FUSION POLYPEPTIDE COMPRISING THE EXTRACELLULAR BINDING DOMAIN OF GROWTH HORMONE RECEPTOR

Abstract

The invention relates to fusion polypeptides comprising the extracellular domain of growth hormone linked either directly or indirectly to a polypeptide wherein the polypeptide is not growth hormone.

Claims

1. A fusion polypeptide comprising: the extracellular binding domain of growth hormone receptor, or active binding part thereof, linked directly or indirectly, to a polypeptide that is not the growth hormone.

2. (canceled)

3. The fusion polypeptide according to claim 1, wherein said extracellular binding domain of growth hormone receptor comprises or consists of the amino acid sequence of SEQ ID NO:1.

4. The fusion polypeptide according to claim 1, wherein said extracellular binding domain of growth hormone receptor, or active binding part thereof, is modified by addition, deletion or substitution of at least one amino acid residue, wherein said modified extracellular binding domain of growth hormone receptor, or active binding part thereof substantially lacks growth hormone binding activity or has reduced growth hormone binding activity.

5. The fusion polypeptide according to claim 4, wherein the extracellular binding domain of growth hormone receptor comprises a modification in the growth hormone binding domain.

6. The fusion polypeptide according to claim 4,5 wherein said modification is at one or more of amino acids: W169, R43, E44, I103, W104, 1105, P106, I164 and D165.

7. The fusion polypeptide according to claim 6, wherein said modification comprises or consists of deletion of W104 of SEQ ID NO: 1 or SEQ ID NO: 21.

8. The fusion polypeptide according to claim 6, wherein said W104 is substituted for one or more amino acid residues.

9. The fusion polypeptide according to claim 8, wherein W104 is substituted for alanine as shown in SEQ ID NO: 2 or SEQ ID NO: 22.

10. The fusion polypeptide according to claim 4, wherein said modification comprises modification of amino acid residues 125-131 of SEQ ID NO 1 or SEQ ID NO: 21.

11. The fusion polypeptide according to claim 10, wherein said modification is the deletion of all or part of amino acid residues 125-131 of SEQ ID NO 1 or SEQ ID NO: 22.

12. The fusion polypeptide according to claim 1, wherein the polypeptide that is not the growth hormone is linked to the extracellular binding domain of growth hormone receptor and is positioned amino terminal to a growth receptor binding domain in said fusion polypeptide.

13. The fusion polypeptide according to claim 1, wherein said polypeptide that is not the growth hormone is linked to the extracellular binding domain of growth hormone receptor and is positioned carboxyl terminal to a growth receptor binding domain in said fusion polypeptide.

14. The fusion polypeptide according to claim 1, wherein said polypeptide that is not the growth hormone is linked to the receptor binding domain by a peptide linker.

15. (canceled)

16. The fusion polypeptide according to claim 14, wherein said peptide linker comprises at least one copy of the peptide Gly Gly Gly Gly Ser.

17. The fusion polypeptide according to claim 16 wherein said peptide linker comprises 2, 3, 4, 5, 6 or 7 copies of the peptide Gly Gly Gly Gly Ser.

18. The fusion polypeptide according to claim 1, wherein said fusion polypeptide does not comprise a peptide linker and is a direct fusion of the polypeptide indirectly, to a polypeptide that is not the growth hormone and the extracellular binding domain of growth hormone receptor.

19. The fusion polypeptide according to claim 1, wherein said fusion polypeptide comprises a cytokine.

20. The fusion polypeptide according to claim 19 wherein said fusion polypeptide comprises GCSF.

21. The fusion polypeptide according to claim 20 wherein said fusion polypeptide comprises the amino acid sequence in SEQ ID NO: 3.

22. The fusion polypeptide according to claim 19, wherein said fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 5, 7, 9, 11, 19, 20, 24, 26, 28, 30, 32, 34, 36 or 38.

23.-27. (canceled)

28. The fusion polypeptide according to claim 1, wherein said fusion polypeptide comprises a non-native amino terminal signal peptide.

29. The fusion polypeptide according to claim 28 wherein said non-native amino terminal signal peptide comprises the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA [SEQ ID NO: 39].

30. A nucleic acid molecule that encodes the fusion polypeptide of claim 1.

31. A vector comprising the nucleic acid molecule according to claim 30.

32. The vector according to claim 31 wherein said vector is an expression vector.

33. An isolated cell transfected or transformed with the nucleic acid molecule of claim 30.

34. A pharmaceutical composition comprising the fusion polypeptide of claim 1 and an excipient or carrier.

35. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] An embodiment of the invention will now be described by example only and with reference to the following figures:

[0083] FIG. 1A is the amino acid sequence of human GHR extracellular domain (SEQ ID NO 21); and FIG. 1B (SEQ ID NO 22) is the human GHR extracellular domain modified at W104; signal peptides are shown in bold and are optional; FIG. 10 is GCSF (SEQ ID NO 3) and FIG. 1D is Leptin (SEQ ID NO 4); FIG. 1E (SEQ ID NO: 1) is the amino acid sequence of human GHR extracellular domain without signal sequence; and FIG. 1F (SEQ ID NO: 2) is the amino acid sequence of human GHR extracellular domain without signal sequence and modified at W104.

[0084] FIG. 2A provides a schematic of Leptin-link-GHBP (2N1).

[0085] FIG. 2B provides the amino acid sequence of 2N1 (SEQ ID NO: 5).

[0086] FIG. 2C provides the nucleotide sequence of 2N1 (SEQ ID NO: 6).

[0087] FIG. 3A provides a schematic of Leptin-link-GHBP-His (2N1-Hist).

[0088] FIG. 3B shows the amino acid sequence of 2N1-Hist (SEQ ID NO:7).

[0089] FIG. C shows the nucleotide sequence of 2N1-Hist (SEQ ID NO:8).

[0090] FIG. 4A is a western blot of transient expression of 2N1 and 2N1-Hist. 1. leptin (50 ng/lane), 2. leptin (10 ng/lane), 3. MW standards, 4. 2N1 (clone 1_1), 5. 2N1 (clone 1_3), 6. 2N1-Hist (clone 2_2), 7. negative control.

[0091] FIG. 4B is a western blot of expression of 2N1 from a stable CHO Flp In cell line. (An antibody that recognised leptin was used for both western blots).

[0092] FIG. 5: In vitro bioassay for 2N10.2 ml medium from cells transiently expressing 2N1 was used in a 1 ml reaction volume;

[0093] FIG. 6A provides a schematic of GCSF-link-GHBP.

[0094] FIG. 6B provides the amino acid sequence of GCSF-link-GHBP ([SEQ ID NO: 9) and the amino acid sequence of GCSF linked to GHBP without a signal sequence (SEQ ID NO: 17).

[0095] FIG. 6C provides the nucleotide sequence of GCSF-link-GHBP (SEQ ID NO: 10), with the signal sequence underlined.

[0096] FIG. 7A provides a schematic of GCSF-link-GHBP (W104A).

[0097] FIG. 7B provides the amino acid sequence of GCSF-link-GHBP (W104A) (SEQ ID NO: 11); the W104A mutation is shown in bold and underlined and the amino acid sequence of GCSF-link-GHBP (W104A) without the N-terminal sequence (SEQ ID NO: 18).

[0098] FIG. 7C provides the nucleotide sequence of GCSF-link-GHBP (W104A) (SEQ ID NO:12); the signal sequence is underlined, the W104A mutation is shown in bold and underlined.

[0099] FIG. 8A provides a western blot of transiently expressed GCSF-link-GHBP and GCSF-link-GCSF(W104A). 1. GCSF-link-GHBP #1, 2. GCSF-link-GHBP #2, 3. GCSF-link-GHBP(W104A) #1, 4. GCSF-link-GHBP(W104A) #2, 5. Positive control (GCSF-link-GCSFR) #1, 6. Positive control (GCSF-link-GCSFR) #2, 7. Negative control (media only) #1, 8. Negative control (media only) #2.

[0100] FIG. 8B provides a western blot of stably expressed GCSF-link-GHBP and GCSF-link-GCSF(W104A). 1. GCSF-link-GHBP from adherent cells, 2. GCSF-link-GHBP(W104A) from adherent cells, 3. GCSF-link-GHBP from suspension cells, 4. GCSF-link-GHBP(W104A) from suspension cells.

[0101] FIG. 9: Purification of GCSF-link-GCSF(W104A). The fusion protein was purified by IMAC using a Ni-resin column. The picture shows the SDS-PAGE gel of the relevant elution fractions from this purification;

[0102] FIG. 10: Purification of GCSF-link-GCSF(W104A). The fusion protein was purified by IMAC using a Ni-resin column. The picture shows the SDS-PAGE gel of the relevant elution fractions from this purification;

[0103] FIG. 11: Temperature stability of GCSF fusion polypeptide (non-reduced gel);

[0104] FIG. 12: Temperature stability (reduced gel); Lane 1: Untreated GCSF_GHBP, Lane 2: Room temperature, Lane 3: 4 C., Lane 4: 80 C. (Freeze/Thaw), Lane 5: Untreated GCSF_W104A_GHBP, Lane 6: Room temperature, Lane 7: 4 C., Lane 8: 80 C. (Freeze/Thaw);

[0105] FIG. 13: Extended stability study (non-reduced gel); Lane 1: Untreated GCSF_GHBP, Lane 2: Room temperature, Lane 3: 4 C., Lane 4: 80 C. (Freeze/Thaw), Lane 5: Untreated GCSF_W104A_GHBP, Lane 6: Room temperature, Lane 7: 4 C., Lane 8: 80 C. (Freeze/Thaw);

[0106] FIG. 14: AML-193 proliferation assay. AML 193 (peripheral blood; acute monocytic leukemia) cells proliferate when stimulated with IL-3, GM-CSF or GCSF;

[0107] FIG. 15 FACS analysis; % neutrophils in blood measured at 24h, 48h, 72h and 96h after injection of control, Filgrastim and 4F1;

[0108] FIG. 16 FACS analysis; % neutrophils in bone marrow (BM) measured at 24h, 48h, 72h and 96h after injection of control, Filgrastim and 4F1;

[0109] FIG. 17: FACS analysis; mobilisation of HPC to blood measured at 24h, 48h, 72h and 96h after injection of control, Filgrastim and 4F1;

[0110] FIG. 18 FACS analysis; mobilisation of HPC in BM measured at 24h, 48h, 72h and 96h after injection of control, Filgrastim and 4F1;

[0111] FIG. 19: PK study; concentration of Filgrastm and 4F1 measured in the serum over 50h;

[0112] FIG. 20: Coomassie stained 10% SDS-PAGE (non-reducing); Analysis of purified product 2N2 (from run#2), 1: Final purified product (5 microg per lane), 2: Broad range BioRad standards;

[0113] FIG. 21: Bioactivity of 2N2 analysed in dual luciferase reporter assay;

[0114] FIG. 22: Protein sequence of 2N2 comprising the W104A transition (SEQ ID NO 19);

[0115] FIG. 23: Protein sequence of 2N2 (SEQ ID NO: 20);

[0116] FIG. 24: nucleotide sequence of 2N3 (SEQ ID NO: 23);

[0117] FIG. 25: translated protein sequence of 2N3 (SEQ ID NO: 24);

[0118] FIG. 26: nucleotide sequence of 2N4 (SEQ ID NO 25);

[0119] FIG. 27: translated protein sequence of 2N4. (SEQ ID NO 26);

[0120] FIG. 28: nucleotide sequence of 2N5 (SEQ ID NO 27);

[0121] FIG. 29: translated protein sequence of 2N5 (SEQ ID NO 28);

[0122] FIG. 30: nucleotide sequence of 2N6 (SEQ ID No 29);

[0123] FIG. 31: translated protein sequence of 2N6. (SEQ ID NO 30);

[0124] FIG. 32: nucleotide sequence of 4F2 (SEQ ID NO 31);

[0125] FIG. 33: translated protein sequence of 4F2 (SEQ ID NO 32);

[0126] FIG. 34: nucleotide sequence of 4F2_W104A (SEQ ID NO: 33);

[0127] FIG. 35: translated amino acid sequence of 4F2_W104A (SEQ ID NO 34);

[0128] FIG. 36: nucleotide sequence of 4F3 (SEQ ID NO: 35);

[0129] FIG. 37: translated protein sequence of 4F3 (SEQ ID NO 36);

[0130] FIG. 38: nucleotide sequence of 4F3_W104A (SEQ ID NO 37);

[0131] FIG. 39: translated amino acid sequence of 4F3_W104A (SEQ ID NO 38)

[0132]

TABLE-US-00001 TABLE 1 Full plasmid Name Description Status pObsecTag_2N1 Ob-(G.sub.4S).sub.4- Leptin linked to GHBP Constructed GHBP via a (Gly.sub.4Ser).sub.4 linker, and Contains leptin signal crude media sequence tested: pObsecTag_2N2 Ob-(G.sub.4S).sub.4- As above but GHBP Biologically GHBP contains the W104A active (W104A) mutation

TABLE-US-00002 TABLE 2 Full plasmid Name Description pOBsecTag_2N3 leptin linked to GHBP (contains (G4S)5 linker) pOBsecTag_2N4 leptin linked to GHBP (contains (G4S)5 linker) pOBsecTag_2N5 leptin linked to GHBP (No linker) pOBsecTag_2N6 leptin linked to GHBP (No linker)

TABLE-US-00003 TABLE 3 Full plasmid Name Description pGCSFsecTag_4F2 GCSF linked to GHBP (contains (G4S)5 linker) pGCSFsecTag_4F2_W104 GCSF linked to GHBP (contains (G4S)5 linker) pOBsecTag_4F3 GCSF linked to GHBP (No linker) pOBsecTag_4F3_W104A GCSF linked to GHBP (No linker)

Materials and Methods

Generation of the Expression Plasmids

[0133] Both the leptin and GCSF fusion proteins were expressed from genes which had been constructed by using the polymerase chain reaction (PCR) to introduce NheI and NotI restriction sites at either end of the ligand gene and then inserting this into the gene for GH-link-GHBP to replace the GH sequence in this gene. The gene manipulations were carried out in the plasmid vector pGHSecTag1B8v0 (+/His6-tag) (pSecTag/FRT/V5-His TOPO (Invitrogen) containing the gene for GH-link-GHBP); using the following primers.

TABLE-US-00004 LEPTINPRIMERS Forwardprimer (SEQID13) 5-GGGAAAGCTAGCCACCATGCATTGGGGAACCCTGTG-3 Reverseprimer (SEQID14) 5-ATTGATTAGCGGCCGCCGCACCCAGGGCTGAGGTCC-3 GCSFPRIMERS Forwardprimer (SEQID15) 5-AAGCTGGCTAGCCACCATGGC-3 Reverseprimer (SEQID16) 5-AATTAATAGCGGCCGCCGGGCTGGGCAAG-3

[0134] The W104A mutation was introduced into the fusion protein by digestion, of a previously synthesised, GHBPW104A gene using AvrII and EcoRV, the fragment containing the mutation was then inserted into GHBP of the fusion protein sequences between the same restriction sites.

[0135] Plasmids were maintained in E. coli XL1 Blue cells and expression was carried out in mammalian Chinese Hamster Ovary (CHO) cells.

Expression of the Fusion Proteins

[0136] Transient expression of the fusion proteins were carried out by using a transfection agent (Mirius or Fugene-6) to introduce the expression plasmid into CHO Flp-In cells (Invitrogen). CHO cells were first seeded into the wells of a 24-well culture plate at 2105 cells/ml using 1 ml/well, the plate was incubated overnight at 37 C./5% CO.sub.2 to allow the cells to attach. The following day 6 l of Fugene-6 or Mirius was added to 100 l serum free culture media and mixed; 4 g plasmid DNA was then added [transfectant:DNA ratio of 3:2] and the solution mixed. After 15 minutes at room temperature the transfection mixture was added drop-wise into the individual wells containing the CHO cells. The cells were incubated overnight 37 C./5% CO.sub.2 and the culture media replaced with serum free media. The media was harvested after 72 hours and analysed for expression of the protein of interest.

[0137] Stable expression of the fusion proteins was carried out in the same way as for transient expression; however after the overnight incubation post-transfection the media was replaced with media containing 600 g/ml Hygromycin B. This selective pressure was maintained until most of the cells had died off and only the cells which had been stably transfected started to regrow. Once a stably expressing population of CHO cells had been obtained this was maintained using a lower concentration of Hygromycin B. The cells were then subsequently adapted to suspension growth in Hyclone SFM4CHO Utility culture medium and this suspension culture used to produce fusion protein for purification.

Western Blot/Immunoblot

[0138] Protein samples were separated by SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) and then the protein on the gels transferred to PVDF or nitrocellulose membrane by electro-transfer. After blocking with 5% (w/v) milk powder the membrane was probed with a primary antibody which recognized the protein of interest (leptin or GCSF). The membrane was then washed and then a secondary HRP-conjugated antibody used which bound to the primary antibody. After another wash the membrane was exposed to ECL reagent and exposed to x-ray film in a dark room. This caused black bands to appear where the antibodies had sequentially bound at the location of the protein of interest.

Detection/Quantification Using ELISA

[0139] A sandwich Enzyme Linked Immuno-Sorbant Assay (ELISA) was used to detect and quantify both leptin and GCSF fusion proteins. In the case of leptin; an anti-leptin capture antibody was bound to the wells of a 96-well plate, the plate was then blocked with 3% (w/v) bovine serum albumin, the protein samples (standard curve and unknowns) were added to the wells, and then another anti-leptin detection antibody added to the wells. A secondary HRP-conjugated antibody was then added to the wells and the amount of leptin measured using a colourmetric reagent which reacts to horse radish peroxidase (HRP). The colourmetric change is proportional to the amount of leptin in the protein samples. The GCSF fusion was measured in the same way but anti-GCSF capture and detection antibodies were used.

Protein Purification

[0140] A CHO cell line stably expressing the protein of interest was seeded at a density of 0.5106/ml and grown at 37 C./5% CO.sub.2 in roller bottles with a maximum volume of 500 ml/bottle. Once the viability of the cells had dropped to 20-30% the culture media was harvested and concentrated approximately 10-fold. The concentrated culture media was diluted with an equal volume of 40 mM phosphate buffer, pH 7.4, 1M NaCl, 20% glycerol, 20 mM Imidazole and loaded onto a 5 ml Ni-chelate column which had been pre-equilibrated with 10 column volumes of 20 mM phosphate buffer, pH 7.4, 0.5M NaCl, 10% glycerol (Buffer A). The column was washed with 10 column volumes of Buffer A with 10 mM imidazole, followed 10 column volumes of 20 mM Na acetate buffer, 0.5 M NaCl, 10% glycerol, pH 6 (Buffer B). Bound protein was then eluted using Buffer B containing increasing concentrations of imidazole. All buffers were filtered prior to use and the sample was clarified by centrifugation prior to loading.

Leptin Bioassay

[0141] The bioactivity of the leptin-link-GHBP fusion was measured using a dual-luciferase bioassay. Briefly, HEK293 cells were transfected with a plasmid expressing the leptin receptor, a plasmid expressing firefly luciferase under the control of SIE (reporter plasmid), a plasmid expressing Renilla luciferase under the control of a CMV promoter (control plasmid) and a plasmid expressing STAT3 (to increase endogenous STAT3 levels in the cells). These were grown in starvation media overnight and then stimulated with different concentrations of leptin or media from cells expressing the leptin-link-GHBP fusion; increasing levels of stimulation led to increased expression of firefly luciferase via the SIE reporter plasmid.

[0142] After 6 hours stimulation the cells were lysed and the levels of firefly and Renilla luciferase activities measured using the Dual-Luciferase Assay Kit (Promega) in a luminometer. The firefly luciferase signal was divided by the Renilla luciferase signal to correct for sample-to-sample variation and then this divided by the signal for an unstimulated sample to provide a fold induction over background.

GCSF Bioassay

[0143] The bioactivity of GCSF-link-GHBP was measured using a cell proliferation assay. Briefly, AML-193 cells were washed with PBS and then resuspended in culture medium (no GM-CSF) at 5105 cells/ml. 50 l of the cells were pipetted into the wells of a 96 well-plate and then stimulated with 50 l of a dose range of GCSF or GCSF-link-GHBP. The plate was then incubated at 37 C./5% CO2 for 3 days. 20 l of CellTitre 96 AQueousNon-Radioactive Proliferation Assay Reagent (Promega) was then added to the wells. The colourmetric change caused by the CellTitre reagent was measured using a spectrophotometric plate reader; the OD measured was proportional to the number of cells in the well which in turn is proportional to the activity of GCSF or the fusion molecule.

Protein Stability Studies

[0144] Stability studies were undertaken on protein constructs to look for degradation and formation of higher order structures over an 8 day period. Samples of each construct were kept at room temperature, 4 degrees Celsius (fridge) and 80 degrees Celsius (freeze/thaw). Analysis was undertaken using SDS PAGE and native PAGE followed by coomassie staining and western blotting.

Extended Stability Study

[0145] To assess stability of protein samples over a longer period of time, 4C, RmT and 80C F/T samples were also analysed after 3 months incubation. Samples from this experiment were only analysed by SDS PAGE followed by coomassie staining.

Animals

[0146] Specific pathogen-free (BDF1) male mice at (6-9) weeks of age are used for efficacy and pharmacokinetics studies. BDF1 mice were chosen for these studies because the pharmacokinetics of G-CSF are known to depend on the strain of mice used, and BDF1 mice have been shown to have a robust response to G-CSF (Haan et al 2000, Halpern et al 2002, Lord et al 2001, Molineux et al 1999). Mice are allowed to acclimate for 1 week prior to the start of the experiment. All animal handling and experimental procedures will be carried out under Home Office License according to the provisions of the United Kingdom Animals.

Protein Preparations

[0147] Proteins to be tested and their concentrations are given in Table 1. 4F1-W104 samples are in PBS buffer.

TABLE-US-00005 TABLE 4 Proteins and their concentration Concentration Protein (g/ml) Total Protein (mg) Total Protein (nM) Filgrastim 300 4F1-W104 450 13.95 9595 nM

[0148] The working concentrations of 4-F1-W104 for pharmacokinetic and pharmacodynamic studies are obtained by diluting as necessary in phosphate-buffered saline (PBS). The working concentrations of Filgrastim are obtained by diluting as necessary in 5% Dextrose.

Protein Administration

[0149] Molar equivalence of the test proteins is based upon an average molecular weight of 46.9 kD for 4F1-W104 and reported MW of 19 kD for Filgrastim. Table 5 summarizes the moles (in nmol) for the dose administration used in these studies.

TABLE-US-00006 TABLE 5 Molar Equivalence Molar Equivalent Protein Dose administrated (mg/kg) (nmol/kg) Filgrastim 0.25 13.150 1.25 65.750 4F1-W104 0.25 5.33 1.25 26.6

Analysis the Samples for Pharmacodynamic Studies

[0150] As G-CSF is a haematopoietic cytokine that acts on neutrophil lineage cells and activates mature neutrophils, recombinant G-CSF has been widely used for adjuvant chemotherapy. The biological activity of the constructs can be evaluated by determining G-CSF activity of increasing WBC counts.

Peripheral Blood Smear

[0151] Blood is collected from the tail vein into capillary tubes pre-treated with heparin and transferred into Capiject ethylene diamine tetra acetic acid (EDTA) tubes. For total white blood cell (WBC) count 10 l of blood is added directly to 10 mL of Isoton II buffer containing four drops of Zapoglobin II for red blood cell (RBC) lysis. WBC count is determined via Coulter Counter.

[0152] The number of granulocytes and hematopoietic progenitor cells was assessed by flow cytometry using Gr-1 (granulocytes) and c-kit (hematopoietic progenitor cells) antibody markers. For staining, 20-50 l of whole blood is added to an antibody cocktail containing FITC conjugated Gr.1/8C5, c-kit/CD117 conjugated to R-PE, and Mac-1/CD11b conjugated to PE-Cy5. Cells were incubated 20 min at room temperature.

[0153] Erythrocytes are lysed by a brief incubation with 0.5 mL ACK Lysing Buffer. The lysing reaction is stopped with 2 mL of FACS buffer, and the cells are pelleted by centrifugation. The cells are resuspended in FACS buffer (d-PBS with 0.1% sodium azide and 0.1% BSA) and acquired on the FACScan (Halpern et al 2002).

Bone Marrow Biopsy

[0154] Bone marrow was prepared by extensive pipetting of bone marrow.

Pharmacokinetics Studies

[0155] Mice are injected subcutaneously (S.C) in the mid-scapular region with 0.25 mg/kg of filgrastim, 4F1-W104, and PBS for vehicle control mice. Blood is collected via the inferior vena cava at 2, 6, 12, 24, 36, 48, 72 and 96 h after injection. Blood samples were centrifuged at 3000 rpm for 15 min (16000 g for 10 min) to obtain serum and then stored at 80 C. until analysis. The serum concentrations of G-CSF and 4F1-W104 were determined by human G-CSF ELISA kit in the presence of mouse serum.

[0156] Vehicle control mice received a single SC injection of PBS. Experimental mice received a single administration of Filgrastim and 4F1-W104 at a dose level of 0.25 mg/kg. The number of peripheral granulocytes and hematopoietic progenitor cells in mice is evaluated daily for 5 consecutive days (0, 24, 48, 72, 96h).

Expression and Purification of 2N2

[0157] A stable CHO cell line was produced and protein expressed as a secreted product in roller bottle culture. Protein was purified from cell culture media using an anti-GHBP antibody column, dialysed in to PBS and concentrated.

Example 1

Leptin-link-GHBP (2N1)

[0158] The protein fusion construct leptin-link-GHBP (2N1) was designed without (FIG. 2b; SEQ ID NO 5) and with a His6 tag. The expression gene for these constructs were generated and inserted into the expression vector, pSecTag, using conventional molecular biology techniques e.g. PCR, restriction digestion and ligation.

[0159] Expression was first established as transient transfections in CHO Flpln cells, using Fugene-6 as the transfectant and a DNA:transfectant ratio of 2:3the manufacturer's instructions were followed to achieve transfection. Expression was confirmed by western blot (FIG. 4A) using media from the adherent cell culture 72 hours post-transfection. The probe used for the western blot was anti-leptin antibody.

[0160] A stable cell line expressing 2N1 was then established by growing transfected CHO Flpln cells in the presence of Hygromycin B. The selective pressure of Hygromycin B ensured that only the CHO Flpln cells which had been stably transfected with the expression vector would survive. Expression from these cells was also confirmed by western blotting (FIG. 4B).

[0161] To demonstrate that the fusion protein had leptin-like bioactivity media from transiently expressing CHO Flpln cells was used in a leptin bioassay. Cells expressing the leptin receptor and containing a firefly luciferease reporter plasmid were stimulated with 0.2 ml media from cells transiently expressing 2N1. Stimulation of the leptin receptors initiated a signal cascade which resulted in the production of firefly luciferase from the reporter plasmid; production of the firefly luciferase was proportional to the stimulation of the leptin receptor. A constitutively expressing plasmid which produced Renilla luciferase was used to correct for inter-assay variability. Leptin was also used in the assay as a positive control and also to show a dose response. This dual-luciferase assay was measured using a luminometer, and the activity of leptin and 2N1 calculated as fold induction over the background luminosity (FIG. 5).

[0162] Thus, we show that leptin-link-GHBP can be produced and that this fusion protein has bioactivity.

Example 2: Leptin-Link-GHBP (W104A in GHBP) (2N2)

[0163] Purified protein from 2 purification runs (Run #1 & Run#2) were analysed in the dual luciferase reporter assay. Both preparations show biological activity in the assay compared to PBS controls. A sample concentration of 450 nM was used in the assay. Both preparations show biological activity (FIG. 22).

Example 3: GCSF-Link-GHBP

[0164] The protein fusion construct GCSF-link-GHBP was designed without (FIG. 6B; SEQ ID NO 9) and with a W104A mutation in GHBP (FIG. 7B, SEQ ID NO 11). The expression gene for these constructs were generated and inserted into the expression vector, pSecTag, using conventional molecular biology techniques e.g. PCR, restriction digestion and ligation.

[0165] Expression was first established as transient transfections in CHO Flpln cells, using Fugene-6 or Mirus transfection reagent as the transfectant and a DNA:transfectant ratio of 2:3the manufacturer's instructions were followed to achieve transfection. Expression was confirmed by western blot (FIG. 8A) using media from the adherent cell culture 72 hours post-transfection. The probe used for the western blot was anti-GCSF antibody.

[0166] A stable cell line expressing GCSF-link-GHBP+/W104A was then established by growing transfected CHO Flpln cells in the presence of Hygromycin B. The selective pressure of Hygromycin B ensured that only the CHO Flpln cells which had been stably transfected with the expression vector would survive. Expression from these cells was also confirmed by western blotting (FIG. 8B).

[0167] His-tagged GCSF-link-GHBP(W104A) was produced from a large volume of stably expressing CHO Flpln cells grown in suspension culture. The GCSF-link-GHBP(W104A) was then purified by immobilised metal ion chromatography (IMAC) using Ni-resin (FIG. 9).

[0168] To demonstrate that the fusion protein had GCSF-like bioactivity a range of concentrations of the fusion protein were used to stimulate the growth of AML-193 cells. After 72 hours stimulation the proliferation of cells was measured using CellTitre reagent (Promega). The CellTitre reagent causes a colourmetric change proportional to the number of cells in the sample; the number of cells in the sample is in turn proportional to the activity of GCSF. A dose range of GCSF was also used as a positive control and also to compare the activity of the fusion protein. The activities of GCSF and GCSF-link-GHBP(W104A) were plotted as absorbance against protein concentration (FIG. 10) and the EC50s calculated; GCSF and GCSF-link-GHBP(W104A) have EC50s of 4 ng/ml (215 pM; Mw of 18.6 kDa) and 2 ng/ml (43 pM; assuming a Mw of 46.7 kDa), respectively. These calculations show that the fusion protein is approximately 5 times more active than native GCSF.

[0169] Thus, we show that GCSF-link-GHBP+/W104A can be produced and that GCSF-link-GHBP(W104A) can be purified and has bioactivity comparable to that of GCSF.

Example 4: Protein Stability Studies

[0170] A. Non-reduced gel: Test samples were incubated at room temperature, 4 C., and 80 C. (Freeze/Thaw). Samples were taken on day 0, 2, 4 and 8 and analysed by SDS-PAGE followed by Coomassie staining (5 g protein loaded per lane). Both GCSF_GHBP (Top) and GCSF_W104A_GHBP (Bottom) showed no visible signs of degradation under all conditions studied over the 8 day period (FIG. 11).

[0171] B. Reduced Gel: Test samples were incubated at room temperature, 4 C., and 80 C. (Freeze/Thaw). Samples were taken on day 8 and analysed by native PAGE followed by Coomassie staining (4 g protein loaded per lane). No visible signs of degradation under all conditions (FIG. 12).

[0172] C. Non-reduced gel: Test samples were incubated at room temperature, 4 C., and 80 C. (Freeze/Thaw) for 3 months. Samples were analysed by SDS-PAGE followed by Coomassie staining (4 g protein loaded per lane). No visible signs of degradation for 80 C. samples. Minimal degradation for room temperature, and 4 C. samples (FIG. 13).

Example 5: In Vitro Bioactivity Evaluation of Proteins Using an AML-193 Cell-Based Proliferation Assay

[0173] AML 193 (peripheral blood; acute monocytic leukemia) cells proliferate when stimulated with IL-3, GM-CSF or GCSF. Proliferation is dose dependent and can be used to evaluate the activity of the GCSF solutions. Both purified proteins showed increased bioactivity with all standard curves shifted to the left in comparison to native GCSF (FIG. 14, Table 6).

TABLE-US-00007 TABLE 6 EC50 values for each construct Construct EC50 (nM) SEM Native GCSF 0.056 (n = 12) 0.009 GCSF_GHBP 0.024 (n = 6) 0.004 GCSF_W104A_GHBP 0.020 (n = 6) 0.004

Example 6: In Vivo Analysis

FACS Analysis

[0174] A: For Filgrastim group, the maximum mobilisation of peripheral neutrophils occurred on day 1 after injection, but on day 2 for 4F1 (FIG. 15).

[0175] B: After a single administration of Filgrastim, the neutrophil count in the BM decreased at 24 hr, then returned to normal levels at 48 hrs. After a single administration of 4F1, the neutrophil count in the BM started to decrease at 24h. It reached their lowest count at 48 hrs. Then returned to normal levels at 96 hrs, (FIG. 16) The maximum mobilisation of HPC occurred on day 1 for Filgrastim, while on day 2 for 4F1 group. HPC returned to normal levels by day 2 for Filgrastim, while on day 3 for 4F1 group. (FIG. 17). After a single administration of Filgrastim and 4F1, HPC count in the BM decreased at 24 hrs, then started to return to normal levels by 96 hrs. (FIG. 18).

PK Study

[0176] FIG. 19 shows that 4F1-W104 has a much better delayed clearance compared to Filgrastim, with a peak concentration around 12-24 hours and also shows that it might also have a slow rate of absorbance.

TABLE-US-00008 TABLE 7 Summary of SEQ ID NOs. SEQ ID NO: 1 amino acid sequence of human GHR extracellular domain SEQ ID NO: 2 amino acid sequence of human GHR extracellular domain W104 mutation SEQ ID NO: 3 amino acid sequence of GCSF SEQ ID NO: 4 amino acid sequence of leptin SEQ ID NO: 5 amino acid sequence of leptin linked to GHBP SEQ ID NO: 6 nucleotide sequence of leptin linked to GHBP SEQ ID NO: 7 amino acid sequence of leptin linked to GHBP SEQ ID NO: 8 nucleotide sequence of leptin linked to GHBP with histidine tag SEQ ID NO: 9 amino acid sequence of GCSF linked to GHBP with signal sequence SEQ ID NO: 10 nucleotide sequence of GCSF linked to GHBP with signal sequence SEQ ID NO: 11 amino acid sequence of GCSF linked to GHBP with W104 mutation SEQ ID NO: 12 nucleotide sequence of GCSF linked to GHBP with W104 mutation SEQ ID NO: 13 leptin forward primer SEQ ID NO: 14 leptin reverse primer SEQ ID NO: 15 GCSF forward primer SEQ ID NO: 16 GCSF reverse primer SEQ ID NO: 17 amino acid sequence of GCSF linked to GHBP without signal sequence SEQ ID NO: 18 amino acid sequence of GCSF linked to GHBP with W104 mutation, SEQ ID NO: 19 2N2 (W104) SEQ ID NO: 20 2N2 SEQ ID NO: 21 amino acid sequence human GHR/ECD with signal sequence SEQ ID NO: 22 amino acid sequence human GHR/ECD with W104 and signal sequence SEQ ID NO: 23 nucleotide sequence of 2N3 construct (leptin fusion protein) SEQ ID NO: 24 amino acid sequence of 2N3 construct (leptin fusion protein) SEQ ID NO: 25 nucleotide sequence of 2N4 construct (leptin fusion protein) SEQ ID NO: 26 amino acid sequence of 2N4 construct (leptin fusion protein) SEQ ID NO: 27 nucleotide sequence of 2N5 construct (leptin fusion protein) SEQ ID NO: 28 amino acid sequence of 2N5 construct (leptin fusion protein) SEQ ID NO: 29 nucleotide sequence of 2N6 construct (leptin fusion protein) SEQ ID NO: 30 amino acid sequence of 2N6 construct (leptin fusion protein) SEQ ID NO: 31 nucleotide sequence of 4F2 construct (GCSF fusion protein) SEQ ID NO: 32 amino acid sequence of 4F2 construct (GCSF fusion protein) SEQ ID NO: 33 nucleotide sequence of 4F2 construct (GCSF fusion protein) W104 SEQ ID NO: 34 amino acid sequence of 4F2 construct (GCSF fusion protein) W104 SEQ ID NO: 35 nucleotide sequence of 4F3 construct (GCSF fusion protein) SEQ ID NO: 36 amino acid sequence of 4F3 construct (GCSF fusion protein) SEQ ID NO: 37 nucleotide sequence of 4F3 construct (GCSF fusion protein) W104 SEQ ID NO: 38 amino acid sequence of 4F3 construct (GCSF fusion protein) W104