comprises a growth hormone binding domain polypeptide, wherein the growth hormone binding domain polypeptide is located at the amino-terminus of the fusion polypeptide.

31. the fusion polypeptide of claim 1, wherein the fusion polypeptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.

32. The fusion polypeptide of claim 31, wherein the fusion polypeptide comprises or consists of the amino acid sequence shown as SEQ ID No. 15 or 20.

33. The fusion polypeptide of claim 1, wherein the fusion polypeptide comprises or consists of the amino acid sequence shown as SEQ ID NO 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.

34. the fusion polypeptide of any one of claims 1-33, wherein the fusion polypeptide comprises a propeptide.

35. The fusion protein of any one of claims 1-33, wherein the fusion polypeptide further comprises a peptide secretion signal.

36. The fusion polypeptide of any one of claims 1-33, wherein the fusion polypeptide comprises parathyroid hormone, a fragment or analog thereof, and is linked to the receptor polypeptide directly or indirectly through a peptide linker.

37. The fusion polypeptide of any one of claims 1-33, wherein the parathyroid hormone, fragment or analogue is directly linked to the receptor polypeptide as an in-frame translational fusion.

38. A nucleic acid molecule encoding the fusion polypeptide of any one of claims 1-37.

39. A vector comprising the nucleic acid molecule of claim 38.

40. An isolated cell transfected or transformed with the nucleic acid molecule or vector of claim 38 or 39.

41. A method for producing a fusion polypeptide, the method comprising the steps consisting of:

i) Providing a cell and cell culture medium according to claim 40;

ii) culturing the cell; and

iii) isolating the fusion polypeptide according to the invention from the cell or the culture medium.

42. A pharmaceutical composition comprising the fusion polypeptide of any one of claims 1-37, comprising an excipient or carrier.

43. A fusion polypeptide according to any one of claims 1 to 37 for use in the treatment of hypoparathyroidism in a subject.

44. The fusion polypeptide of any one of claims 1-37 for use in treating osteoporosis in a human subject.

45. The fusion polypeptide for use according to claim 44, wherein the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO. 40.

46. The fusion polypeptide for use according to claim 44 or 45, wherein the fusion polypeptide is administered in combination with calcium carbonate and/or a vitamin D supplement such as calciferol or alcalcitol.

Technical Field

The present disclosure relates to long-acting parathyroid hormone fusion polypeptides comprising a receptor polypeptide, wherein the receptor polypeptide is a parathyroid hormone receptor and/or a growth hormone receptor; a nucleic acid molecule encoding the fusion polypeptide; a vector suitable for expressing the fusion polypeptide; a cell transformed or transfected with said nucleic acid or vector; and the use of said polypeptides in the treatment of hypoparathyroidism.

Background

Hypoparathyroidism is characterized by low parathyroid hormone (PTH) levels, a rare condition, and is either congenital or, more commonly, acquired after cervical surgery. PTH is secreted by four parathyroid glands located in the neck, parathyroid, and controls calcium homeostasis, vitamin D-dependent calcium absorption, renal calcium reabsorption, and renal phosphate clearance. PTH stimulates calcium release from bone and enhances calcium absorption in the intestine. Reduced levels of PTH can lead to hypocalcemia and include symptoms such as neuromuscular hypersensitivity, including paresthesia, muscle twitches, laryngeal spasms, tetany and seizures, and can lead to death if left untreated.

The current standard therapy is high dose calcium and active vitamin D; however, many patients show an increased incidence of depression, and an increased risk of infection and renal complications (such as calcification and renal insufficiency) despite the fluctuations in calcium levels caused by treatment. PTH is an 84 amino acid long polypeptide that contains a 34 amino acid long N-terminal bioactive domain that has been found to be a potent PTH receptor agonist.

Recombinant PTH is currently administered as daily subcutaneous injections. PTH is known to be unstable in vitro and to have a short half-life in vivo, leading to fluctuations in PTH levels, which are associated with nausea and vomiting. US7550434, US7144861, US6770623, WO2006/129995 or WO2013/108235 disclose compositions for in vitro stabilization of PTH, and WO2011143406 discloses recombinant PTH analogues with enhanced pharmacokinetics and pharmacodynamics comprising modified PTH fragments of up to 36 amino acids. However, there is a need in the art for long-acting PTH biologics that increase control of serum calcium levels and reduce side effects to minimize the need for daily subcutaneous injections.

Recombinant proteins and peptides used in medicine often suffer from increased serum clearance. There are two components of factors that lead to the removal of the administered protein from the circulation; renal filtration and proteolysis. Typically, proteins with molecular weights above 70kDa cannot be eliminated by glomerular filtration because they are too large to be filtered, whereas proteins with small molecular weights can be filtered by glomeruli and found in urine. One method of increasing the effective molecular weight of a protein and producing a product with reduced immunogenicity is to coat the protein with polyethylene glycol (PEG). PEG is believed to slow renal clearance by providing increased hydrodynamic volume in pegylated proteins (Maxfield et al, Polymer,16: 505-. However, pegylation of a protein results in a decrease in affinity for its receptor, thereby decreasing biological activity. An alternative approach to improving PK and PD of protein biologics is disclosed in WO 2009/013461. Human growth hormone fused to the extracellular domain of human Growth Hormone Receptor (GHR) increased PK and PD, resulting in an approximately 200-fold increase in PK, when compared to growth hormone. In the currently unpublished PCT/GB2016/053218, the effect of GHR on non-growth hormone polypeptides is disclosed, wherein leptin and granulocyte colony stimulating factor (GSCF) have increased PK and PD when fused to GHR.

the present disclosure relates to PTH fusion polypeptides in which PTH fused to a receptor (e.g., its cognate receptor and/or GHR) has increased PK and PD. The PTH fusion polypeptides are useful in the treatment of conditions resulting from aberrant PTH activity, including hypoparathyroidism, as well as in the treatment of conditions benefiting from PTH therapy, including osteoporosis.

Summary of The Invention

according to one aspect of the invention, there is provided a fusion polypeptide comprising

A polypeptide comprising the amino acid sequence of parathyroid hormone or a biologically active fragment or analogue thereof,

a polypeptide comprising the amino acid sequence of a receptor polypeptide or a fragment or analog thereof, wherein said parathyroid hormone or a biologically active fragment or analog thereof is linked directly or indirectly to said receptor polypeptide as a translational fusion.

By "analog" is meant a parathyroid hormone that binds to a parathyroid hormone receptor or a variant of the receptor polypeptide amino acid sequence. Parathyroid hormone analogs include, but are not limited to, amino acid sequences encoding parathyroid hormone-related protein and variants thereof.

In a preferred embodiment of the invention, the fusion polypeptide comprises parathyroid hormone comprising or consisting of the amino acid sequence shown in SEQ ID NO 10.

In a preferred embodiment of the invention said fusion polypeptide comprises a fragment of SEQ ID NO 10 comprising amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34 or 1-35.

In a preferred embodiment of the invention, the fusion polypeptide comprises parathyroid hormone comprising the sequence shown in SEQ ID NO 9.

In a preferred embodiment of the invention said fusion polypeptide comprises a fragment of SEQ ID NO 9 comprising the amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34 or 1-35.

In a preferred embodiment of the invention, the fusion polypeptide comprises parathyroid hormone comprising or consisting of the amino acid sequence shown as SEQ ID NO 54.

In a preferred embodiment of the invention said fusion polypeptide comprises a fragment of SEQ ID NO 54 comprising the amino acids 1-28, 1-29, 1-30, 1-31, 1-32 or 1-33.

In a preferred embodiment of the invention, the fusion polypeptide comprises a parathyroid hormone analogue or fragment thereof, said parathyroid hormone analogue comprising or consisting of: amino acid sequence of formula (I)

Xaa01-Val-Xaa03-Glu-Ile-Gln-Leu-Xaa08-His-Xaa10-Xaa11-Xaa12-Xaa13- Xaa14-Leu-Xaa16-Xaa17-Xaa18-Arg-Arg-Arg-Xaa22-Phe-Leu-Xaa25-Xaa26-Leu-Ile- Ala-Glu-Ile-His-Thr-Ala-Glu-Ile(SEQ ID NO:8)

(I) Wherein Xaa01 is Ser or Ala; xaa03 is Ser or Ala; xaa08 is Met or Leu; xaa10 is Asn, Ala, Val, Asp, Glu or Gln; xaa11 is Leu, Ala, Val, Met, Lys, Arg, or Trp; xaa12 is Gly, Ala, His, or Arg; xaa13 is Lys, Ala, Leu, Gln, Arg, His, or Trp; xaa14 is His, Leu, Arg, Phe, Trp, or Ser; xaa16 is Gln or Asn; xaa17 is Asp or Ser; xaa18 is Ala, Leu, Met, Glu, Ser, or Phe; xaa22 is Ala, Phe, Glu, Ser, Leu, Asn, Trp, or Lys; xaa25 is His, Arg, Leu, Trp, or Lys; and Xaa26 is Lys, His, Ala, Ser, Asn, or Arg, or a fragment thereof comprising amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35 of formula (I), as long as Xaa18 is not Leu or Met, Xaa22 is not Phe, and Xaa26 is not His.

In a preferred embodiment of the invention, the fusion polypeptide comprises parathyroid hormone and parathyroid hormone analogue amino acid sequences comprising or consisting of the amino acid sequence shown in SEQ ID NO 37.

In an alternative embodiment of the invention, the fusion polypeptide comprises a parathyroid hormone-related protein or a biologically active fragment or analogue thereof comprising an amino acid sequence shown in SEQ ID NO: 40.

In a preferred embodiment of the invention said fusion polypeptide comprises a fragment of SEQ ID NO 40 comprising amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34 or 1-35.

In a preferred embodiment of the invention, the fusion polypeptide comprises a receptor polypeptide comprising the extracellular domain of the parathyroid hormone receptor.

In a preferred embodiment of the invention, the fusion polypeptide comprises a parathyroid hormone receptor extracellular domain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO 3.

in another preferred embodiment of the invention, the fusion polypeptide comprises a parathyroid hormone receptor extracellular domain or fragment thereof comprising an amino acid sequence from 10% to 99% identical to the full length amino acid sequence set forth in SEQ ID NO 3, and wherein the domain or fragment binds parathyroid hormone, a fragment or analogue thereof.

in another preferred embodiment of the invention, the fusion polypeptide comprises a parathyroid hormone receptor extracellular domain or fragment thereof comprising an amino acid sequence 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 3, and wherein said domain binds to parathyroid hormone, a fragment or analogue thereof.

In a preferred embodiment of the invention, the fusion polypeptide comprises a modified amino acid sequence encoding the extracellular domain of a parathyroid hormone receptor, wherein the modification is one or more amino acid substitutions selected from the group consisting of: I107K, D109A, P104L or L159A shown in SEQ ID NO 3.

In another preferred embodiment of the invention, the fusion polypeptide comprises the extracellular domain of parathyroid hormone receptor comprising the I07K substitution shown in SEQ ID NO 3.

in a preferred embodiment of the invention said fusion polypeptide comprises or consists of amino acid residues 1 to 14 of the sequence shown in SEQ ID NO 10.

In an alternative embodiment of the invention, the fusion polypeptide comprises a modified parathyroid hormone receptor extracellular domain, wherein the modification is by addition, deletion or substitution of at least one amino acid residue, wherein the modified polypeptide substantially lacks parathyroid hormone binding activity or has reduced parathyroid hormone binding activity.

In one embodiment of the invention said fusion polypeptide comprising a receptor polypeptide comprises alternatively or additionally a growth hormone binding domain polypeptide of a growth hormone receptor.

In a preferred embodiment of the invention, the fusion polypeptide comprises an extracellular growth hormone binding domain polypeptide of human growth hormone receptor.

In another embodiment of the invention said fusion polypeptide comprises an extracellular growth hormone binding domain polypeptide comprising the amino acid sequence shown as SEQ ID NO 5.

In an alternative embodiment of the invention said fusion polypeptide comprises a modified extracellular growth hormone binding domain polypeptide, wherein said modification is made by addition, deletion or substitution of at least one amino acid residue, wherein said modified polypeptide substantially lacks growth hormone binding activity or has reduced growth hormone binding activity.

In a preferred embodiment of the invention, the fusion polypeptide comprises one or more modifications of an amino acid residue selected from the group consisting of: w169, R43, E44, I103, W104, I105, P106, I164 and D165 as shown in SEQ ID NO 5.

in a preferred embodiment of the invention, the fusion polypeptide comprises the deletion of the amino acid residue tryptophan 104 of the amino acid sequence shown as SEQ ID NO. 5.

In an alternative embodiment of the invention, the fusion polypeptide comprises a substitution of tryptophan 104 of the amino acid sequence shown as SEQ ID NO. 5.

In a preferred embodiment of the invention said fusion polypeptide comprises an alanine to tryptophan 104 substitution as shown in SEQ ID NO 7.

In another preferred embodiment of the invention said fusion polypeptide comprises or consists of a fragment of parathyroid hormone, said fragment comprising or consisting of amino acid residues 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34 or 1-35 of SEQ ID NO 8.

In another preferred embodiment, the fusion polypeptide comprises a parathyroid hormone or an analogue thereof comprising or consisting of an amino acid sequence as set forth in SEQ ID NO 8, 9 or 10, wherein the parathyroid hormone amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue, and wherein the modified fusion polypeptide retains parathyroid hormone activity.

In another preferred embodiment, said fusion polypeptide comprises a parathyroid hormone or an analogue thereof comprising or consisting of an amino acid sequence as set forth in SEQ ID NO 54, wherein said parathyroid hormone amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue, and wherein said modified fusion polypeptide retains parathyroid hormone activity.

In another preferred embodiment, said fusion polypeptide comprises a parathyroid hormone analogue comprising or consisting of an amino acid sequence as set forth in SEQ ID NO 37 or 40, wherein said parathyroid hormone amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue, and wherein said modified fusion polypeptide retains parathyroid hormone activity.

In another preferred embodiment, the fusion polypeptide comprises a modified amino acid sequence which is 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the full length of the amino acid sequence set forth in SEQ ID NO 8, 9 or 10.

In another preferred embodiment, the fusion polypeptide comprises a modified amino acid sequence which is 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the full length of the amino acid sequence shown as SEQ ID NO 40 or 37.

In another preferred embodiment, the fusion polypeptide comprises a modified amino acid sequence that is 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the full length of the amino acid sequence shown as SEQ ID NO: 54.

In a preferred embodiment of the invention, the fusion polypeptide comprises a parathyroid receptor domain polypeptide, wherein the parathyroid receptor domain polypeptide is at the carboxy terminus of the fusion polypeptide.

In an alternative embodiment of the invention, the fusion polypeptide comprises a parathyroid receptor domain polypeptide, wherein the parathyroid receptor domain polypeptide is located at the amino-terminus of the fusion polypeptide.

In an alternative embodiment of the invention, said fusion polypeptide comprises a growth hormone binding domain polypeptide, wherein said growth hormone binding domain polypeptide is located at the carboxy terminus of said fusion polypeptide.

in one embodiment of the invention, the fusion polypeptide comprises a growth hormone binding domain polypeptide, wherein the growth hormone binding domain polypeptide is located at the amino-terminus of the fusion polypeptide.

In a preferred embodiment of the invention said fusion polypeptide comprises or consists of the amino acid sequence as shown in SEQ ID NO 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.

In a preferred embodiment of the invention, the fusion polypeptide comprises or consists of the amino acid sequence shown as SEQ ID NO 15 or 20.

In a preferred embodiment of the invention said fusion polypeptide comprises or consists of the amino acid sequence as shown in SEQ ID NO 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.

In a preferred embodiment of the invention, the fusion polypeptide comprises a propeptide.

In another preferred embodiment of the invention said propeptide comprises or consists of SEQ ID NO 11.

In an alternative preferred embodiment of the invention said propeptide comprises or consists of SEQ ID NO 4.

In a preferred embodiment of the invention, the fusion polypeptide further comprises a peptide secretion signal.

In a preferred embodiment of the invention, the fusion polypeptide comprises a parathyroid hormone peptide secretion signal.

in a preferred embodiment of the present invention, said parathyroid hormone peptide secretion signal comprises an amino acid sequence as set forth in SEQ ID NO 1.

In a preferred embodiment of the invention, the fusion polypeptide comprises a parathyroid hormone-related protein peptide secretion signal.

In a preferred embodiment of the present invention, said parathyroid hormone-related protein peptide secretion signal comprises an amino acid sequence shown in SEQ ID NO 41.

In an alternative preferred embodiment of the invention, the fusion polypeptide comprises a growth hormone peptide secretion signal.

In a preferred embodiment of the invention, the fusion polypeptide comprises a growth hormone secretion signal comprising the amino acid sequence shown as SEQ ID NO. 2.

In a preferred embodiment of the invention, the fusion polypeptide comprises parathyroid hormone, a fragment or analogue thereof and is linked to the receptor polypeptide either directly or indirectly via a peptide linker.

In an alternative embodiment of the invention, the parathyroid hormone, fragment or analogue is directly linked to the receptor polypeptide as an in-frame translational fusion.

preferably, the peptide linker comprises amino acid sequence Gly Gly Gly Gly Ser.

In another preferred embodiment, the peptide linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units of a peptide comprising amino acid sequence Gly Gly Gly Gly Ser.

In another preferred embodiment, the peptide linker comprises 4 repeat units of amino acid sequence Gly Gly Gly Gly Ser.

According to a further aspect of the invention there is provided a nucleic acid molecule encoding a fusion polypeptide according to the invention.

According to another aspect of the invention, there is provided a vector comprising a nucleic acid molecule of the invention.

In a preferred embodiment of the invention, the vector is an expression vector suitable for expressing the nucleic acid molecule of the invention.

Vectors comprising the nucleic acids of the invention need not include promoters or other regulatory sequences, particularly where the vector is to be used to introduce the nucleic acid into a cell for recombination into the genome for stable transfection. Preferably, the nucleic acid in the vector is operably linked to a suitable promoter or other regulatory element for transcription in a host cell. The vector may be a bifunctional expression vector that functions in a variety of hosts. "promoter" refers to a nucleotide sequence upstream of the transcription initiation site that contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental promoters or other promoters for expression in eukaryotic or prokaryotic cells. "operably linked" refers to being joined as part of the same nucleic acid molecule, appropriately positioned and oriented so as to initiate transcription from a promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation of the promoter.

In a preferred embodiment, the promoter is a constitutive, inducible or regulatable promoter.

According to another aspect of the invention, there is provided a cell transfected or transformed with a nucleic acid molecule or vector of the invention.

Preferably, the cell is a eukaryotic cell.

in a preferred embodiment of the invention, the cell is selected from the group consisting of: fungal cells (e.g., Pichia spp, Saccharomyces spp, Neurospora spp); insect cells (e.g., certain species of the genus Spodoptera spp); mammalian cells (e.g., COS cells, CHO cells); a plant cell.

In an alternative embodiment of the invention, the cell is a prokaryotic cell.

According to one aspect of the invention, there is provided a method of producing a fusion polypeptide according to the invention, the method comprising the steps consisting of:

i) Providing the cells and cell culture media of the invention;

ii) culturing the cell; and

iii) isolating the fusion polypeptide according to the invention from the cell or the culture medium.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a fusion polypeptide according to the present invention, said pharmaceutical composition comprising an excipient or carrier.

In a preferred embodiment of the invention, the pharmaceutical composition is combined with another therapeutic agent.

When administered, the pharmaceutical compositions of the present invention are administered in a pharmaceutically acceptable formulation. Such formulations may typically contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents, such as chemotherapeutic agents.

The pharmaceutical compositions of the present invention may be administered by any conventional route, including injection. For example, administration and application may be subcutaneous.

The pharmaceutical compositions of the present invention are administered in an effective amount. An "effective amount" refers to the amount of drug/composition that alone or in combination with other dosages or co-drugs produces the desired response. This may involve only temporarily slowing the progression of the disease, although more preferably it involves permanently halting the progression of the disease. This can be monitored by conventional methods, but also according to diagnostic methods.

The dosage of the pharmaceutical composition to be administered to the subject may be selected according to different parameters, in particular according to the mode of administration used and the state of the subject (i.e. age, sex). When administered, the pharmaceutical compositions of the present invention are used in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. In addition, pharmaceutically acceptable salts may be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts.

The pharmaceutical composition may be combined with a pharmaceutically acceptable carrier, if desired. The term "pharmaceutically acceptable carrier" as used herein refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a human. The term "carrier" denotes a natural or synthetic organic or inorganic ingredient combined with an active ingredient for ease of use. The components of the pharmaceutical composition can also be co-mixed with the molecules of the present invention and intermixed such that there are no interactions that would significantly impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents including: acetic acid in salt; citric acid in salt; boric acid in salt; and phosphoric acid in salt. The pharmaceutical compositions may also optionally contain suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active agent with the carrier which constitutes one or more accessory ingredients. Generally, compositions are prepared by bringing into association the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for subcutaneous administration conveniently comprise a sterile aqueous or non-aqueous formulation which is preferably isotonic with the blood of the recipient. The formulations may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-parenterally acceptable non-toxic diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any stable fixed oil may be used, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Carrier formulations suitable for subcutaneous administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing co.

According to one aspect of the invention there is provided a fusion polypeptide according to the invention for use in the treatment of hypoparathyroidism in a subject.

According to another aspect of the present invention there is provided a method of treating a human subject suffering from hypoparathyroidism, said method comprising administering an effective amount of a fusion polypeptide according to the present invention, thereby treating hypoparathyroidism.

According to one aspect of the invention there is provided a fusion polypeptide according to the invention for use in the treatment of osteoporosis in a human subject.

according to another aspect of the present invention there is provided a method of treating a human subject suffering from osteoporosis, the method comprising administering an effective amount of a fusion polypeptide according to the present invention, thereby treating osteoporosis.

In a preferred embodiment or method of the invention, the fusion polypeptide comprises the amino acid sequence shown as SEQ ID NO. 40.

In a preferred embodiment or method of the invention, the fusion polypeptide is administered in combination with calcium carbonate and/or a vitamin D supplement such as calciferol or calcipotriol.

In a preferred embodiment or method of the invention, the hypoparathyroidism is caused by thyroid or cervical surgery, autoimmune disease, radiation therapy, cancer, Addison's disease, or dijorge syndrome (Di-George syndrome).

in a preferred embodiment or method of the invention, the fusion polypeptide is administered at least once a day.

In one embodiment or method of the invention, the fusion polypeptide is administered at least once or twice weekly.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Embodiments of the present invention will now be described, by way of example only, with reference to the following figures.

Brief Description of Drawings

FIG. 1: we have linked PTH (residues 1-34) to the N-terminal PTH receptor domain (PTHrExt) and Growth Hormone Binding Protein (GHBP). GHBP is an inert moiety aimed at increasing molecular weight and delaying clearance. The binding of PTH to PTHrExt protects PTH from degradation and creates an "inactive" PTH pool that balances with active PTH, resulting in a more physiologically active PTH exposure. The molecule will be expressed under the control of a PTH secretion signal (with or without a propeptide) or a growth hormone secretion signal to allow efficient processing in CHO cell lines;

FIG. 2: SDS-PAGE analysis of the purified proteins. A) Purified 14a1, B) purified 14 A2B. The protein was expressed in a CHO cell line and purified from Hyclone SFM4CHO Utility medium as a secreted product using a combination of Q-sepharose and anti-growth hormone receptor affinity chromatography. The protein was isolated at about 75-100kDa and was intact with no signs of degradation;

FIG. 3: fig. 14a1 and 14a2 proteins. Purified proteins were tested for their ability to stimulate production of cAMP by the PTH-responsive cell line UMR-106 (rat osteoblast-like cell line). Cells were stimulated for 15 min in the presence of test molecules and cAMP levels in cell lysates were measured using cAMP-specific Elisa. Positive controls, PTH1-34 (100nM), Forskolin (Forskolin) (100 μm), and negative controls (cell only, buffer only, and control protein, erythropoietin) were included in the assay. Data are expressed in pmol cAMP/ml +/-standard deviation;

FIG. 4 Parathyroid hormone, locus NM-000315834 bp mRNA linear PRI 2016, 13.6.2016, defining homo sapiens parathyroid hormone (PTH), transcript variant 1, mRNA, accession NM-000315, form NM-000315.3, keyword RefSeq, derived homo sapiens (human). a) Signal peptide underlined, propeptide in italics/lower case, mature protein (1-34) in uppercase/bold, b) origin, signal peptide underlined (116-;

FIG. 5 PTH1-34 for the fusion protein; a) amino acid sequence of PTH1-34, b) nucleotide sequence (102bp), c) amino acid sequence of PTH signal peptide and propeptide sequence for fusion protein (propeptide shown in lower case/italics), d) nucleotide sequence of PTH signal peptide and propeptide sequence for fusion protein (propeptide shown in lower case/italics);

Figure 6 human parathyroid hormone receptor 1, a) locus NM 0011847442007 bp mRNA linear PRI 2016, 10 months and 6 days, defining homo sapiens parathyroid hormone 1 receptor (PTH1R), transcript variant 2, mRNA, accession NM _001184744, form NM _001184744.1, keyword RefSeq, source homo sapiens (human), signal peptide underlined, mature extracellular domain shown in bold (D29-I187); b) the signal peptide is underlined and the mature extracellular domain is shown in bold;

FIG. 7 PTH receptor extracellular domain for fusion proteins

a) Amino acid sequence (amino acids D29-L187), b) nucleotide sequence (477bp)

FIG. 8 human Growth Hormone Binding Protein (GHBP), a) the GHBP portion of the fusion consists of amino acid residues 1-238 (extracellular domain) and includes the W104A mutation, the GHBP nucleotide sequence (714 bp); b) amino acid sequence (amino acids 1-238);

FIG. 9 GH secretion signal, a) amino acid sequence b) nucleotide sequence;

FIG. 10 PTH- (g4s)4-PTHrEx- (g4s)4-GHBP (code No. 14A 1); PTH signal peptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (amino acids 1-159): bold/lowercase, GHBP (amino acids 1-238): capitalization; connector sub-region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 11 PTH- (g4s)4-PTHrExt- (g4s)4-GHBP (code No. 14A2), PTH signal peptide and propeptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (amino acids 1-159): lower case/bold, GHBP (amino acids 1-238): upper case, connection sub-region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 12 PTH- (g4s)4-PTHrExt- (g4s)4-GHBP (code No. 14A3), GH signal peptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (amino acids 1-159): lower case/bold, GHBP (amino acids 1-238): upper case, connection sub-region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 13 PTH- (g4s)4-GHBP (code No. 14A4), GH signal peptide: lower case, PTH (amino acids 1-34): capital/underlined, GHBP (amino acids 1-238): upper case, connection sub-region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 14 PTH- (g4s)4-PTHrExt _ Hist (code No. 14A5_ Hist), GH signal peptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (amino acids 1-159): lower case/bold, linker region (g4s) 4: upper case/bold, C-terminal 6 × Hist tag: capitalization/underlining, a) nucleotide sequences, b) protein sequences;

FIG. 15 PTH- (g4s)4-PTHrExt (code No. 14A5), GH signal peptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (amino acids 1-159): lower case/bold, linker region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 16 PTH- (g4s)4-PTHrExt _ Hist (code No. 14A6_ Hist), PTH signal peptide and propeptide: lower case), PTH (amino acids 1-34): upper case/underline, PTHrExt (amino acids 1-159): lower case/bold, linker region (g4s) 4: upper case/bold, C-terminal 6 × Hist tag: capitalization/underlining; a) a nucleotide sequence, b) a protein sequence;

FIG. 17 PTH- (g4s)4-PTHrExt _ Hist (code No. 14A6), PTH signal peptide and propeptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (amino acids 1-159): lower case/bold, linker region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 18 PTH- (g4s)4-GHBP (code No. 14A7), PTH signal peptide and propeptide: lower case, PTH (amino acids 1-34): capital/underlined, GHBP (amino acids 1-238): upper case, connection sub-region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 19 PTH- (g4s)4-PTHrExt (I135K) - (g4s)4-GHBP (code No. 14A8), PTH signal peptide and propeptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (I135K) (amino acids 1-159): lower case/bold (I135K variation underlined). GHBP (amino acids 1-238): upper case, connection sub-region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 20 PTH- (g4s)4-GHBP (code No. 14A9), GH signal peptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (I135K) (amino acids 1-159): lower case/bold (I135K variation underlined), GHBP (amino acids 1-238): upper case, connection sub-region (g4s) 4: upper case/bold; a) a nucleotide sequence, b) a protein sequence;

FIG. 21 PTH- (g4s)4-PTHrExt (I135K) -Hist (code No. 14A10), PTH signal peptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (I135K) (amino acids 1-159): lower case/bold (I135K variation underlined), connecting sub-region (g4s) 4: upper case/bold, C-terminal 6 × Hist tag: capitalization/underlining; a) a nucleotide sequence, b) a protein sequence;

FIG. 22 PTH- (g4s)4-PTHrExt (I135K) -Hist (code No. 14A11-Hist), GH signal peptide: lower case, PTH (amino acids 1-34): upper case/underline, PTHrExt (I135K) (amino acids 1-159): lower case/bold (I135K variation underlined), connecting sub-region (g4s) 4: upper case/bold, C-terminal 6 × Hist tag: capitalization/underlining; a) a nucleotide sequence, b) a protein sequence;

FIG. 23 PTH sequence (amino acids 1-84);

FIG. 24(SEQ ID NO 40): PTHrP (1-36);

FIG. 25(SEQ ID NO 41): a PTHrP signal sequence;

FIG. 26(SEQ ID NO 4): a PTHrP propeptide;

FIG. 27 is a schematic view showing: a schematic model of the structure of the PTH fusion molecule is shown. 1A: PTH (purple) is linked to PTHrExt (green), which in turn is linked to GHBP (orange). The linker is shown in grey lines. W104A change is shown in blue on GHBP. An equilibrium state is assumed to exist between PTH bound to PTHrExt (inactive state: a) and PTH released and able to bind PTHR1 (active state, B). 1B: PTH (purple) is linked to GHBP (orange). The linker is shown in grey lines. W104A change is shown in blue on GHBP;

Fig. 28A and B: analysis of purified PTH fusion molecules by 10% SDS-PAGE under non-reducing conditions (Coomassie staining). FIG. 2A: lane 1: 5 μ g 14A2c lane 2: 5 μ g of 14A3 b. 14A2c (containing PTH pro-peptide) was isolated from 60-100kDa as two spreading bands. 14A3b (GHss) is resolved by a single diffusion band of about 75-100 kDa. FIG. 2B: lane 3: 6 μ g 14A7 lane 4: 3 μ g of 14A 4. Protein purity was judged to be > 90%. Purified protein was obtained from 1 liter roller bottle cultures at about 10mg 14A2c, about 4mg 14A3b, 1.42mg 14a7, and 0.29mg 14a 4. C: western blotting (Western blot) against PTH 14A2c and 14A3 b. Analysis of purified PTH fusion molecules by 10% SDS-PAGE under non-reducing conditions. Samples were transferred to PVDF membrane and probed with anti-human PTH (1-34) -specific antibody. Lanes 1-3: 14A2c with loads of 125ng, 250ng and 500 ng. Lanes 4-6: 14A3b with loads of 125ng, 250ng and 500 ng. 14A2c was more sensitive to the detection of anti-PTH antibodies. Represents 3 independent western blot experiments;

Fig. 29A: in vitro induction of cAMP. UMR-106 cells were challenged with 500nM PTH fusion or 100nM human PTH1-34 for 15 min at 37 ℃/5% CO 2. Cells were lysed and cAMP levels were measured using R & D systems parameter cAMP Elisa. Data are shown as log pmol cAMP/ml. + -. SD. cAMP levels of 14A2c were found to be 11-fold greater than 14A3 b: mean. + -. SD 264. + -.12 vs 25. + -. 0.95pmol cAMP/ml. Both fusions showed reduced biological activity compared to PTH 1-34: mean. + -. SD 2551. + -. 186pmol cAMP/ml. N-1 mean of experiments performed in duplicate. B: dual luciferase reporter assay for cAMP activity. UMR-106 cells were transfected with reporter plasmid pGL4.29/CRE/Luc2/Hygro and transfection control plasmid phRL (Renilla) and challenged with PTH fusion or human PTH1-34 at 37 ℃/5% CO2 for 5 hours. Cells were lysed and luciferase activity was measured using the Promega dual-luciferase assay kit. Data are shown as fold induction ± SD of control. 14A2c was more biologically active than 14A3b, corresponding to approximately 1.3 to 1.6 fold increase at 100nM and 500nM, respectively: mean fold induction ± SD, 16.5 ± 1.7, 63 ± 5.9 pairs 12.4 ± 1.0, 40.3 ± 3.1. Average of the cubic values. Both fusions showed reduced biological activity compared to the equivalent concentration of 100nM PTH 1-34: mean. + -. SD, 67.3. + -. 1.6. Three separate experiments are represented. C: dual luciferase reporter assay to compare cAMP activity of all 4 PTH fusion molecules. UMR-106 cells were transfected with reporter plasmid pGL4.29/CRE/Luc2/Hygro and transfection control plasmid phRL (Renilla) and challenged with PTH fusion or human PTH1-34 at 37 ℃/5% CO2 for 5 hours. Cells were lysed and luciferase activity was measured using the Promega dual-luciferase assay kit. Data are shown as fold induction ± SD of control. The activity pattern was similar to that previously determined for 14A2c and 14A3b for PTH. Both PTH-GHBP fusion molecules (14A4, 49 + -4.17 and 14A7, 44 + -0.42) were more active at 100nM than PTH-PTHrExt-GHBP fusion molecules (14A2c, 15 + -0.09 and 14A3b, 7.7 + -3.13). At 100nM, 14A4 and 14A7 both had comparable PTH activity (54. + -. 2.7). D: EC50 values for PTH and PTH fusions were determined using a dual luciferase reporter assay. PTH had an EC50 of 32 ± 10.67nM (n ═ 4 experiments), potency was about 18 times that of 14A2c (EC50 ═ 579 ± 138nM, n ═ 3 experiments), potency was 28 times that of 14A3b (EC50 ═ 896nM, n ═ 1), and potency was 4.7 times that of 14a7(EC50 ═ 153nM, n ═ 1 experiments).

Detailed Description

Code (SEQID)	Description of the molecules
		14A1(12)	PTHss-PTH-(g4s)4-PTHrExt-(g4s)4-GHBP
14A2(13)	PTHss-pp-PTH-(g4s)4-PTHrExt-(g4s)4-GHBP
		14A3(14)	GHss-PTH-(g4s)4-PTHrExt-(g4s)4-GHBP
14A4(15)	GHss-PTH-(g4s)4-GHBP
		14A5(16)	GHss-PTH-(g4s)4-PTHrExt
14A5_Hist(17)	GHss-PTH-(g4s)4-PTHrExt-Hist
		14A6_Hist(18)	PTHss-pp-PTH-(g4s)4-PTHrExt-Hist
14A6(19)	PTHss-pp-PTH-(g4s)4-PTHrExt
		14A7(20)	PTHss-pp-PTH-(g4s)4-GHBP
14A8(21)	PTHss-pp-PTH-(g4s)4-PTHrExt(I135K)-(g4s)4-GHBP
		14A9(22)	GHss-PTH-(g4s)4-PTHrExt(I135K)-(g4s)4-GHBP
14A10_Hist(23)	PTHss-pp-PTH-(g4s)4-PTHrExt(I135K)-Hist
		14A11_Hist(24)	GHss-pp-PTH-(g4s)4-PTHrExt(I135K)-Hist
14A12(25)	PTHss-pp-LA：PTH-(g4s)4-PTHrExt-(g4s)4-GHBP
		14A13(26)	GHss-LA：PTH-(g4s)4-PTHrExt-(g4s)4-GHBP
14A15(27)	PTHss-pp-LA：PTH-(g4s)4-GHBP
		14A16(28)	GHss-LA：PTH-(g4s)4-GHBP
14A17(29)	PTHss-pp-LA：PTH-(g4s)4-PTHrExt-Hist
		14A18(30)	GHss-LA：PTH-(g4s)4-PTHrExt-Hist
14A19(31)	PTHss-pp-PTH(1-84)-(g4s)4-PTHrExt-(g4s)4-GHBP
		14A20(32)	GHss-PTH(1-84)-(g4s)4-PTHrExt-(g4s)4-GHBP
14A21(33)	PTHss-pp-PTH(1-84)-(g4s)4-GHBP
		14A22(34)	GHss-PTH(1-84)-(g4s)4-GHBP
14A23(35)	PTHss-pp-PTH(1-84)-(g4s)4-PTHrExt-Hist
		14A24(36)	GHss-PTH(1-84)-(g4s)4-PTHrExt-Hist

TABLE 1

pths ═ parathyroid hormone secretion signal; pp ═ propeptide; GHss ═ growth hormone secretion signal; PTH ═ (amino acids 1-34 or 1-84 or fragments thereof); PTHrExt ═ PTH receptor extracellular domain; GHBP ═ growth hormone binding protein (amino acids 1-238); (g4s)4 ═ 4 repeats of the amino acid GGGGS; hist mark is hhhhhhhh; isoleucine-135 mutation to lysine in I135K ═ PTHrExt; LA: PTH ═ long acting PTH [ fusion of PTH 1-14 with PTHrP as described in the text ].

The linker region in the above fusions is composed of multiple GGGGS. In the example given in table 1, the linker region consists of 4 x GGGGS, but variable polyploid forms may be used.

The I135K changes present in PTHrExt in selected constructs have been shown to reduce PTH binding to the receptor. Other amino acid changes may also be used in combination or as single point mutations (such as D137A, P132L, and L187A). (SEQ ID NO: 56).

The numbering of PTHrExt in the following sequence refers to the mature protein processed at alanine 28, so that D29-I187 is referred to as amino acids 1-159 in the following sequence (SEQ ID NO: 3).

All of the above PTH sequences can be replaced with PTH 1-84 and its variables, if desired.

Table 2: list of all proposed PTHrP fusion constructs (SEQ ID 42-53)

	Description of the molecules
		14A25(42)	PTHrPss-pp-PTHrP-(g4s)4-PTHrExt-(g4s)4-GHBP
14A26(43)	GHss-PTHrP-(g4s)4-PTHrExt-(g4s)4-GHBP
		14A27(44)	GHss-PTHrP-(g4s)4-GHBP
14A28(45)	GHss-PTHrP-(g4s)4-PTHrExt
		14A29_Hist(46)	GHss-PTHrP-(g4s)4-PTHrExt-Hist
14A30_Hist(47)	PTHrPss-pp-PTHrP-(g4s)4-PTHrExt-Hist
		14A31(48)	PTHrPss-pp-PTHrP-(g4s)4-PTHrExt
14A32(49)	PTHrPss-pp-PTHrP-(g4s)4-GHBP
		14A33(50)	PTHrPss-pp-PTHrP-(g4s)4-PTHrExt(I135K)-(g4s)4-GHBP
14A34(51)	GHss-PTHrP-(g4s)4-PTHrExt(I135K)-(g4s)4-GHBP
		14A35_Hist(52)	PTHrPss-pp-PTHrP-(g4s)4-PTHrExt(I135K)-Hist
14A36_Hist(53)	GHss-pp-PTHrP-(g4s)4-PTHrExt(I135K)-Hist

PTHrPss ═ parathyroid hormone-related protein secretion signal; pp ═ propeptide; GHss ═ growth hormone secretion signal; PTHrP ═ (amino acids 1 to 36 or fragments thereof); PTHrExt ═ PTH receptor extracellular domain; GHBP ═ growth hormone binding protein (amino acids 1-238); (g4s)4 ═ 4 repeats of the amino acid GGGGS; hist mark is hhhhhhhh; isoleucine-135 mutation to lysine in I135K-PTHrExt

TABLE 3 summary of SEQ ID numbers

Materials and methods

Construction of PTH fusions: the molecules were constructed by a combination of gene synthesis (Eurofin MWG) and standard DNA manipulation techniques. The recombinant gene encoding the full-length PTH fusion was cloned into the modified mammalian expression plasmid PSecTag/FRT/V5/Hist-TOPO (Invitrogen). Stable cell lines were generated in CHO Flp-ln cell line (Invitrogen) and adapted to serum-free medium in Hyclone SFM4CHO Utility (thermo scientific) according to the manufacturer's instructions. PTH fusions are under secretory expression of PTH or GH signal peptides.

Expression and purification: cells were maintained in Hyclone SFM4CHO Utility media in roller bottle culture, passaged every 2-3 days, maintaining cell densities between 0.25X 106 viable cells/ml (VCPM) and 1.5X 106 VCPM. For expression studies, roller bottles were inoculated at 0.5X 106VCPM, grown at 37 deg.C under 5% CO2, and brought to 1X 106 VPCM. Valproic acid was added to a final concentration of 2mM and the temperature was lowered to 31 ℃. Cells were grown for up to 8-10 days with viability still around 70%, at which time the harvest was clarified by centrifugation at 22,000 Xg for 20 minutes at 4 ℃ using Beckman JLA 16-25 rotors. EDTA and benzamidine hydrochloride were added to final concentrations of 5mM and 10mM, respectively, and the medium was concentrated using a Vivaflow 200 tangential flow concentrator and stored frozen at-20 ℃. The target protein was purified from this concentrate by anion exchange (Q-sepharose FF, GE Healthcare) and affinity chromatography (anti-GHBP antibody column). Protein concentration was measured by the Bradford protein assay and samples were analyzed by SDS-PAGE under non-reducing conditions and either stained with coomassie blue or protein blotted with a commercially available anti-PTH 1-34 antibody (Abeam 14493) or an internally developed anti-GHBP antibody. The purified samples were aliquoted and stored at-80 ℃.

in vitro biological activity: purified proteins were tested for their ability to stimulate production of cAMP by the PTH-responsive cell line UMR-106 (rat osteoblast-like cell line). Cells were stimulated for 15 minutes in the presence of test molecules and cAMP levels in cell lysates were measured using cAMP-specific Elisa (R & D systems).

Animal model of hypoparathyroidism

Shimizu et al used an animal model of hypoparathyroidism in the study of LA-PT. In this model, rats were subjected to Thyroid Parathyroidectomy (TPTX) prior to treatment. Briefly: surgical TPTX was performed on 6 week old rats obtained from Charles River Laboratories Japan, Inc. After surgery, each cage was supplied with pellet feed (CE-2; CLEA Japan, Inc., Tokyo, Japan) containing 1.10% calcium and 1.09% phosphate moistened with tap water for easy access and digestion in sham surgery and TPTX rats. From the next day, post-operative rats exhibiting sCa below 8.0mg/dL 5 days post-TPTX surgery were selected for subsequent peptide injection studies.

Example 1

According to crystal structure analysis of PTH using N-domain PTH receptor [1], PTH is shown to be located in the groove formed by the N-terminal receptor moiety. It is this mode of interaction that is hypothesized to protect PTH from degradation and to create an "inactive pool" of PTH, thereby prolonging the bioactivity of said PTH to produce long-acting PTH. It has been proposed that a novel molecule (see FIG. 1) would be a fusion between PTH (residues 1-34), the N-terminal PTH receptor domain (PTHrExt, most commonly residues D29-L187, but not limited to other combinations) and growth hormone binding protein (GHBP, residues 1-238). GHBP is an inert moiety designed to increase Mw and thus delay scavenging. It will contain the W104A mutation to prevent interaction with GH in the circulation.