BONE DELIVERY CONJUGATES AND METHOD OF USING SAME TO TARGET PROTEINS TO BONE

Abstract

A bone delivery conjugate having a structure selected from the group consisting of: A) X-D.sub.n-Y-protein-Z; and B) Z-protein-Y-D.sub.n-X, wherein X is absent or is an amino acid sequence of at least one amino acid; Y is absent or is an amino acid sequence of at least one amino acid; Z is absent or is an amino acid sequence of at least one amino acid; and D.sub.n is a poly aspartate wherein n=10 to 16. Compositions comprising same and methods of use thereof.

Claims

1. A bone delivery conjugate having a structure selected from the group consisting of: A) X-D.sub.n-Y-protein-Z; and B) Z-protein-Y-D.sub.n-X, wherein X is absent or is an amino acid sequence of at least one amino acid; Y is absent or is an amino acid sequence of at least one amino acid; Z is absent or is an amino acid sequence of at least one amino acid; and D.sub.n is a poly aspartate wherein n=10 to 16.

2. A bone delivery conjugate as recited in claim 1 wherein the protein is a soluble alkaline phosphatase (sALP).

3. A bone delivery conjugate as recited in claim 2 wherein said structure is: Z-sALP-X-D.sub.n-Y.

4. A bone delivery conjugate as recited in claim 3, wherein the sALP is encoded by the sequence as set forth in FIG. 16A.

5. A bone delivery conjugate as recited in claim 3, wherein the sALP has the sequence as set forth in FIG. 16B.

6. A bone delivery conjugate as recited in claim 3, wherein n=10.

7. A bone delivery composition comprising a bone delivery conjugate as recited in claim 1, and a pharmaceutically acceptable carrier.

8. A method of delivering a protein to bone tissue of a mammal comprising administering to said mammal an effective amount of a bone delivery conjugate as recited in claim 1.

9. A method of delivering ALP to bone tissue of a mammal in need thereof comprising administering to said mammal an effective amount of a bone delivery conjugate as recited in claim 2.

10. A method of treating a condition or disease related to a bone defect characterized by a lack of or an insufficient amount of functional alkaline phosphatase comprising administering to a mammal in need thereof a conjugate as recited in claim 2, said conjugate being in a pharmaceutically acceptable carrier.

11. A method as recited in claim 10, wherein the condition or disease is hypophosphatasia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In the appended drawings:

[0062] FIG. 1 presents the purity status of GST and GST-D.sub.10 proteins on an SDS polyacrylamide gel after CL-4B chromatography;

[0063] FIG. 2 shows the promotion of GST binding to bone by D.sub.6, D.sub.10 and D.sub.16 peptide motifs through the percentage of the injected dose of recombinant GST found associated with specific tissues;

[0064] FIG. 3 provides a schematic representation of the plasmid pCDNA3-RSV-D.sub.10sPHEX-NEO vector;

[0065] FIG. 4 presents a chromatographic profile of 280 nm detection of PHEX flow for the SP-Sepharose HP (A) and the blue-Sepharose HP (B). Straight line represents buffer ratio;

[0066] FIG. 5 presents a Sypro-ruby stained SDS-PAGE analysis of the different fractions collected throughout D.sub.10sPHEX purification procedure;

[0067] FIG. 6 shows the variation in serum alkaline phosphatase levels (ALP) observed in Hyp mice injected daily with i.v. doses of sPHEX and D.sub.10-sPHEX for 14 days. U/I values represent the decrease observed between day 3 (corresponding in the graphic to 0 U/I) and day 15. of the injection regimen and are the mean of measures made on 6 animals;

[0068] FIG. 7 shows the nucleotide sequence of a recombinant DNA sequence encoding a protein cleavable so as to produce D.sub.10-sPHEX (SEQ ID NO: 1);

[0069] FIG. 8 shows the amino acid sequence encoded by the D.sub.10-sPHEX of FIG. 7 (SEQ ID NO: 2);

[0070] FIG. 9 compares the binding to the mineral phase of bone of proteins (A. GST B. sPHEX) with that of their deca-aspartate fused counterparts;

[0071] FIG. 10 shows the nucleotide sequence of a native (or membrane-bound) PHEX (SEQ ID NO: 3);

[0072] FIG. 11 shows the amino acid sequence (SEQ ID NO: 4) of a D.sub.10-sPHEX conjugate produced by cleavage of the recombinant cleavable protein of FIG. 8;

[0073] FIG. 12 schematically illustrates the structure and activities of various secPHEX constructs;

[0074] FIG. 13 graphically illustrates through fluorimetric measurement of the alkaline phosphatase activity in the soluble cell extract and spent medium of HEK293 transiently transfected with expression vectors encoding sALP-D.sub.10 and sALP;

[0075] FIG. 14 graphically illustrates the detection of sALP and sALP-D.sub.10 by Western blotting with the specific B4-78 antibody in the spent media and cell extract of HEK-293 after transient transfection. (Panel A: Ponceau red staining; Panel B: Blot a-B4-78). Shown on the left are the sizes of the molecular weight markers;

[0076] FIG. 15 graphically shows the binding to bone mineral phase of a deca-aspartate fused to secreted alkaline phosphatase;

[0077] FIG. 16 shows A. the nucleotidic sequence (SEQ ID NO: 5) of a soluble alkaline phosphatase; and B. the amino acid sequence (SEQ ID NO: 6) of that soluble alkaline phosphatase;

[0078] FIG. 17 shows A. the nucleotidic sequence (SEQ ID NO: 7) encoding a conjugate of the present invention, namely sALP-D.sub.10; and B. the amino acid sequence (SEQ ID NO: 8) of that conjugate; and

[0079] FIG. 18 graphically shows the effect of D10-sALP on PPi-mediated mineralization inhibition.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0080] The present invention showed that specific poly-aspartic peptides fused in frame to a protein, as exemplified herein by the gluthatione-S-transferase protein (GST), used as a reporter protein, by sPHEX and by sALP, can significantly increase the bone binding capacity of these proteins.

[0081] The present invention is illustrated in further details by the following non-limiting examples.

[0082] Table 1 presents the sequence of oligonucleotides used in Examples 1 to 7.

TABLE-US-00001 TABLE1 SEQUENCEOFSYNTHETICOLIGONUCLEOTIDESUSEDINEXAMPLES1TO7 D.sub.6 SEQIDNO:9. 5-GATCCGATGACGATGACGATGACGC-3 SEQIDNO:10. 5-GGCCGCGTCATCGTCATCGTCATCG-3 D.sub.10 SEQIDNO:11. 5-GATCCGATGACGATGACGATGACGATGACGATGACGC-3 SEQIDNO:12. 5-GGCCGCGTCATCGTCATCGTCATCGTCATCGTCATCG-3 D.sub.16 SEQIDNO:13. 5-GATCCGATGACGATGACGATGACGATGACGATGACGATGACGATGACGATGACGC-3 SEQIDNO:14. 5-GGCCGCGTCATCGTCATCGTCATCGTCATCGTCATCGTCATCGTCATCGTCATCG-3 hMEPE SEQIDNO:15. 5-GATCCGATGACAGTAGTGAGTCATCTGACAGTGGCAGTTCAAGTGAGAGCGATGGTGACGC-3 SEQIDNO:16. 5-GGCCGCGTCACCATCGCTCTCACTTGAACTGCCACTGTCAGATGACTCACTACTGTCATCG-3 hStatherin SEQIDNO:17. 5-GATCCGATTCATCTGAAGAGAAATTTTTGCGTAGAATTGGAAGATTCGGTGC-3 SEQIDNO:18. 5-GGCCGCACCGAATCTTCCAATTCTACGCAAAAATTTCTCTTCAGATGAATCG-3 hMGP SEQIDNO:19. 5-GATCCTGTTATGAATCACATGAAAGCATGGAATCTTATGAACTTAATCCCTTCATTGC-3 SECIDNO:20. 5-GGCCGCAATGAAGGGATTAAGTTCATAAGATTCCATGCTTTCATGTGATTCATAACAG-3 hOsteopontin SEQIDNO:21. 5-GATCCCAGAATGCTGTGTCCTCTGAAGAAACCAATGACTTTAAAGC-3 SEQIDNO:22. 5-GGCCGCTTTAAAGTCATTGGTTTCTTCAGAGGACACAGCATTCTGG-3 hBSP2 SEQIDNO:23. 5- GATCCGGCAGTAGTGACTCATCCGAAGAAAATGGAGATGACAGTTCAGAAGAGGAGGAGGAAGC 3 SEQIDNO:24. 5-GGCCGCTTCCTCCTCCTCTTCTGAACTGTCATCTCCATTTTCTTCGGATGAGTCACTACTGCCG- 3 hIGFBP5 SEQIDNQ:25. 5-GATCCCGCAAAGGATTCTACAAGAGAAAGCAGTGCAAACCTTCCCGTGGCCGCAAGCGTGC-3 SEQIDNO:26. 5-GGCCGCACGCTTGCGGCCACGGGAAGGTTTGCACTGCTTTCTCTTGTAGAATCCTTTGCGG-3 M81736CBS SEQIDNO:27. 5-AGTCGGGATCCGGAACAAGCAGCGTGTTCTAC-3 SEQIDNO:28. 5-AGATCGCGGCCGCTCAATTGTGCACGGTGTGATTAAAGG-3 D.sub.10 SEQIDNO:29. 5-CCGGAGATGACGATGACGATGACGATGACGATGACT-3 SEQIDNO:30. 3-TCTACTGCTACTGCTACTGCTACTGCTACTGAGGCC-5

EXAMPLE 1

Bone Binding of GST-D.SUB.6., GST-D.SUB.10 .and GST-D.SUB.16

[0083] Recombinant DNA technology was used to generate a plasmid containing a nucleic acid encoding GST followed in frame by a nucleic acid encoding a D.sub.6, D.sub.10 or D.sub.16 acidic peptide. To obtain the GST-D.sub.6, GST-D.sub.10 and GST-D.sub.16 conjugates, the oligonucleotide of SEQ ID NO:9 (see Table 1) was first mixed with the oligonucleotide of SEQ ID NO:10, oligonucleotide of SEQ ID NO:11 mixed with oligonucleotide of SEQ ID NO:12, and oligonucleotide of SEQ ID NO:13 mixed with oligonucleotide of SEQ ID NO:14. This procedure generated duplex oligonucleotides coding for D.sub.6, D.sub.10 and D.sub.16, respectively, and having extremities compatible with cloning in the pGEX3T-4 plasmid (Pharmacia biotechnology) pre-digested with restriction endonucleases BamHI and NotI. pGEX3T-4 vectors were transformed into AP401 protease minus E. coli bacteria strain (Ion::mini tetR ara-lac-pro naIA argEam rifR thiI [F pro AB lacIq Z M15]).

[0084] Positive bacterial colonies were used to seed a 10 ml pre-culture of double YT media and 100 mg/litre ampicilin. Bacteria were grown overnight at 37 C. in an orbital shaker set at 250 rpm. The pre-culture was added to 500 ml of fresh double YT ampicilin media in a 2 litres Erlenmeyer flask. Bacteria were let to grow at 37 C. under orbital shaking until a 595 nm optical density of 0.7 was reached. Protein expression was then induced by adding 500 l of 0.1 M IPTG solution and the bacteria put back to incubation for 2 hours. Bacteria were spun down at 8000g for 10 minutes at 4 C. The pellet was suspended in 25 ml of ice-cold PBS containing Complete-EDTA caplet protease inhibitor (Boehringer Mannheim) and frozen at 20 C.

[0085] Bacteria cells were thawed and disrupted on ice with 6 pulses of sonication every 50 seconds prior to centrifugation at 12000g for 10 minutes at 4 C. Supernatant was mixed with 500 l of GS-4B wet resin (Amersham Pharmacia Biotech) equilibrated with PBS. The resin was kept as a suspension during the overnight incubation at 4 C. The resin was rinsed with PBS until 280 nm optical density was below 0.01. Resin was then laid on an empty column and proteins eluted with 10 mM glutathione dissolved in PBS. The pooled elution fractions were dialyzed against 1 mM sodium PO.sub.4 pH 7.4 and 150 mM NaCl. Dialyzed proteins were filtered in a sterile environment on 0.22 m PES membrane and kept at 4 C. Typically 40 and 60 mg of pure proteins were recovered per litre of culture respectively. FIG. 1 shows an example of an SDS-PAGE analysis of the purified GST and GST-D.sub.10. Purified proteins were iodinated using Iodo-Beads Iodination Reagent (Pierce).

[0086] GST and peptide-fused GST were dialyzed against PBS and concentration set to 2 mg/ml. Iodination reaction was initiated by adding 2 PBS-rinsed Iodo-Beads to 2 mCi of Na125I (100 Ci/l, ICN) dissolved in 500 l of PBS. Beads were incubated at room temperature for five minutes before adding 1 mg of dialyzed protein. The iodination reaction proceeded for 15 minutes before the bead was removed and rinsed in 500 ml of PBS. To the final 1.5 ml of iodinated protein solution, 15 l of 6 mM NaI was added to dilute non-specific radioactivity. The mixture was then desalted using PD-10 gel filtration columns (Amersham Pharmacia Biotech) equilibrated with PBS. Proteins eluted in the void volume. They were concentrated and dialysed against the in vivo buffer (1 mM sodium PO4 pH 7.4 and 150 mM NaCl) using Centriprep-YM10 cartridges (Amicon). Radioactivity was measured using a gamma radiation counter, protein concentration was assessed by the Bradford assay and .sup.125I chemical linkage to proteins was revealed by autoradiography of dried SDS-PAGE. Iodinated samples were kept at 4 C.

Bone Binding Ability of GST-Poly-Aspartic Peptides Fusion Proteins Compared to that of GST Alone

[0087] The iodinated GST-fusion proteins were injected to mice under isoflurane anesthesia as an intravenous bolus through the subclavian vein. A dose of 1 mg of iodinated protein/per kg body weight was injected. The maximum dose volume was set at 10 ml/kg. Duration of treatment was sixty minutes. Ten and sixty minutes after injection, blood samples (0.1 to 0.2 ml) were collected via the subclavian vein under anesthesia into serum/gel clotting activator Microvette tubes (Sarstedt, #20.1291). At necropsy, blood samples were collected and animals were sacrificed by exsanguination from the heart under isoflurane anesthesia. Organs (kidneys, liver, femurs, tibias and thyroid) were collected, rinsed in saline 0.9% USP, blotted on gauze and transferred into gamma counter tubes. Serum samples and organs were weighted and radioactivity was measured. Results were expressed as percentage of injected dose. Neither D.sub.10-GST nor D.sub.16-GST promoted binding to other organs than bone. This showed the specificity of these conjugates to bone (Data not shown).

[0088] FIG. 2 shows that GST-D.sub.6 fusion protein did not bind more to tibia or femur than GST alone. In contrast, D.sub.10 and D.sub.16 peptide motifs promoted GST binding to bones.

[0089] The fact that D.sub.6, a peptide shown to successfully deliver small molecules to bone could not successfully deliver a protein, namely GST, to bone shows that it is not predictable whether a specific acidic peptide known to effectively deliver a small molecule to bone will also be effective in delivering a protein to bone.

EXAMPLE 2

Binding Ability of GST Fused with Various Peptides

[0090] Human matrix extracellular phosphoglycoprotein (hMEPE) is a protein synthesized by osteoblasts that shows major similarities to a group of bone and teeth mineral matrix phosphor-glycoproteins, proteins known to naturally bind to bone matrix (8). Of particular importance, hMEPE presents at its carboxy-terminus a sequence of 18 amino acid residues (DDSSESSDSGSSSESDGD) (SEQ ID NO: 31) similar to acidic peptides found in dentin phosphorin and dentin sialophosphoprotein, both known to bind to bone matrix (8).

[0091] Human Statherin (hStatherin) is a protein synthesized by salivary glands, which similarly to histatin directly modulates hydroxyapatite nucleation and/or growth. Of particular importance, hStatherin presents a sequence of 15 amino acid residues at positions 20 to 34 (DSSEEKFLRRIGRFG) (SEQ ID NO: 32) that was shown to bind tightly to hydroxyapatite (9).

[0092] Human Matrix Gla Protein (hMGP) is a protein synthesized by vascular smooth muscle cells and chondrocytes that functions as an inhibitor of hydroxyapatite polymerization by binding to crystal nuclei. Of particular importance, hMGP presents at its amino-terminus a sequence of 17 amino acid residue at positions 19 to 35 of the open reading frame (CYESHESMESYELNPFI) (SEQ ID NO: 33) similar to phosphorylated gamma carboxyglutamic acidic peptides found in osteocalcin known to bind to bone matrix, and thought to promote binding to bone matrix (10).

[0093] Human osteopontin (hOPN) is a protein synthesized by osteoblasts that regulates hydroxyapatite crystal growth. This protein belongs to the bone sialophosphoprotein family. Of particular importance, hOPN presents a sequence of 13 amino acid residue (QNAVSSEETNDFK) (SEQ ID NO: 34) at positions 58 to 70 of the open reading frame. This sequence shows a high level of homology among mammal species. Secondary structure prediction makes this sequence appropriate to solvent exposure and this sequence was shown to be phosphorylated at its serine residues. This latter characteristic is thought to affect binding to bone matrix (11).

[0094] Human Bone SialoProtein II (hBSP2) is a protein synthesized by osteoblasts that shows major similarities to a group of bone and teeth mineral matrix phosphor-glycoproteins, proteins known to naturally bind to bone matrix. Of particular importance, hBSPII presents at its amino-terminus a sequence of 18 amino acid residues at positions 62 to 79 of the open reading frame (GSSDSSEENGDDSSEEEE) (SEQ ID NO: 35) similar to acidic peptides found in dentin phosphorin and MEPE, and thought to promote binding to bone matrix (8).

[0095] Human Insulin-like Growth Factor binding protein-5 (hIGFBP5) is synthesized by osteoblasts. This protein, similarly to proteins of the IGFBP family, is thought to regulate osteoblast function in the bone remodeling process. Of particular importance, hIGFBP5 presents a sequence of 18 amino acid residues at positions 221 to 238 of the open reading frame (RKGFYKRKQCKPSRGRKR) (SEQ ID NO: 36) that was shown to bind tightly to hydroxyapatite (12).

[0096] Staphylococcus aureus collagen adhesin (M81736) is a protein expressed at the surface of S. aureus that promotes bacteria binding to collagen matrix of mammalian bone and cartilageneous tissues. Such a binding was reported to be instrumental in the development of pathogenesis such as osteomyelitis and infectious arthritis. Of particular importance, the collagen binding domain (CBS) of this adhesin was reported to encompass 151 amino acid residues (G168 to N318) of the open reading frame of the protein (13, 14). The amino acid primary sequence being the following:

TABLE-US-00002 (SEQIDNO:37) GTSSVFYYKTGDMLPEDTTHVRWFLNINNEKSYVSKDITIKDQI QGGQQLDLSTLNINVTGTHSNYYSGQSAITDFEKAFPGSKITVDNTKNTI DVTIPQGYGSYNSFSINYKTKITNEQQKEFVNNSQAWYQEHGKEEVNGKS FNHTVHN.

[0097] Plasmids containing the acidic peptide sequences derived from hMEPE, hStatherin, hMGP, hOPN, hBSP2, hIGFBP5 and CBS following GST in frame were constructed to determine whether they could promote bone targeting of a recombinant protein. Recombinant DNA technology as described in Example 1 was used to generate plasmids for hMEPE, hStatherin, hMGP, hOPN, hBSP2 and hIGFBP5 derived peptides. The oligonucleotide pairs identified in Table 1 for each of these peptides were mixed to obtain the corresponding GST-acidic peptide fusion protein. This procedure generated duplex oligonucleotides coding for these acidic peptides and having extremities compatible with cloning in the pGEX3T-4 (Pharmacia biotechnology) plasmid pre digested with restriction endonucleases BamHI and NotI.

[0098] A CBS-containing plasmid was constructed as follows. A synthetic gene corresponding to the CBS sequence was obtained from Bio S&T (Montreal) and inserted in plasmid pLIV Select. Oligonucleotides of SEQ ID NO: 27 and 28 were used as primers in PCR reactions with plasmid pLIV Select containing the CBS gene to amplify the CBS specific sequences. pGEX-4T-3 vectors were transformed into AP401 protease minus E. coli bacteria strain (Ion::mini tetR ara-lac-pro naIA argEam rifR thiI [F pro AB lacIq Z M15]).

[0099] Protein production and purification, and pharmacodistribution of the iodinated fusion protein were performed as described in Example 1.

[0100] None of these GST-acidic peptides was shown to bind to bones (result not shown).

[0101] The fact that the peptide derived from statherin, a peptide shown to successfully deliver a small portion of osteopontin to bone, could not successfully deliver the GST protein to bone shows that it is not predictable whether a specific acidic peptide known to effectively deliver a small peptide to bone will also be effective in delivering a protein to bone.

EXAMPLE 3

D.SUB.10 .Increases sPHEX's Ability to Correct Alkaline Phosphatase Levels in Mice

[0102] PHEX is a metallopeptidase that is widely believed to control the level of bone peptide factors involved in the regulation of mineralization and kidney phosphate homeostasis. PHEX is expressed at the surface of osteoblasts and osteocytes in contact with or imbedded in the bone matrix. This example provides data on the design, production and purification of an extended form of sPHEX containing at its N-terminus a sequence of 10 aspartic acid residues designed to anchor itself to the bone matrix.

D.SUB.10.sPHEX Expression Vector

[0103] A BspEI endonuclease restriction site was inserted by site directed mutagenesis (QuickChange, Stratagene) into the pCDNA3-RSV-sPHEX-NEO vector (Boileau G. et al., Biochem. J. (2001) 355, 707-13) using the following oligonucleotide primers:

TABLE-US-00003 (SEQIDNO:38) 5- CAGTCAAGGTCTCTTATCCGGAAGTCTCCAAGCTAAACAGG-3 and (SEQIDNO:39) 5- CTGTTTAGCTTGGAGACTTCCGGATAAGAGACCTTGACTGG-3.

[0104] The hexamer BspEI sequence (underlined) was inserted in frame with and upstream of the sPHEX DNA sequence. This construct encodes a recombinant protein which is cleavable between the leucine and serine at positions 41 and 42, respectively in FIG. 8. It is constituted therefore of two exogenous amino acids, followed downstream by a deca-aspartate, which is in turn followed by two additional exogenous amino acids. These 4 exogenous amino acids derive from the cloning strategy used to produce the conjugate. These exogenous amino acids were shown not to defeat the enzymatic activity of the conjugate (See FIG. 12 showing the specific activity of this construct) but may be dispensed with. Downstream of these exogenous amino acids is an ectodomain fragment of the native PHEX starting therefore with the serine at position 46 of the sequence presented in FIG. 10. The modified pCDNA3-RSV-NEO vector was cleaved with BspEI and then digested with alkaline phosphatase to remove the 5 phosphate moieties. An oligonucleotide duplex coding for deca-aspartate: [5-CCGGAGATGACGATGACGATGACGATGACGATGACT-3 (SEQ ID NO: 29) and 3-TCTACTGCTACTGCTACTGCTACTGCTACTGAGGCC-5 (SEQ ID NO: 30)] was first phosphorylated on its 5 ends with T4 polynucleotide kinase and ligated to the BspEI digested vector. This yielded the pCDNA3-RSV-D.sub.10sPHEX-NEO vector (FIG. 3). This vector comprised the sequence presented in FIG. 7 which encodes the recombinant cleavable PHEX having the amino acid sequence presented in FIG. 8.

Expression of Recombinant D.SUB.10.sPHEX

[0105] To induce the stable expression of the D.sub.10sPHEX protein, the pCDNA3-RSV-D.sub.10sPHEX-NEO vector was transfected in LLC-PK1 cells (Porcine Kidney cells; ATCC No. CRL-1392) using the Lipofectamine-Plus liposome transfection kit (Invitrogen). Transfected cells were selected by adding 400 g/ml G-418 (Life Technologies) to the medium. Clones of G-418 resistant cells were screened for D.sub.10sPHEX expression using the PHEX fluorescent enzymatic assay [Campos M. et al. Biochem. J. (2003) 373, 271-9]. The apparent molecular weight of the protein recovered in the spent medium was estimated by immunoblotting using a monoclonal antibody raised against a recombinant human PHEX fragment (K121-E294) as described previously (Ruchon A F et al. J. Bone Miner. Res. (2000) 15, 1440-1450). A G-418 resistant clone expressing 1 to 2 mg of D10sPHEX per litre was used for protein production. Cells were seeded in Cellstack-10 (Corning) at a density of 710.sup.7 in 1.75 litres of media (199 media, 6% FBS, 1 mM NaPyruvate, Penicillin 110.sup.5 U/litre, Streptomycin 100 mg/litre and 1% G-418. D.sub.10sPHEX expression was increased by incubating the cells in 1.75 litre of DMEM+10 mM sodium butyrate for four days at 37 C. and 5% CO.sub.2 prior to harvest of the spent medium.

Purification and Characterization

[0106] Cell supernatant was centrifuged at 500g for 5 minutes at 4 C. and filtered on fiberglass (Fisher, APFC09050) and concentrated 10 to 40 times using an Ultrasette 30 tangential flow filtration device (Pall Canada). The pH of the solution was brought to 5.6 with 1 M acetic acid before an overnight dialysis at 4 C. against 50 mM sodium acetate, 100 mM NaCl pH 5.6 (SP-buffer). The dialyzed supernatant was loaded, at a flow rate of 4 ml/min, on a 20 ml SulfoPropyl-Sepharose cation-exchange column (Amersham Pharmacia Biotech) previously equilibrated with SP-buffer. The column was washed with the same buffer at the same flow rate until 280 nm absorbance baseline was reached. Most of the contaminant proteins were then eluted with a 226 mM NaCl step in the SP buffer. D.sub.10sPHEX was then eluted with a 280 mM NaCl step (FIG. 4A). Fractions were analyzed by SDS-PAGE and with the PHEX enzymatic activity assay. Fractions containing sPHEX were pooled and extensively dialyzed against 20 mM MOPS pH 7, 250 mM NaCl prior to loading on a 5 ml Blue-Sepharose HP (Amersham Pharmacia) column at 5 ml/min. The column was rinsed, at the same flow rate with the same buffer and most of the D.sub.10sPHEX protein was recovered by increasing the NaCl concentration stepwise to 350 mM (FIG. 4B). Purity of the final fraction was greater than 95%. Alternatively, the Blue-Sepharose could be replaced by Heparin-Sepharose (Amersham Pharmacia) on which D.sub.10sPHEX binds tightly over a range of pH (5 to 8). D10sPHEX was eluted by using NaCl gradient. Purity was determined to be above 90%. D.sub.10sPHEX was concentrated and dialyzed against 1 mM sodium PO4 pH 7.4, 150 mM NaCl using Centriprep-50 cartridges. Dialyzed sample was filtered in a sterile environment on 0.22 m membrane. Purified D.sub.10sPHEX was shown to remain stable over months at 4 C. Protein concentrations were determined using the Bradford method (DC protein assay kit; Biorad) with bovine serum albumin (BSA) as a standard. Protein purity was assessed by Sypro-Ruby (Molecular Probes) staining of proteins resolved on SDS-PAGE 4-12% (FIG. 3). D.sub.10sPHEX enzymatic activity was determined using the fluorigenic substrate.

Effect of sPHEX and D.sub.10-sPHEX Injections on Circulating Levels of Alkaline Phosphatase in Hyp Mice

[0107] The X-linked Hyp mice harbors a large deletion in 3 region of the PHEX gene and is the murine homologue of human X-linked hypophosphatemia (XLH). These mice therefore represent a useful model to study the pathophysiology of XLH as well as a to test the efficacy of therapeutic agents in preclinical studies.

[0108] The potential therapeutic effect of D.sub.10sPHEX and sPHEX was thus investigated with bolus intravenous injection to Hyp/Y mice over a 2 week period.

[0109] D.sub.10sPHEX and sPHEX were dialyzed against vehicle and the solutions were filtered through 0.22 m low binding protein filter. The solutions were aliquoted and re-assayed for enzymatic activity and concentration by fluorogenic enzymatic assay and Bradford method, respectively.

[0110] Each mouse was anesthetized with vaporized Isoflurane (2%) and D.sub.10sPHEX, or sPHEX were injected as an intravenous bolus through the subclavian vein. The dose was 5 mg/kg of body weight for each group. The animals were treated once daily for 14 consecutive days. Blood samples (0.1-0.2 ml) were collected via the subclavian vein under anesthesia on study days 3 and +15 (before necropsy, 24 hours after last injection). Total Alkaline phosphatase (ALP) levels were assayed in diluted serum (30 l of serum sample with 90 l of 0.9% saline USP). Although, appropriate dosages for human patients are not proportional to those used in mice, these dosages are predictive of the dosages ranges that could be suitable in humans using published tables.

[0111] As seen in FIG. 6 the D.sub.10-extended form of sPHEX induced a larger decrease in alkaline phosphatase levels than the normal sPHEX form.

EXAMPLE 4

D.SUB.10 .Fusion to Recombinant GST Increases its Binding to the Mineral Phase of Bone In Vitro

Fluorescein Labelling of Purified Proteins

[0112] Recombinant purified proteins were labelled with fluorescein-isothiocyanate (FITC, Molecular Probes F143). Reaction was carried out by adding proteins to 10 mM sodium phosphate, 50 mM NaCl buffer pH 7 at a final protein concentration of 1 mg/ml. Labelling reaction was started by adding FITC dissolved in DMSO at a concentration of 20 mg/ml to reach 20:1 molar ratio with respect to the protein concentration. The mixture was left to react at room temperature for an hour. Labelled protein was separated from the free fluorescein on a PD-10 column (Pharmacia) prior to dialysis in the binding buffer (1 mM sodium phosphate 150 mM NaCl, pH 7.4).

Preparation of the Mineral Phase of Bones

[0113] Long bones were dissected from a rat and crushed to powder in a liquid nitrogen cooled mortar. The powder was either kept at 80 C. or directly used. An aliquot of the powder (300 mg) was washed 3 times with 8 ml of PBS and 8 ml of 1 M HCl were added. The mixture was kept in suspension on a rotating mixer for 1 hour at room temperature. The insoluble fraction was spun down and the clear acidic supernatant collected. This acidic solution was stable at room temperature for at least two weeks.

Binding Reaction

[0114] Aliquots of 20 l of the acidic bone extract were mixed with 2 l of 10 M NaOH and the precipitate was pelleted 10,000g for 3 minutes at room temperature. The pellet was rinsed twice by resuspending in 100 l of binding buffer. The bone extract was then mixed with 100 l of a solution containing 5 to 45 g of fluorescein-labelled protein in the binding buffer to which phosphate was added to reach a final concentration of 80 mM. The samples were incubated for 30 minutes at room temperature on the rotating wheel to keep the mineral phase in suspension. The samples were then centrifuged for 3 minutes at room temperature. The pellet containing the bound protein was dissolved in 200 l of 0.5 M EDTA pH 8. To estimate the amount of free protein present, 100 l of 0.5 M EDTA .sub.pH 8 was added to the supernatant. Fluorescence of the different samples was measured on a 96 wells plate reader set at 494 nm for excitation and 516 nm for emission.

Results

[0115] Samples containing 50 g of fluorescein-labelled GST and GST-D.sub.10 were used in the binding assay described above. FIG. 9A shows that fusion of the D.sub.10 sequence to GST caused a 6-fold increase in binding to the mineral phase of bone.

EXAMPLE 5

D.SUB.10 .Fusion to sPHEX Increases its Binding to Bone

[0116] Using a procedure analogous to that described in Example 4 above, samples containing 50 g of fluorescein-labelled sPHEX and D.sub.10sPHEX were used in a binding assay. FIG. 9B shows that fusion of the D.sub.10 sequence to sPHEX caused a 4.3 increase in binding to the mineral phase of bone.

[0117] In contrast, D.sub.6-sPHEX was constructed and tested after in vivo injection in animals (as described in Example 1 above) and did not promote binding of recombinant proteins to bone (Data not shown).

EXAMPLE 6

D.SUB.10 .Fusion to a Soluble Form of Alkaline Phosphatase Increases its Targeting to the Mineral Phase of Bone

[0118] Construction of Expression Vectors Encoding Human Recombinant Soluble Phosphatase Alkaline, sALP and sALP-D.sub.10

[0119] The human full length cDNA encoding tissue non-specific alkaline phosphatase (ALP) was obtained from bone marrow polyA RNA (Clonetech) by RT-PCR. Briefly, 20 ng of polyA was reverse transcribed with SuperscriptII and an oligo dT.sub.12-18 using the First Strand Synthesis System (Invitrogen). An aliquot representing 1/20.sup.th of the RT step was used directly in a PCR reaction with ALP specific oligos (forward 5-gataaagcaggtcttggggtgcacc-3 (SEQ ID NO: *); reverse 5-gttggcatctgtcacgggcttgtgg-3 (SEQ ID NO: *)) and the Expand High Fidelity Enzyme Kit (Roche). The resulting ALP specific product (1644 bp) was separated on and purified from an agarose gel (1%) using the Qiaquick Gel Extraction Kit (QIAGEN). The ALP cDNA was then ligated into the pCR4-blunt-TOPO vector (Invitrogen), transformed into Top10 bacteria (Invitrogen), and a positive clone identified by colony PCR. The identity of the cDNA was verified by automated DNA sequencing.

[0120] Secreted forms of ALP (sALP) having the GPI anchor signal removed were constructed by PCR using Expand High Fidelity Enzyme Kit. They comprised residues 1-502 followed by either a stop codon (sALP) or a deca aspartate targeting motif and a stop codon (sALP-D10). In both cases the forward primer (5-tggatccaccatgatttcaccattcttagtac-3 (SEQ ID NO: 40)) covered the initiator methionine (underlined) and included a BamHI site (italicized). The reverse primers (sALP: 5-ttctagactacgagctggcaggagcacagtggccg-3 (SEQ ID NO: 41); sALP-D.sub.10 5-ttctagactagtcgtcatcatcgtcatcatcgtcgtcatccgagctggcaggagcacagtggccg-3 (SEQ ID NO: 42)) contained a stop codon (underlined) and an XbaI site (italicized). The PCR products were digested with BamHI and XbaI and cloned into the pCDNA3.1-RSV that had been pre-digested with the same enzymes. Plasmid DNA were sequenced.

ALP Fluorescent Enzymatic Assay

[0121] Enzymatic activity of sALP and sALP-D.sub.10 was assayed using 4-methylumbelliferyl phosphate (MUP, Molecular Probes, M8425) as a fluorigenic substrate according to Gee K R et al. (Anal. Biochem. 273, 41-48 (1999)) Typically, the assay was carried out at 37 C. in 96-well plates in a final volume of 200 l with 10 M of MUP. Readings were recorded using a Spectramax Gemini (Molecular Devices) plate reader every minute for 30 minutes at 450 nm upon excitation at 360 nm. Emission wavelength cut-off was set at 435 nm. ALP initial speed rate was estimated by linear regression fitting (with r.sup.2 equal or greater than 0.98).

Expression of Recombinant sALP and sALP-D.sub.10 Proteins

[0122] In order to determine whether the recombinant sALP and sALP-D.sub.10 proteins were secreted, each construct (pCDNA3-RSV-sALP-NEO and pCDNA3-RSV-sALP-D.sub.10-NEO) was transiently transfected in HEK-293S cells (Human Embryonic Kidney cells; ATCC No. CRL-1392) using the Lipofectamine-Plus liposome transfection kit (Invitrogen). HEK-293S cells were also mock-transfected as a negative control. The day after transfection, cells were incubated for 24 h in serum-free DMEM. The conditioned media were collected and centrifuged at 14000 RPM for 5 min at 4 C. to remove dead cells and debris. The supernatants were assayed for sALP or sALP-D.sub.10 enzymatic activity and expression using the ALP fluorescent enzymatic assay and Western blotting respectively. For Western blotting, the spent media were precipitated for 1 h on ice with trichloroacetic acid (final concentration 10% (v/v)). The precipitated proteins were spun down at 14000 RPM for 20 min at 4 C., washed once with chilled acetone, dried, and resuspended in 60 l 1 Laemmli sample buffer with DTT and boiled for 5 min.

[0123] To evaluate the intracellular content of sALP and sALP-D.sub.10 the cells were washed 3 times with PBS and lysed with 200 l Tris-HCl 50 mM (pH 8) containing 150 mM NaCl and 1% NP-40 on ice for 20 min. The lysates were spun down and the supernatant was assayed for sALP or sALP-D.sub.10 enzymatic activity and expression using the ALP fluorescent enzymatic assay and Western blotting, respectively. For Western blotting, 50 l aliquots were mixed with 10 l 6 Laemmli sample buffer with DTT and boiled for 5 min.

[0124] Samples were loaded on a Novex precast 4-12% Tris-Glycine polyacrylamide gel (Invitrogen) and transferred onto 0.45 m nitrocellulose (Protran, Schleicher&Schuell, Keene, N.H.) with Tris-glycine containing 10% methanol. The membrane was stained with Ponceau red and blocked for 1 h at room temperature with PBS containing 0.05% Tween 20 (PBST) and 5% dried milk. The membrane was then sequentially incubated at room temperature with the anti-hBAP antibody (mAb 4B-78, Developmental Studies Hybridoma Bank) (1:1000 in PBST with 5% dried milk) and a rabbit anti-mouse IgG coupled to horseradish peroxidase (Sigma) (1:12000 in PBST with 5% dried milk). The signal was developed with the Western Lightning Chemiluminescence Reagent Plus (PerkinElmer).

[0125] The ALP enzymatic activity measured in the conditioned media of HEK293 after transient transfection was very high and of similar magnitude for pCDNA3-RSV-sALP-NEO (sALP) and pCDNA3-RSV-sALP-D.sub.10-NEO (sALP-D.sub.10) (FIG. 13). This activity was specific to the plasmid DNA transfected as it was undetectable in mock-transfected cells (mock). The relative activity measured in the media was 35-times greater than that measured in the cell extracts thus attesting to the secretory nature of sALP and sALP-D.sub.10. Accordingly, for both sALP and sALP-D.sub.10, immunoblotting using a monoclonal antibody raised against recombinant tissue non-specific human alkaline phosphatase (mAb 4B-78, Developmental Studies Hybridoma Bank) revealed a much stronger signal in the conditioned media than in the cell extracts (FIG. 14B, compare lanes 2, 3 vs. 5, 6). No signal was visualized in the mock-transfected samples (FIG. 14B, lanes 4 and 7). The signal appearing in the mock-transfected cells consists of BSA trace. The apparent molecular weight of the protein detected was estimated to be 70 kDa in the cell extracts (arrow) and slightly higher in the conditioned media (arrowhead). Ponceau red staining of the membrane was performed to monitor the uniform loading of samples (FIG. 14A).

Generation of HEK293 Cells Constitutively Secreting sALP and sALP-D.sub.10

[0126] To induce the stable expression of the sALP and sALP-D.sub.10 proteins, the pCDNA3-RSV-sALP-NEO and pCDNA3-RSV-sALP-D.sub.10-NEO vectors was transfected separately in HEK-293S cells using the Lipofectamine-Plus liposome transfection kit (Invitrogen). Transfected cells were selected by adding 800 g/ml G418 (Life Technologies) to the medium. For each transfection a pool of G-418 resistant cells were analyzed for sALP or sALP-D.sub.10 expression in the spent culture media using the ALP fluorescent enzymatic assay. The conditioned media collected from the stable cell lines were used for the binding assay study on the bone mineral.

Binding to Reconstituted Mineral Phase of Bone

[0127] Aliquots of 20 l of the acidic bone extract were mixed with 2 l of 10 M NaOH and the precipitate was pelleted at 10,000g for 3 minutes at room temperature. The pellet was rinsed twice in 100 l of buffer (1 mM sodium phosphate pH 7.4+150 mM NaCl). The resultant mineral phase of bone (equivalent to 0.37 mg of dried powder) was then mixed with 100 l of a solution containing sALP or sALP-D.sub.10 proteins in the binding buffer (80 mM sodium phosphate pH 7.4+150 mM NaCl). The samples were incubated for 30 minutes at room temperature on the rotating wheel to keep the mineral phase in suspension. The samples were then centrifuged for 3 minutes at room temperature. The pellet containing the bound protein was mixed with 180 l of the ALP enzymatic assay buffer containing 0.1% BSA and the reaction initiated by adding 20 l of 100 M MUP. To allow for more homogeneous assay, conditions the 96 wells plate was shaken for 10 seconds every minute for the duration of the assay.

[0128] Enzymatic activity retained on reconstituted mineral bone phase was compared to the equivalent enzymatic activity added in the binding assay. Values of 0.98% and 13.3% of total protein activity bound to the bone mineral phase were calculated for sALP and sALP-D.sub.10 respectively. A binding difference of more than 13 times in favour of sALP-D.sub.10 suggests that the C-terminal fused deca-aspartate sequence directly targets sALP to the mineral phase of bone. Furthermore, the fact that it was possible to measure directly ALP activity bound to the mineral phase of bone indicates that the enzyme is bound in a catalytically competent form to hydroxyapatite crystals.

[0129] Such fusion protein can be targeted directly to bones where the accumulation of PPi inhibits skeletal mineralization.

EXAMPLE 7

D.SUB.10.-ALP Decreases Inhibitory Effect of Pyrophosphate on Bone Mineralization

[0130] UMR106 cells were grown to confluence. They were then cultured for a further 7 days in media containing 10 mM -glycerophosphate to induce mineralization. Throughout this 7-day culture period, cells were treated with or without 75 M pyrophosphate (PPi), a mineralization inhibitor and a alkaline phosphatase substrate. To assess the ability of alkaline phosphatase to rescue the PPi-induced mineralization inhibition, cells treated with or without PPi were cultured with varying concentrations of semi-purified D.sub.10-sALP produced from HEK293, human embryonic kidney cells. Mineralization was assessed by .sup.45Ca uptake. Parameters used for this experiment are presented in table 2 below.

TABLE-US-00004 TABLE 2 PARAMETERS USED IN D.sub.10-ALP ON PPi-INDUCED MINERALIZATION INHIBITION [ALP] -GP PPi ALP (Units/well) l ALP/well (mM) (M) 0 0 10 0 0 0 10 75 D.sub.10-sALP 1.5 0.5 10 0 D.sub.10-sALP 1.5 0.5 10 75 D.sub.10-sALP 3 1.0 10 0 D.sub.10-sALP 3 1.0 10 75 D.sub.10-sALP 4.5 1.5 10 0 D.sub.10-sALP 4.5 1.5 10 75 D.sub.10-sALP 6 2 10 0 D.sub.10-sALP 6 2 10 75

[0131] 7-days of treatment with PPi resulted in a 43% decrease in mineralization. Co-treatment of cultures with D.sub.10sALP resulted in a dose-responsive rescue of this mineralization inhibition. Treatment with 1.5 units of D.sub.10-sALP resulted in a 30% decrease, 3 and 4.5 units a 24% decrease and 6 units resulted in a 15% decrease in mineralization, corresponding to a 65% rescue of PPi-induced mineralization inhibition.

[0132] These results show that the treatment of mineralizing osteoblast with D.sub.10-sALP dose-responsively rescues mineralization inhibition induced by PPi.

[0133] The above Examples shows that a poly-aspartate fusion to recombinant proteins increases their binding to the mineral phase of bone or to bone tissue and increases the ability of the protein to perform its biological activity as compared to when it is administered alone.

[0134] Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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