PARATHYROID HORMONE FUSION POLYPEPTIDE

Abstract

The disclosure relates to long acting parathyroid or parathyroid hormone like fusion polypeptides comprising a receptor polypeptide and its use in the treatment of hypoparathyroidism and osteoporosis.

Claims

1. A fusion polypeptide comprising a polypeptide comprising an amino acid sequence of a parathyroid hormone or biologically active fragment or analogue thereof, a polypeptide comprising an amino acid sequence of a growth hormone binding domain of growth hormone receptor or fragment or analogue thereof, wherein said parathyroid hormone or biologically active fragment or analogue thereof is linked either directly or indirectly as a translational fusion to said growth hormone binding domain of growth hormone receptor.

2. The fusion polypeptide according to claim 1, wherein said fusion polypeptide comprises a parathyroid hormone comprising or consisting of: the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 9; or a fragment of SEQ ID NO: 10 including amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35 of SEQ ID NO: 10, or a fragment of SEQ ID NO: 9 including amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35 of SEQ ID NO: 9.

3. The fusion polypeptide according to claim 2 wherein said fusion polypeptide comprises a parathyroid hormone comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 54.

4. The fusion polypeptide according to claim 1 wherein said fusion polypeptide comprises a parathyroid hormone analogue comprising or consisting of the amino acid sequence of formula (I): TABLE-US-00005 (SEQ ID NO: 8) 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 (I) where 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 including amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35 of formula (I), with the proviso that at least one of Xaa18 is not Leu or Met, Xaa22 is not Phe, and Xaa26 is not His.

5. The fusion polypeptide according to claim 1 wherein said fusion polypeptide comprises: a parathyroid hormone and a parathyroid hormone analogue amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 37; or a parathyroid hormone related protein or biologically active fragment or analogue thereof, comprising the amino acid sequence set forth in SEQ ID NO: 40.

6. The fusion polypeptide according to claim 1 wherein said fusion polypeptide comprises: the extracellular growth hormone binding domain polypeptide of human growth hormone receptor; or an extracellular growth hormone binding domain polypeptide comprising of the amino acid sequence set forth in SEQ ID NO: 5.

7. The fusion polypeptide according to claim 6 wherein said fusion polypeptide comprises: a modified extracellular growth hormone binding domain polypeptide wherein said modification is 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; a modification of one or more of the amino acid residues selected from the group consisting of: W169, R43, E44, I103, W104, I105, P106, I164 and D165 as set forth in SEQ ID NO: 5; a deletion or substitution of amino acid residue tryptophan 104 of the amino acid sequence set forth in SEQ ID NO: 5; or a substitution of tryptophan 104 for alanine as set forth in SEQ ID NO: 5.

8. The fusion polypeptide according to claim 1 wherein said fusion polypeptide comprises or consists of a parathyroid hormone fragment comprising or consisting of the 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.

9. The fusion polypeptide according to claim 8 wherein said fusion polypeptide comprises or consists of the amino acid sequence 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.

10. The fusion polypeptide according to claim 1 wherein said fusion polypeptide comprises a growth hormone binding domain polypeptide wherein said growth hormone binding domain polypeptide is located at the carboxyl terminal end or amino terminal end of said fusion polypeptide.

11. The fusion polypeptide according to claim 1 wherein said fusion polypeptide comprises a pro-peptide and/or a peptide secretion signal.

12. The fusion polypeptide according to claim 1 wherein said fusion polypeptide comprises a parathyroid hormone, fragment or analogue thereof and is linked directly or indirectly to said growth hormone binding domain polypeptide of growth hormone receptor by a peptide linker and/or is directly linked to said receptor polypeptide as an in-frame translational fusion.

13. A nucleic acid molecule that encodes a fusion polypeptide according to claim 1.

14. A vector comprising a nucleic acid molecule according to claim 13.

15. An isolated cell transfected or transformed with a nucleic acid molecule according to claim 13 or a vector comprising the nucleic acid molecule.

16. A method for the production of a fusion polypeptide comprising the steps consisting of: i) providing a cell transfected or transformed with a nucleic acid molecule encoding the fusion polypeptide of claim 1 or a vector comprising the nucleic acid molecule and cell culture medium; ii) culturing said cell; and iii) isolating the fusion polypeptide from said cell or medium.

17. A pharmaceutical composition comprising a fusion polypeptide according to claim 1 including an excipient or carrier.

18. A method of treating hypoparathyroidism in a subject comprising administering the fusion polypeptide according to claim 1 to the subject.

19. A method of treating osteoporosis in a human subject comprising administering the fusion polypeptide according to claim 1 to the human subject.

20. The method of claim 19 wherein said fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

21. The method of claim 19 wherein said fusion polypeptide is administered to the subject in combination with calcium carbonate and/or vitamin D supplements such as calcitrol or alfacalcitrol.

22. The fusion polypeptide according to claim 1, wherein said fusion polypeptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 20, 27, 28, 33 or 34.

23. The fusion polypeptide according to claim 1, wherein said fusion polypeptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 20.

24. The fusion polypeptide according to claim 1, wherein said fusion polypeptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 27.

Description

BRIEF SUMMARY OF THE DRAWINGS

[0103] FIG. 1: We have linked PTH (residues 1-34) to the N-terminal PTH receptor domain (PTHrExt) and the growth hormone binding protein (GHBP). GHBP is an inert moiety designed to increase molecular weight and delay clearance. The binding of PTH to PTHrExt protects PTH from degradation and produces a pool of “inactive” PTH that is in equilibrium with active PTH generating a more physiological PTH exposure. Molecules will be expressed under the control of either the PTH secretion signal (with or without the inclusion of the propeptide) or the growth hormone secretion signal to allow for efficient processing in a CHO cell line;

[0104] FIGS. 2A and 2B: SDS-PAGE Analysis of Purified Protein. FIG. 2A) Purified 14A1, FIG. 2B) Purified 14A2b. Proteins were expressed in a CHO cell line and purified as a secreted product from Hyclone SFM4CHO Utility media using a combination of Q-Sepharose and anti-growth hormone receptor affinity chromatography. Protein separates at ˜75-100 kDa and is intact with no sign of degradation;

[0105] FIG. 3: In Vitro Bioactivity of 14A1 and 14A2 proteins. Purified proteins were tested for their ability to stimulate cAMP production from the PTH responsive cell line, UMR-106 (rat osteoblastic like cell line). Cells were stimulated for 15 minutes in the presence of test molecules and cAMP levels measured from cell lysates using a cAMP specific Elisa. Both positive controls, PTH 1-34 (100 μM), Forskolin (100 μM) and negative controls (Cells only, Buffer only and Control protein, Erythropoetin) were included in the analysis. Data is presented as pmol cAMP/ml+/−Standard deviation;

[0106] FIGS. 4A and 4B: Parathyroid Hormone, LOCUS NM_000315 834 bp mRNA linear PRI 13 Jun. 2016, DEFINITION Homo sapiens parathyroid hormone (PTH), transcript variant 1, mRNA., ACCESSION NM_000315, VERSION NM_000315.3, KEYWORDS RefSeq., SOURCE Homo sapiens (human). FIG. 4A) Signal peptide underlined, propeptide italics/lowercase, mature protein (1-34) shown in uppercase/bold, FIG. 4B) origin, Signal peptide is underlined (116-190 bp), Propeptide in lowercase/italics, Mature protein in uppercase/bold (209-460 bp);

[0107] FIGS. 5A-5D: PTH 1-34 used in fusion proteins; FIG. 5A) Amino acid sequence of PTH 1-34, FIG. 5B) nucleotide sequence (102 bp), FIG. 5C) Amino acid sequence of PTH signal peptide and propeptide sequence used in fusion proteins (propeptide in lowercase/italics), FIG. 5D) Nucleotide sequence of PTH signal peptide and propeptide sequence used in fusion proteins (propeptide in lowercase/italics);

[0108] FIGS. 6A and 6B: Human parathyroid hormone receptor 1, FIG. 6A) LOCUS NM_001184744 2007 bp mRNA linear PRI 6 Oct. 2016, DEFINITION Homo sapiens parathyroid hormone 1 receptor (PTH1R), transcript variant 2, mRNA. ACCESSION NM_001184744 VERSION NM_001184744.1, KEYWORDS RefSeq., SOURCE Homo sapiens (human), Signal peptide underlined, Mature extracellular domain in bold (D29-I187); FIG. 6B) Signal peptide underlined, Mature extracellular domain in bold;

[0109] FIGS. 7A and 7B: PTH receptor extracellular domain used in fusion proteins. FIG. 7A) Amino acid sequence (aa D29-L187), FIG. 7B) Nucleotide sequence (477 bp);

[0110] FIGS. 8A and 8B: Human growth hormone binding protein (GHBP), FIG. 8A) GHBP portion of the fusion is composed of amino acid residues 1-238 (extracellular domain) and includes a W104A mutation, Nucleotide sequence of GHBP (714 bp); FIG. 8B) Amino acid sequence (aa 1-238);

[0111] FIGS. 9A and 9B: GH Secretion signal, FIG. 9A) amino acid sequence, FIG. 9B) nucleotide sequence;

[0112] FIGS. 10A and 10B: PTH-(g4s)4-PTHrEx-(g4s)4-GHBP (Code #14A1); PTH Signal peptide: Lowercase, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (aa 1-159): bold/Lowercase, GHBP (aa 1-238): Uppercase; Linker regions (g4s)4: Uppercase/Bold; FIG. 10A) nucleotide sequence, FIG. 10B) protein sequence;

[0113] FIGS. 11A and 11B: PTH-(g4s)4-PTHrExt-(g4s)4-GHBP (Code #14A2), PTH Signal peptide and propeptide: Lowercase, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (aa 1-159): Lowercase/Bold, GHBP (aa 1-238): Uppercase, Linker regions (g4s)4: Uppercase/Bold; FIG. 11A) nucleotide sequence, FIG. 11B) protein sequence;

[0114] FIGS. 12A and 12 B: PTH-(g4s)4-PTHrExt-(g4s)4-GHBP (Code #14A3), GH Signal peptide: Lowercase, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (aa 1-159): Lowercase/Bold, GHBP (aa 1-238): Uppercase, Linker regions (g4s)4: Uppercase/Bold; FIG. 12A) nucleotide sequence, FIG. 12B) protein sequence;

[0115] FIGS. 13A and 13B: PTH-(g4s)4-GHBP (Code #14A4), GH Signal peptide: Lower case, PTH (aa 1-34): Uppercase/Underlined, GHBP (aa 1-238): Uppercase, Linker region (g4s)4: Uppercase/Bold; FIG. 13A) nucleotide sequence, FIG. 13B) protein sequence;

[0116] FIGS. 14A and 14B: PTH-(g4s)4-PTHrExt_Hist (Code #14A5_Hist), GH Signal peptide: Lower case, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (aa 1-159): Lowercase/Bold, Linker region (g4s)4: Uppercase/Bold, C-terminal 6× Hist tag: Uppercase/Underlined, FIG. 14A) nucleotide sequence, FIG. 14B) protein sequence;

[0117] FIGS. 15A and 15B: PTH-(g4s)4-PTHrExt (Code #14A5), GH Signal peptide: Lowercase, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (aa 1-159): Lowercase/Bold, Linker region (g4s)4: Uppercase/Bold; FIG. 15A) nucleotide sequence, FIG. 15B) protein sequence;

[0118] FIGS. 16A and 16B: PTH-(g4s)4-PTHrExt_Hist (Code #14A6_Hist), PTH Signal peptide & propeptide: Lowercase), PTH (aa 1-34): Uppercase/Underlined, PTHrExt (aa 1-159): Lowercase/Bold, Linker regions (g4s)4: Uppercase/Bold, C-terminal 6× Hist tag: Uppercase/Underlined; FIG. 16A) nucleotide sequence, FIG. 16B) protein sequence;

[0119] FIGS. 17A and 17B: PTH-(g4s)4-PTHrExt_Hist (Code #14A6), PTH Signal peptide & propeptide: Lower case, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (aa 1-159): Lowercase/Bold, Linker regions (g4s)4: Uppercase/Bold; FIG. 17A) nucleotide sequence, FIG. 17B) protein sequence;

[0120] FIGS. 18A and 18B: PTH-(g4s)4-GHBP (Code #14A7), PTH Signal peptide & propeptide: Lowercase, PTH (aa 1-34): Uppercase/Underlined, GHBP (aa 1-238): Uppercase, Linker region (g4s)4: Uppercase/Bold; FIG. 18A) nucleotide sequence, FIG. 18B) protein sequence;

[0121] FIGS. 19A and 19B: PTH-(g4s)4-PTHrExt (I135K)-(g4s)4-GHBP (Code #14A8), PTH Signal peptide & propeptide: Lowercase, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (I135K) (aa 1-159): Lowercase/Bold (I135K change underlined). GHBP (aa 1-238): Uppercase, Linker regions (g4s)4: Uppercase/Bold; FIG. 19A) nucleotide sequence, FIG. 19B) protein sequence;

[0122] FIGS. 20A and 20B: PTH-(g4s)4-GHBP (Code #14A9), GH Signal peptide: Lower case, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (I135K) (aa 1-159): Lowercase/Bold (I135K change underlined), GHBP (aa 1-238): Uppercase, Linker regions (g4s)4: Uppercase/Bold; FIG. 20A) nucleotide sequence, FIG. 20B) protein sequence;

[0123] FIGS. 21A and 21B: PTH-(g4s)4-PTHrExt (I135K)-Hist (Code #14A10), PTH Signal peptide: Lowercase, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (I135K) (aa 1-159): Lowercase/Bold (I135K change underlined), Linker region (g4s)4: Uppercase/Bold, C-terminal 6× Hist tag: Uppercase/Underlined; FIG. 21A) nucleotide sequence, FIG. 21B) protein sequence;

[0124] FIGS. 22A and 22B: PTH-(g4s)4-PTHrExt (I135K)-Hist (Code #14A11-Hist), GH Signal peptide: Lowercase, PTH (aa 1-34): Uppercase/Underlined, PTHrExt (I135K) (aa 1-159): Lowercase/Bold (I135K change underlined), Linker region (g4s)4: Uppercase/Bold, C-terminal 6× Hist tag: Uppercase/Underlined; FIG. 22A) nucleotide sequence, FIG. 22B) protein sequence;

[0125] FIG. 23 PTH Sequence (AA 1-84);

[0126] FIG. 24 (SEQ ID NO: 40): PTHrP (1-36);

[0127] FIG. 25 (SEQ ID NO: 41): PTHrP signal sequence;

[0128] FIG. 26 (SEQ ID NO: 4): PTHrP propeptide;

[0129] FIGS. 27A and 27B: Schematic models showing the structure of the PTH fusion molecules. FIG. 27A: PTH (Purple) linked to PTHrExt (Green) which in turn is linked to GHBP (Orange). Linkers shown as grey lines. W104A change shown in blue on GHBP. It is hypothesised that a state of equilibrium exists between PTH bound to PTHrExt (Inactive State: A) and PTH being released and able to bind PTHR1 (Active State, B). FIG. 27B: PTH (Purple) linked to GHBP (Orange). Linker shown as grey line. W104A change shown in blue on GHBP;

[0130] FIGS. 28A, 28B, and 29C: Analysis of purified PTH fusion molecules by 10% SDS-PAGE under non-reducing conditions (Coomassie Stained). FIG. 28A: Lane 1: 5 μg 14A2c Lane 2: 5 μg 14A3b. 14A2c (contains PTH prepropeptide) separates as two diffuse bands from 60-100 kDa. 14A3b (GHss) resolves as a single diffuse band ˜75-100 kDa. FIG. 28B: Lane 3: 6 μg 14A7 Lane 4: 3 μg 14A4. Proteins judged to be >90% pure. Obtained ˜10 mg 14A2c, ˜4 mg 14A3b, 1.42 mg 14A7 and 0.29 mg 14A4 purified protein from a 1 litre roller bottle culture.

[0131] FIG. 28C: Western blot anti PTH 14A2c & 14A3b. Analysis of purified PTH fusion molecules by 10% SDS-PAGE under non-reducing conditions. Samples were transferred to a PVDF membrane and probed with anti-human PTH (1-34) specific antibody. Lanes 1-3: 14A2c at a loading of 125 ng, 250 ng and 500 ng. Lanes 4-6: 14A3b at a loading of 125 ng, 250 ng and 500 ng. 14A2c is more sensitive to detection by anti-PTH antibody. Representative of 3 independent western blot experiments;

[0132] FIG. 29A: In vitro induction of cAMP. UMR-106 cells were challenged with either 500 nM PTH fusion or 100 nM human PTH 1-34 for 15 minutes at 3TC/5% CO.sub.2. Cells were lysed and cAMP levels measured using an R & D systems Parameter cAMP Elisa. Data are presented as log pmol cAMP/ml ±SD. An 11-fold increase in cAMP levels was found for 14A2c over 14A3b: mean±SD 264±12 vs 25±0.95 pmol cAMP/ml. Both Fusions showed reduced biological activity when compared to PTH 1-34: mean±SD 2551±186 pmol cAMP/ml. Average of n=1 experiment carried out in duplicate.

[0133] FIG. 29B: Dual Luciferase Reporter Assay for cAMP Activity. UMR-106 cells were transfected with the reporter plasmid pGL4.29/CRE/Luc2/Hygro and transfection control plasmid phRL (Renilla) and challenged with either PTH fusion or human PTH 1-34 for 5 hours at 3TC/5% CO.sub.2. Cells were lysed and Luciferase activity measured using the Promega Dual Luciferase Assay kit. Data are presented as fold induction of control ±SD. 14A2c is more bioactive than 14A3b equating to a ˜1.3 to 1.6 fold increase at 100 & 500 nM respectively: mean fold induction±SD, 16.5±1.7, 63±5.9 vs 12.4±1.0, 40.3±3.1. Average of triplicate values. Both Fusions showed reduced biological activity when compared to PTH 1-34 at an equivalent concentration of 100 nM: mean±SD, 67.3±1.6. Representative of 3 separate experiments.

[0134] FIG. 29C: Dual Luciferase Reporter Assay for cAMP Activity Comparison of all 4 PTH fusion molecules. UMR-106 cells were transfected with the reporter plasmid pGL4.29/CRE/Luc2/Hygro and transfection control plasmid phRL (Renilla) and challenged with either PTH fusion or human PTH 1-34 for 5 hours at 37° C./5% CO.sub.2. Cells were lysed and Luciferase activity measured using the Promega Dual Luciferase Assay kit. Data are presented as fold induction of control ±SD. Pattern of activity is similar to previous assays for 14A2c & 14A3b vs PTH. Both PTH-GHBP fusion molecules (14A4, 49±4.17 & 14A7, 44±0.42) are more active than the PTH-PTHrExt-GHBP fusion molecules (14A2c, 15±0.09 & 14A3b, 7.7±3.13) at 100 nM. At 100 nM both 14A4 and 14A7 have comparable activity to PTH (54±2.7).

[0135] FIG. 29D: Determination of EC.sub.50 values for PTH & PTH fusions using the Dual Luciferase Reporter Assay. PTH has an EC.sub.50 of 32±10.67 nM (n=4 experiments) and is ˜18-fold more potent than 14A2c (EC.sub.50=579±138 nM, n=3 experiments), 28-fold more potent than 14A3b (EC.sub.50=896 nM, n=1), and 4.7-fold more potent than 14A7 (EC.sub.50=153 nM, n=1 experiment).

SEQUENCE LISTING

[0136] The Sequence Listing is submitted as an ASCII text file in the form of the file name “Sequence.txt,” 163,840 bytes, created on Mar. 18, 2022, which is incorporated by reference herein.

TABLE-US-00002 TABLE 1 Code (SEQ ID) Molecule Description 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 PTHss = Parathyroid Hormone secretion signal; pp = proppeptide; GHss = Growth hormone secretion signal; PTH = (aa 1-34 or 1-84 as stated or fragments thereof); PTHrExt = PTH receptor extracellular domain; GHBP = Growth hormone binding protein (aa 1-238); (g4s)4 = 4 repeats of amino acids GGGGS (residues 66-85 of SEQ ID NO: 20); Hist tagged = HHHHHH (residues 242-247 of SEQ ID NO: 17); I135K = mutation of Isoleucine-135 to lysine in PTHrExt:LA:PTH = long acting PTH [fusion of PTH 1-14 with PTHrP as described in text].

[0137] Linker regions in the above fusions are composed of multiples of GGGGS. In the examples given in Table 1 the linker regions are composed of 4×GGGGS (residues 66-85 of SEQ ID NO: 20), but variable multiples can be used.

[0138] The I135K change present in PTHrExt in selected constructs has been shown to reduce the binding of PTH for the receptor. Other amino acid changes can also be used in combination or as single point mutations such as D137A, P132L & L187A. (SEQ ID NO: 56).

[0139] The numbering for the PTHrExt in the sequences below refer to the mature protein processed at Alanine 28 and therefore D29-I187 is thus referred to as amino acids 1-159 (SEQ ID NO: 3) in the following sequences.

[0140] All of above PTH sequences can be replaced with PTH 1-84 if desired and variables thereof.

TABLE-US-00003 TABLE 2 List of all Proposed PTHrP Fusion Constructs (SEQ ID 42-53) Molecule Description 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-(g45)4-GHBP 14A28 (45) GHss-PTHrP-(g45)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-(g45)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

[0141] PTHrPss=Parathyroid Hormone related protein secretion signal; pp=proppeptide; GHss=Growth hormone secretion signal; PTHrP=(aa 1-36 or fragments thereof); PTHrExt=PTH receptor extracellular domain; GHBP=Growth hormone binding protein (aa 1-238); (g4s)4=4 repeats of amino acids GGGGS (residues 66-85 of SEQ ID NO: 20); Hist tagged=HHHHHH (residues 242-247 of SEQ ID NO: 17); I135K=mutation of Isoleucine-135 to lysine in PTHrExt

TABLE-US-00004 TABLE 3 SEQ ID NUMBER SUMMARY SEQ ID NO Name 1 PTH Signal Peptide: MIPAKDMAKVMIVMLAICFLTKSDG 2 GH Secretion signal: MATGSRTSLLLAFGLLCLPWLQEGSA 3 PTH receptor ECD (1-159): DDVMTKEEQIFLLHRAQAQCEKRLKEVLQRPASIMESDKGWTSASTSGKPRK DKASGKLYPESEEDKEAPTGSRYRGRPCLPEWDHILCWPLGAPGEVVAVPCP DYIYDFNHKGHAYRRCDRNGSWELVPGHNRTWANYSECVKFLTNETREREV FDRL 4 PTHrP propeptide: rsveglsrrl 5 GH receptor ECD (1-238): FSGSEATAAILSRAPWSLQSVNPGLKTNSSKEPKFTKCRSPERETFSCHWTD EVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIW IPYCIKLTSNGGTVDEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEA PRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVR VRSKQRNSGNYGEFSEVLYVTLPQMSQ 7 GH ECD substitution: w104a substitution FSGSEATAAILSRAPWSLQSVNPGLKTNSSKEPKFTKCRSPERETFSCHWTD EVHHGTKNLGPIQLFYIRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIAI PYCIKLTSNGGTVDEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEA PRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVR VRSKQRNSGNYGEFSEVLYVTLPQMSQ 8 PTH formula (I): general AA formula (1-36) 9 PTH 1-84: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPR KKEDNVLVESHEKSLGEADKADVNVLTKAKSQ 10 PTH defined formula (I): Ala-Val-Ala-Glu-Ile-Gln-Leu-Met-His-Gln-Arg-Ala-Lys- Trp-Ile-Gln-Asp-Ala-Arg-Arg-Arg-Ala-Phe-Leu-His-Lys-Leu-Ile-Ala-Glu-Ile-His- Thr-Ala-Glu-Ile, or a fragment thereof including amino acids 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, or 1-35 of said sequence. (1-36) 11 PTH Propeptide: ksvkkr 12-36 Constructs of table 1 37 PTH short (LA PTH): LA-PTH ([Ala1,3,12,Gln10,Arg11,Trp14]PTH(1- 14)/[Ala18,22,Lys26]PTHrP(15-36)COOH) 40 PTHrP (1-36): AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEI 41 PTHrP signal sequence: MQRRLVQQWSVAVFLLSYAVPSCG 42-53 Constructs of table 2 54 PTH 1-34: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF 55 PTH ECD Signal peptide: MGTARIAPGLALLLCCPVLSSAYALVDA 56 PTH receptor full length: MGTARIAPGLALLLCCPVLSSAYALVDADDVMTKEEQIFLLHRAQAQCEKRLKE VLQRPASIMESDKGWTSASTSGKPRKDKASGKLYPESEEDKEAPTGSRYRGR PCLPEWDHILCWPLGAPGEVVAVPCPDYIYDFNHKGHAYRRCDRNGSWELV PGHNRTWANYSECVKFLTNETREREVFDRLGMIYTVGYSVSLASLTVAVLILAY FRRLHCTRNYIHMHLFLSFMLRAVSIFVKDAVLYSGATLDEAERLTEEELRAIAQ APPPPATAAAGYAGCRVAVTFFLYFLATNYYWILVEGLYLHSLIFMAFFSEKKY LWGFTVFGWGLPAVFVAVWVSVRATLANTGCWDLSSGNKKWIIQVPILASIVL NFILFINIVRVLATKLRETNAGRCDTRQQYRKLLKSTLVLMPLFGVHYIVFMATP YTEVSGTLWQVQMHYEMLFNSFQGFFVAIIYCFCNGEVQAEIKKSWSRWTLA LDFKRKARSGSSSYSYGPMVSHTSVTNVGPRVGLGLPLSPRLLPTATTNGHP QLPGHAKPGTPALETLETTPPAMAAPKDDGFLNGSCSGLDEEASGPERPPAL LQEEWETVM 57 Fusion of SEQ ID NO: 1, 11 and 9 58 DNA sequence encoding SEQ ID NO: 57 59 DNA sequence encoding SEQ ID NO: 59 60 Fusion of SEQ ID NO: 1 and 11 61 DNA sequence encoding SEQ ID NO: 60 62 DNA sequence encoding SEQ ID NO: 56 63 DNA sequence encoding SEQ ID NO: 3 64 DNA sequence encoding SEQ ID NO: 7 65 DNA sequence encoding SEQ ID NO: 2 66 DNA sequence encoding SEQ ID NO: 12 67 DNA sequence encoding SEQ ID NO: 13 68 DNA sequence encoding SEQ ID NO: 14 69 DNA sequence encoding SEQ ID NO: 15 70 DNA sequence encoding SEQ ID NO: 17 71 DNA sequence encoding SEQ ID NO: 16 72 DNA sequence encoding SEQ ID NO: 18 73 DNA sequence encoding SEQ ID NO: 19 74 DNA sequence encoding SEQ ID NO: 20 75 DNA sequence encoding SEQ ID NO: 21 39 DNA sequence encoding SEQ ID NO: 22 38 DNA sequence encoding SEQ ID NO: 23 6 DNA sequence encoding SEQ ID NO: 24

Materials and Methods

[0142] Construction of PTH fusions: Molecules were constructed by a combination of gene synthesis (Eurofin MWG) and standard DNA manipulation techniques. Recombinant genes encoding full length PTH fusions were cloned into a modified mammalian expression plasmid, pSecTag/FRT/V5/Hist-TOPO (Invitrogen). Stable cell lines were produced in the CHO Flp-In cell line (Invitrogen) according to manufacturer's instructions and adapted to serum free media in Hyclone SFM4CHO Utility (Thermo Scientific). PTH fusions were under the secreted expression of either the PTH or GH signal peptides.

[0143] Expression and Purification: Cells were maintained in roller bottle cultures in Hyclone SFM4CHO Utility medium with passaging every 2-3 days, keeping cell densities between 0.25×10.sup.6 viable cells/ml (VCPM) and 1.5×10.sup.6 VCPM. For expression studies, roller bottles were seeded at 0.5×10.sup.6 VCPM and grown at 37° C., 5% CO.sub.2 and allowed to reach 1×10.sup.6 VPCM. Valproic acid was added to a final concentration of 2 mM and the temperature reduced to 31° C. Cells were grown for up to 8-10 days with viability still at ˜70% when harvest was clarified by centrifugation at 22,000×g using a Beckman JLA 16-25 rotor for 20 minutes at 4° C. EDTA and Benzamidine-HCl were added to final concentrations of 5 and 10 mM respectively and the medium concentrated using a Vivaflow 200 tangential flow concentrator and stored frozen at −20° C. Target protein was purified from this concentrate by anion exchange (Q-Sepharose FF, GE Healthcare) and affinity chromatography (anti GHBP antibody column). Protein concentrations were measured by Bradford protein assay and samples analysed by SDS-PAGE under non-reducing conditions and either stained with coomassie blue or western blotted using a commercial anti-PTH 1-34 antibody (Abcam 14493) or an in house developed anti GHBP antibody. Purified samples were aliquoted and stored at −80° C.

[0144] In Vitro Bioactivity: Purified proteins were tested for their ability to stimulate cAMP production from the PTH responsive cell line, UMR-106 (rat osteoblastic like cell line). Cells were stimulated for 15 minutes in the presence of test molecules and cAMP levels measured from cell lysates using a cAMP specific Elisa (R&D systems).

[0145] Animal Model for Hypoparathyroidism Shimizu et al have used an animal model for hypoparathyroidism in their studies on LA-PT. In this model rats were thyroparathyroidectomized (TPTX) prior to treatment. Briefly: Surgical TPTX was performed on 6-week-old rats obtained from Charles River Laboratories Japan, Inc. After surgery, pellet food (CE-2; CLEA Japan, Inc., Tokyo, Japan) containing 1.10% calcium and 1.09% phosphate moisturized with tap water was supplied inside each cage for easy access and digestion in sham-operated and TPTX rats. Postsurgical rats exhibiting sCa levels less than 8.0 mg/dL at 5 days after TPTX surgery were selected for subsequent peptide injection studies from the next day.

Example 1

[0146] From the crystal structure analysis of PTH with the N-domain PTH receptor [1], the PTH is shown to sit in a groove formed by the N-terminal receptor portion. It is this mode of interaction that is hypothesised to protect PTH from degradation and to create an “inactive pool” of PTH, thus prolonging its biological activity to create a long acting PTH. It is proposed that the new molecules (See FIG. 1) will be fusions between PTH (residues 1-34), the N-terminal PTH receptor domain (PTHrExt, most commonly residues D29-L187 but not restricted to other combinations) and the growth hormone binding protein (GHBP, residues 1-238). GHBP is an inert moiety designed to increase Mw & therefore delay clearance. It will contain a W104A mutation to prevent interaction with GH in the circulation.

Example 2

[0147] Initial expression studies showed that we are able to express and purify a PTH fusion molecule from a CHO cell line at sufficient levels (˜10 mg/L) to justify further progress. All molecules appear to be intact and >95% pure as judged by SDS-PAGE.

[0148] Both PTH fusion molecules so far tested are biologically activity and produce a dose response in an in vitro cAMP assay.

Example 3

[0149] PTH1-34 has been fused to growth hormone binding protein (GHbp) with or without the extracellular domain of the PTH receptor (PTHextR). Molecules have then been expressed with either the GH (GHss) or PTH (PTHss) signal sequence and propeptide (pp). Thus, the following 4 molecules have been generated: [0150] 14A2c=PTHss-pp-PTH (1-34)-(g4s)4-PTHrExt-(g4s)4-GHbp (SEQ ID NO: 13) [0151] 14A3b=GHss-PTH (1-34)-(g4s)4-PTHrExt-(g4s)4-GHbp (SEQ ID NO: 14) [0152] 14A4=GHss-PTH (1-34)-(g4s)4-GHbp (SEQ ID NO: 15) [0153] 14A7=PTHss-pp-PTH(1-34)-(g4s)4-GHbp (SEQ ID NO: 20)

[0154] Stable clones for all 4 molecules have been generated, all stable clones expressed protein and all 4 proteins have been purified on an affinity column for GHbp. Those using the GHss are expressed at a lower level and SDS-PAGE analysis and bioassays suggest that there may be incomplete processing of the translated product with additional aa at the N-terminus and possibly differential glycosylation of those with the GHss compared to those with the PTHss. (FIG. 28) This would fit with PTH requiring its own signal sequence for complete processing of the preproPTH sequence, which is in the design of the molecules with the PTHss. All molecules show bioactivity in the two bioassays used and evidence suggests that the molecule that includes the PTHextR is less bioactive (FIGS. 29 and 30).

[0155] PTH Fusion Molecules: Design & Hypothesis

[0156] Parathyroid hormone (PTH) is an 84-aa peptide with biological activity residing in residues 1-34. PTH is produced by the parathyroid glands in response to low serum calcium levels. PTH acts on the parathyroid receptor (PTHR1) on bone and kidney promoting the release of calcium from bone, slowing excretion of calcium from kidneys, increasing absorption from intestines and promoting renal excretion of phosphate. In Hypoparathyroidism (HypoPT) the parathyroid glands are either absent or damaged and therefore cannot produce any or sufficient amounts of parathyroid hormone. Initial treatment is with oral calcium and active VD3 supplements. Recently, replacement of PTH in HypoPT with Natpara (PTH 1-84) has been licensed but requires daily sc injections and is complicated by fluctuating calcium levels. Continuous pump therapy is effective but impractical for most patients. There is therefore an unmet need for a long acting PTH molecule that provides constant physiological levels of PTH activity. Previously we have shown that the fusion of growth hormone to its binding protein (GHBP) can generate a long-acting growth hormone (1). Using this technology we have generated a number of PTH fusion molecules (See FIGS. 1A & B). The PTH fusions are predicted to have a prolonged circulating half-life through increased protein size, whilst retaining biological activity. As a further modification of the PTH fusion shown in FIG. 1A, it is hypothesised that PTH will form intramolecular interactions with PTHrExt, be protected from degradation, and provide an intravascular pool of active PTH. To prevent GH binding, a single amino acid change of tryptophan-104 to alanine in the GHBP moiety (W104A) will be introduced in to all PTH fusion molecules. All PTH fusion will be expressed using either the naturally occurring PTH prepropeptide sequence or the GH secretion signal.

REFERENCES

[0157] Shimizu, M., et al., Pharmacodynamic Actions of a Long-Acting PTH Analog (LA-PTH) in Thyroparathyroidectomized (TPTX) Rats and Normal Monkeys. J Bone Miner Res, 2016. 31(7): p. 1405-12