WO2005103263A1 - Bone delivery conjugates and method of using same to target proteins to bone - Google Patents
Bone delivery conjugates and method of using same to target proteins to bone Download PDFInfo
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
- C12N9/6494—Neprilysin (3.4.24.11), i.e. enkephalinase or neutral-endopeptidase 24.11
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24011—Neprilysin (3.4.24.11), i.e. enkephalinase or neutral endopeptidase 24.11
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03001—Alkaline phosphatase (3.1.3.1)
Definitions
- the present invention relates to bone delivery conjugates and method of using same to target proteins to bone. More specifically, the present invention relates to bone delivery compositions comprising peptide motifs, engineered within the structure of a protein through recombinant DNA technology to promote binding to bone matrix.
- ERT enzyme replacement therapy
- hypophosphatasia is a rare, heritable type of rickets or osteomalacia that occurs with an incidence of 1 per 100,000 births for the more severe form of the disease. Milder forms are more prevalent.
- mutations inactivate the gene that encodes the tissue- nonspecific isoenzyme of alkaline phosphatase. It is characterized biochemically by subnormal alkaline phosphatase activity in serum. Alkaline phosphatase deficiency in osteoblasts and chondrocytes impairs skeletal mineralization, leading to rickets or osteomalacia.
- hypophosphatasia There is a very broad range of expressivity of hypophosphatasia, spanning from a perinatal form often causing stillbirth from an unmineralized skeleton, to a milder form featuring only premature loss of teeth. Severely affected infants and children inherit hypophosphatasia as an autosomal recessive trait. There are four main forms of the disease: perinatal, infantile, childhood and adult. Perinatal hypophosphatasia manifests during gestation and most affected newboms survive only briefly. Infantile hypophosphatasia becomes clinically apparent before 6 months of age. About 50% of patients die within a year. Childhood hypophosphatasia varies greatly in severity but most of these patients will suffer from skeletal symptoms throughout their life. Adult hypophosphatasia appears during middle age, with symptoms such as painful recurrent stress fractures having poor healing.
- Osteoblasts and chondrocytes are normally rich in tissue- nonspecific alkaline phosphatase where it is attached to the cell surface.
- the lack of alkaline phosphatase activity results in the extracellular accumulation of three phosphorus-compounds believed to be substrates of the enzyme: phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP).
- PDA phosphoethanolamine
- PPi inorganic pyrophosphate
- PPi is an inhibitor of hydroxyapatite crystal growth, and PPi build-up in the disease accounts for the impaired skeletal mineralization. Consequently, providing active enzyme to patients suffering from hypophosphatasia will decrease extracellular PPi levels and improve skeletal mineralization.
- Bone-targeted proteins could be useful not only for the treatment or prevention of hypophosphatasia (loss of function of alkaline phosphatase) but also for the treatment or prevention of other genetic diseases characterized by defective enzymatic activity involved in bone metabolism, such as X-linked hypophosphatemic rickets (XLH) (loss of function of phosphate regulating gene with homology to endopeptidases on the X chromosome (PHEX)).
- XLH X-linked hypophosphatemic rickets
- PHEX X-linked hypophosphatemic rickets
- XLH is the most prevalent of the familial hypophosphatemias
- OMIM 307800, 307810 OMIM 307800, 307810. It is characterized by reduced phosphate reuptake in the kidney, hypophosphatemia, normocalcemia, normal to low plasma 1 ,25- dihydroxyvitamin D3 (1 ,25(OH)2D, calcitriol) levels, normal parathyroid gland function and elevated plasma alkaline phosphatase activity. These changes are associated with growth retardation, lower extremity deformity, radiologic and histomorphometric evidence of rickets and osteomalacia. This disease appears to result from combined renal defects in tubular phosphate reabsorption and vitamin D metabolism, as well as a functional disorder in bone and teeth.
- XLH results from inactivating mutations in the PHEX gene, a member of the zinc metallopeptidase family of type II integral membrane glycoproteins. These mutations prevent the expression of a functional PHEX enzyme at the cell surface of osteoblasts.
- treatment of XLH patients is restricted to supplementation with oral inorganic phosphate (Pi) supplements in four or five divided doses per day, and co-administration of 1,25(OH)2D to compensate for the inappropriate synthesis of 1 ,25(OH)2D.
- Such high doses of phosphate frequently cause gastrointestinal intolerances, particularly diarrhea, leading to patient non-compliance.
- the phosphate load carries the risk of provoking secondary hyperparathyroidism (which may be severe enough to necessitate parathyroidectomy) while on the other hand, administration of excess 1,25(OH)2D may lead to hypercalciuria, hypercalcemia and nephrocalcinosis.
- PHEX enzyme in XLH patients with a functional enzyme obtained through recombinant DNA technology As the normal PHEX enzyme is anchored in osteoblast plasma membrane by a hydrophobic peptide, the natural form of PHEX cannot be produced and purified in sufficient quantities to be used in a pharmaceutical preparation.
- a soluble form of recombinant PHEX (or sPHEX) was engineered and produced in cell cultures, purified and formulated for intravenous (IV) administration (WO 00/50580). sPHEX was then injected in Hyp mice, a mouse model for XLH, as described in co-pending US application no 10/362,259.
- Biphosphonates are known to present high affinity binding to hydroxyapatite (HA), and has been used to target small molecules (4) and proteins (5) to bones.
- HA hydroxyapatite
- this strategy requires chemical modifications of the purified proteins, and presents several disadvantages including possible interference with protein activity and additional purification steps.
- Another strategy to target small molecules to bone has been to conjugate these entities to acidic peptides such as poly-Asp (6).
- acidic peptides such as poly-Asp (6).
- This strategy was developed after the observation that several proteins synthesized by osteoblasts, the bone forming cells, bind to bone matrix through sequences particularly rich in acidic amino acid residues (Asp and Glu). This is the case of osteopontin (7) and bone sialoprotein, two noncollagenous proteins.
- acidic peptides E2-10 and D2-10) were used to target small molecules (i.e. methotrexate, FITC, Fmoc, biotin, estradiol) to hydroxyapatite in vitro.
- Acidic peptides (E ⁇ and D ⁇ -io) were used to target small molecules (i.e. FITC, Fmoc, estradiol) to hydroxyapatite in vivo.
- small molecules i.e. FITC, Fmoc, estradiol
- E ⁇ was shown to confer to BSA, hemoglobin and IgG the ability to bind hydroxyapatite in vitro.
- linking of the acidic sequence was performed chemically.
- the present invention shows that large and complex molecules such as proteins can be fused with acidic peptides to successfully target bone in vivo.
- the protein in the bone delivery conjugate is a soluble phosphate regulating gene with homology to endopeptidases on the X chromosome (sPHEX).
- the structure of the conjugate is: X-Dn-Y-sPHEX-Z.
- the sPHEX has a sequence selected from the group consisting of amino acids 46 to 749 of Figure 10; 47 to 749 of Figure 10; 48 to 749 of Figure 10; 49 to 749 of Figure 10; 50 to 749 of Figure 10; 51 to 749 of Figure 10; 52 to 749 of Figure 10; 53 to 749 of Figure 10; and 54 to 749 of Figure 10.
- n is 10.
- n is 11.
- n is 12.
- n is 13. In an other specific embodiment of this bone delivery conjugate, n is 14. In an other specific embodiment of this bone delivery conjugate, n is 15. In an other specific embodiment of this bone delivery conjugate, n is 16.
- the protein in the conjugate is a soluble alkaline phosphatase (sALP).
- the structure of the conjugate is: Z-sALP-X-Dn-Y.
- sALP is encoded by the sequence as set forth in Figure 16A.
- sALP has the sequence as set forth in Figure 16B.
- n is 10.
- n is 11.
- n is 12.
- n is 13.
- n is 14.
- n is 15.
- n is 16.
- n 10.
- an isolated nucleic acid molecule comprising a polynucleotide sequence selected from the group consisting of: a polynucleotide encoding a polypeptide comprising an amino acid sequence as set forth in Figure 8; a polynucleotide encoding a polypeptide comprising an amino acid sequence as set forth in Figure 11 ; a polynucleotide comprising the nucleotide sequence as set forth in Figure 7; a nucleotide sequence completely complementary to any of the nucleotide sequences in (a) ,(b) or (c); and a nucleotide sequence which is hybridizable under high stringency conditions to any of the nucleotide sequences in (a), (b), (c) or (d), wherein the high stringency conditions comprise: pre-hybridization and hybridization in 6XSSC, ⁇ XDenhardt's reagent, 0.5% SDS and 100mg/ml of denatured fragment
- a recombinant vector comprising said sequence.
- a recombinant host cell comprising said vector.
- an isolated nucleic acid molecule comprising a polynucleotide sequence selected from the group consisting of: a polynucleotide comprising the nucleotide sequence as set forth in Figure 17A; a polynucleotide encoding a polypeptide comprising an amino acid sequence as set forth in Figure 17B; a nucleotide sequence completely complementary to any of the nucleotide sequences in (a) or (b); and a nucleotide sequence which is hybridizable under high stringency conditions to any of the nucleotide sequences in (a), (b) or (c), wherein the high stringency conditions comprise: pre-hybridization and hybridization in 6XSSC, ⁇ XDenhardt's reagent, 0.5% SDS and 100mg/ml of denatured fragmented salmon sperm DNA at 68°C; and washes in 2XSSC and 0.5% SDS at room temperature for 10 min; in 2X
- an isolated nucleic acid molecule encoding a functional soluble PHEX comprising a polynucleotide sequence selected from the group consisting of: a polynucleotide encoding a sPHEX comprising amino acids 54 to 749 as set forth in Figure 10; a polynucleotide encoding a sPHEX comprising amino acids 53 to 749 as set forth in Figure 10; a polynucleotide encoding a sPHEX comprising amino acids 52 to 749 as set forth in Figure 10; a polynucleotide encoding a sPHEX comprising amino acids 51 to 749 as set forth in Figure 10; a polynucleotide encoding a sPHEX comprising amino acids 50 to 749 as set forth in Figure 10; a polynucleotide encoding a sPHEX comprising amino acids 49 to 749 as set forth in Figure 10; a polynucleotide en
- an isolated sPHEX polypeptide comprising a sequence selected from the group consisting of: amino acids 54 to 749 as set for in Figure 10; amino acids 53 to 749 as set for in Figure 10; amino acids 52 to 749 as set for in Figure 10; amino acids 51 to 749 as set for in Figure 10; amino acids 50 to 749 as set for in Figure 10; amino acids 49 to 749 as set for in Figure 10; amino acids 48 to 749 as set for in Figure 10; amino acids 47 to 749 as set for in Figure 10; and amino acids 46 to 749 as set for in Figure 10.
- a bone delivery composition comprising a bone delivery conjugate of the present invention, and a pharmaceutically acceptable carrier.
- a method of treating a condition or disease related to a bone defect characterized by a lack of or an insufficient amount of functional phosphate regulating gene with homology to endopeptidases on the X chromosome comprising administering to a mammal in need thereof a conjugate of the present invention, said conjugate being in a pharmaceutically acceptable carrier.
- the condition or disease is X-linked hypophosphatemic rickets (XLH).
- 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 of the present invention, said conjugate being in a pharmaceutically acceptable carrier.
- the condition or disease is hypophosphatasia.
- a bone delivery conjugate of the present invention for delivering a protein to bone tissue of a mammal.
- a bone delivery conjugate of the present invention for treating a condition or disease related to a bone defect characterized by a lack of or an insufficient amount of functional phosphate regulating gene with homology to endopeptidases on the X chromosome (PHEX), said conjugate being in a pharmaceutically acceptable carrier.
- PHEX X chromosome
- a bone delivery conjugate of the present invention in the manufacture of a medicament for treating a condition or disease related to a bone defect characterized by a lack of or an insufficient amount of functional phosphate regulating gene with homology.
- PHEX X chromosome
- the condition or disease is X-linked hypophosphatemic rickets (XLH).
- a bone delivery conjugate of the present invention for treating a condition or disease related to a bone defect characterized by a lack of or an insufficient amount of functional alkaline phosphatase, said conjugate being in a pharmaceutically acceptable carrier.
- a bone delivery conjugate of the present invention in the manufacture of a medicament for treating a condition or disease related to a bone defect characterized by a lack of or an insufficient amount of functional alkaline phosphatase, said conjugate being in a pharmaceutically acceptable carrier.
- the condition or disease is hypophosphatasia.
- a method of screening peptides for use in a bone delivery protein-peptide conjugate comprising the steps of: fusing a candidate peptide to a reporter protein to form a protein-peptide conjugate; contacting the conjugate with bone tissue or mineral phase of bone; and wherein the candidate peptide is selected when the presence of the reporter protein on bone tissue or mineral phase of bone is higher when it is conjugated with the candidate peptide than when it is not.
- the sPHEX is fused at its N-terminal to D-io In an other specific embodiment, the sPHEX is fused at its N-terminal to Dn In an other specific embodiment, the sPHEX is fused at its N-terminal to D-i 2 In an other specific embodiment, the sPHEX is fused at its N-terminal to D 13 In an other specific embodiment, the sPHEX is fused at its N-terminal to D1 4 In an other specific embodiment, the sPHEX is fused at its N-terminal to D ⁇ 5 In an other specific embodiment, the sPHEX is fused at its N-terminal to D ⁇ 6 .
- the sALP is fused at its C-terminal to D ⁇ 0 .
- the sALP is fused at its C-terminal to Dn.
- the sALP is fused at its C-terminal to D12
- the sALP is fused at its C-terminal to D13
- the sALP is fused at its C-terminal to D14
- the sALP is fused at its C-terminal to D15
- the sALP is fused at its C-terminal to Die.
- any functional soluble protein may be used in the conjugate of the present invention.
- results for conjugates comprising one specific sPHEX or sALP of the present invention are presented herein, it is understood that any other functional sPHEX or sALP may be so used.
- sPHEX means any soluble biologically active fragment of PHEX or mutein thereof.
- Those of skill in the art may prepare expression constructs other than those expressly described herein for optimal production of sPHEX in suitable cell lines transfected therewith.
- skilled artisans may design fragments of cDNA encoding soluble biologically active fragments and muteins of the naturally occurring PHEX which possess the same or similar biological activity to the naturally occurring full-length enzyme.
- a large series of expression vectors may be constructed and tested for expression of a PHEX cDNA. Based on transient transfection experiments, as well as stable transfections, an expression construct may be identified that provides a particularly high level of expression.
- any sPHEX comprising at least a native
- PHEX ectodomain portion starting with the cysteine at position 54 of the sequence presented at Figure 10 is encompassed by the present invention.
- the conjugates according to specific embodiments of the present invention thus are any sPHEX comprising this 54-749 fragment of the native PHEX, preferably the 53-749 native fragment, more preferably the native 52- 749 fragment, more preferably the native 51-749 fragment, more preferably the 50-749 native fragment, more preferably the 49-749 native fragment, more preferably the 48-749 native fragment, more preferably the 47-749 native fragment, and more preferably the 46-749 native fragment, along with a poly- aspartate selected from the group consisting of D-io to D 16 fused immediately upstream of this fragment.
- the conjugate may further optionally comprise one or more additional amino acids 1) upstream from the poly-aspartate; and/or 2) between the poly-aspartate and the native fragment or functional equivalent.
- These amino acids may be any amino acid. According to specific embodiments, they may be selected independently from the group consisting of any amino acid except for cysteine, proline and tryptophan namely those amino acids known to induce disulfide bond formation or changes in conformation.
- amino acids may be present in the conjugate when for instance the cloning strategy used to produce it introduces them in these locations.
- amino acids located upstream of the poly-aspartate in the recombinant cleavable PHEX can be selected according to known parameters so as to provide an adequate substrate for specific enzymes of the secretory pathway (e.g. furin or signal peptidase) of the host cell that will be used to cleave the produced recombinant cleavable PHEXs into a secreted bone targeting sPHEX.
- the likelihood of a designed sequence being cleaved by the signal peptidase of the host cell can be predicted by an appropriate computer algorithm such as that described in Bendtsen et al. (J Mol Biol.
- amino acids at position -3 and -1 from the cleavage site by the signal peptidase desirably have small and non charged side chains.
- amino acids at position -3 Ala, Ser, Gly, Cys, Thr and occasionally Gin, Pro, and Leu.
- those at position -3 should preferably be: Ala, Ser, Gly, Cys, Thr, lie, Leu, Val.
- amino acids in position -6 and -4 from the cleavage site are desirably those capable of inducing the formation of a beta-turn (such as Pro) residues.
- the present invention hence encompasses conjugates comprising additional amino acids that may be selected on the basis of the cloning strategy used to produce a cleavable recombinant PHEX.
- the cleavable recombinant PHEX disclosed in Examples 3 and 4 below contains such additional amino acids upstream of the poly-aspartate and between the poly-aspartate and the native ectodomain sequence.
- the present invention encompasses a conjugate comprising the secPHEX disclosed in co- pending application no.
- WO 02/15918 prepared by fusing NL-1 N-terminal fragment comprising a furin site to the PHEX native ectodomain with the vector pCDNA3/RSV/NL-1-PHEX, and a secPHEX comprising an immunoglobulin fragment at its N-terminal.
- Figure 12 schematically presents the structure of secPHEXs that comprise additional amino acids upstream of the native 46-749 PHEX ectodomain fragment.
- Constructs no. 1 to 3 and 5 could be fused to a poly-aspartate and be used as conjugates of the present invention.
- Construct no. 4 constitutes a conjugate of the present invention: it comprises a D-io poly-aspartate and a native ectodomain fragment.
- the conjugates of the present invention further also encompass sPHEXs comprising deletions at their C-terminal non detrimental to their enzymatic activity.
- the present invention comprises conjugates wherein the poly-aspartate would be attached at the C-terminal of the native PHEX ectodomain fragment.
- ALP is a membrane-bound protein anchored through a glycolipid to its C-terminal.
- This glycolipid anchor (GPI) is added post translationally after removal of a hydrophobic C-terminal end which serves both as transitional membrane anchor and as a signal for the addition of the GPI.
- GPI glycolipid anchor
- the sALP used in Example 6 herein is constituted of an ALP wherein the first amino acid of the hydrophobic C-terminal sequence, namely alanine, is replaced by a stop codon.
- the soluble ALP so formed contains all amino acids of the native and thus active anchored form of ALP.
- the sALP conjugates according to specific embodiments of the present invention thus are any sALP along with a poly-aspartate selected from the group consisting of D- ⁇ 0 to Die fused immediately downstream of this fragment.
- the conjugate may further optionally comprise one or more additional amino acids 1) upstream from the poly-aspartate; and/or 2) between the poly-aspartate and the native sALP fragment or functional equivalent. This is the case for instance when the cloning strategy used to produce the bone targeting conjugate introduces exogenous amino acids in these locations. However the exogenous amino acids should be selected so as not to provide an additional transamination site. The likelihood of a designed sequence being cleaved by the transaminase of the host cell can be predicted as described by Ikezawa (Biol Pharm. Bull. 2002, 25(4) 409-417).
- the conjugates of the present invention further also encompass sALPs comprising deletions at their N-terminal non detrimental to their enzymatic activity.
- the present invention comprises conjugates wherein the poly-aspartate would be attached at the N-terminal of the native ALP anchored fragment or its biologically active fragment.
- the term "recombinant protein” is used herein to refer to a protein encoded by a genetically manipulated nucleic acid inserted into a prokaryotic or eukaryotic host cell.
- the nucleic acid is generally placed within a vector, such as a plasmid or virus, as appropriate for the host cell.
- E. coli has been used as a host for expressing the conjugates of the present invention in the Examples presented herein, a person of ordinary skill in the art will understand that a number of other hosts may be used to produce recombinant proteins according to methods that are routine in the art. Representative methods are disclosed in Maniatis, et al. Cold Springs Harbor Laboratory (1989).
- "Recombinant cleavable protein” as used herein is meant to refer to a recombinant protein that may be cleaved by a host's enzyme so as to produce a secreted/soluble protein.
- ectodomain fragment is meant herein when used in relation to PHEX is meant to refer to PHEX's fragment that is located outside of the cellular membrane when found in its native form.
- bone tissue is used herein to refer to tissue synthesized by osteoblasts composed of an organic matrix containing mostly collagen and mineralized by the deposition of hydroxyapatite crystals.
- the fusion proteins comprised in the bone delivery conjugates of the present invention are useful for therapeutic treatment of bone defective conditions by providing an effective amount of the fusion protein to the bone.
- the fusion protein is provided in the form of a pharmaceutical composition in any standard pharmaceutically acceptable carrier, and is administered by any standard procedure, for example by intravenous injection.
- pharmaceutically acceptable carrier is used herein to refer, when parenteral administration is elected as the route of administration, to pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions.
- non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters.
- Aqueous solvents include water; water-alcohol solutions; physiological saline; buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, fluid and nutrient replenishers; electrolyte replenishers; Ringer's solution containing lactose, or fixed oils.
- the term "effective amount" is used herein to refer to the minimal amount of a pharmaceutical composition that should be administered to a mammal in order to achieve a significant therapeutic effect.
- the dosages will depend on many factors including the mode of administration.
- the amount of protein contained within a single dose will be an amount that effectively prevents, delays or treats bone related undesired condition without inducing significant toxicity.
- an effective amount of the conjugate and compositions of the present invention will comprise an amount of fusion protein which will cause a significant alleviation of clinical symptoms of the condition.
- the effective amount may be given daily, weekly, monthly or fractions thereof.
- a pharmaceutical composition of the invention can be administered in an amount from about 0.001 mg up to about 500 mg per kg of body weight per day (e.g., 10 mg, 50 mg, 100 mg, or 250 mg). Dosages may be provided in either a single or multiple dosage regimen.
- the effective amount is a dose that ranges from about 1 mg to about 25 grams of the conjugate to be targeted to bone per day, from about 50 mg to about 10 grams of the conjugate to be targeted to bone per day, from about 100 mg to about 5 grams of the conjugate to be targeted to bone per day, about 1 gram of the conjugate to be targeted to bone per day, about 1 mg to about 25 grams of the conjugate to be targeted to bone per week, about 50 mg to about 10 grams of the conjugate to be targeted to bone per week, about 100 mg to about 5 grams of the conjugate to be targeted to bone every other day, and about 1 gram of the conjugate to be targeted to bone once a week.
- high stringency conditions are meant to refer to conditions enabling sequences with a high homology to bind. Without being so limited, examples of such conditions are listed In the handbook "Molecular cloning, a laboratory manual, second edition of 1989 from Sambrook et al.: 6XSSC or 6XSSPE, Denhardt's reagent or not, 0.5% SDS and the temperature used for obtaining high stringency conditions is most often in around 68°C (see pages 9.47 to 9.55 of Sambrook) for nucleic acid of 300 to 1500 nucleotides.
- the optimal temperature to be used for a specific nucleic acid probe may be empirically calculated, and although there is room for alternatives in the buffer conditions selected, within these very well known condition ranges, the nucleic acid captured will not vary significantly. Indeed, Sambrook clearly indicates that the "choice depends to a large extent on personal preference" (see page 9.47).
- Figure 1 presents the purity status of GST and GST-D10 proteins on an SDS polyacrylamide gel after CL-4B chromatography
- Figure 2 shows the promotion of GST binding to bone by D 6 , D 1 0 and Die peptide motifs through the percentage of the injected dose of recombinant GST found associated with specific tissues;
- Figure 3 provides a schematic representation of the plasmid pCDNA3-RSV- D ⁇ 0 sPHEX-NEO vector;
- Figure 4 presents a chromatographic profile of 280 nm detection of PHEX flow for the SP-SepharoseTM HP (A) and the blue-Sepharose HP (B).
- Straight line represents buffer ratio
- Figure 5 presents a Sypro-rubyTM stained SDS-PAGE analysis of the different fractions collected throughout D-iosPHEX purification procedure
- Figure 6 shows the variation in serum alkaline phosphatase levels
- Figure 7 shows the nucleotide sequence of a recombinant DNA sequence encoding a protein cleavable so as to produce D ⁇ 0 -sPHEX (SEQ ID NO: 1);
- Figure 8 shows the amino acid sequence encoded by the D ⁇ 0 - sPHEX of Figure 7(SEQ ID NO: 2);
- Figure 9 compares the binding to the mineral phase of bone of proteins (A. GST B. sPHEX) with that of their deca-aspartate fused counterparts;
- Figure 10 shows the nucleotide sequence of a native (or membrane-bound) PHEX (SEQ ID NO: 3);
- Figure 11 shows the amino acid sequence (SEQ ID NO: 4) of a
- Figure 12 schematically illustrates the structure and activities of various secPHEX constructs
- Figure 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
- Figure 14 graphically illustrates the detection of sALP and sALP-
- Figure 15 graphically shows the binding to bone mineral phase of a deca-aspartate fused to secreted alkaline phosphatase
- Figure 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;
- Figure 17 shows A. the nucleotidic sequence (SEQ ID NO: 7) encoding a conjugate of the present invention, namely sALP-D 10 ; and B. the amino acid sequence (SEQ ID NO: 8) of that conjugate; and
- Figure 18 graphically shows the effect of D10-sALP on PPi- mediated mineralization inhibition.
- 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.
- GST gluthatione-S- transferase protein
- Table 1 presents the sequence of oligonucleotides used in
- 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 ⁇ , D 10 and D 6 , respectively, and having extremities compatible with cloning in the pGEX3T-4 plasmid (Pharmacia biotechnology) pre-digested with restriction endonucleases BamHI and Notl.
- pGEX3T-4 vectors were-transformed into AP401 protease minus E. coli bacteria strain (/orr. :mini tetR ara- Alac-pro nalA argEam rifR thi ⁇ [F' pro AB laclq Z M15).
- 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 8000 x g 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.
- Complete-EDTA caplet protease inhibitor Boehringer Mannheim
- Bacteria cells were thawed and disrupted on ice with 6 pulses of sonication every 50 seconds prior to centrifugation at 12000 x g 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.
- Fig. 1 shows an example of an SDS- PAGE analysis of the purified GST and GST-D-
- 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.
- blood samples (0.1 to 0.2 ml) were collected via the subclavian vein under anesthesia into serum/gel clotting activator MicrovetteTM 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 10 -GST nor Die-GST promoted binding to other organs than bone. This showed the specificity of these conjugates to bone (Data not shown).
- Figure 2 shows that GST-D 6 fusion protein did not bind more to tibia or femur than GST alone. In contrast, D 10 and Die peptide motifs promoted GST binding to bones.
- hMEPE Human matrix extracellular phosphoglycoprotein
- hStatherin Human Statherin is a protein synthesized by salivary glands, which similarly to histatin directly modulates hydroxyapatite nucleation and/or growth.
- 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).
- Human Matrix Gla Protein is a protein synthesized by vascular smooth muscle cells and chondrocytes that functions as an inhibitor of hydroxyapatite polymerization by binding to crystal nuclei.
- 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).
- hOPN Human osteopontin
- QNAVSSEETNDFK 13 amino acid residue
- hBSP2 Human Bone SialoProtein II
- 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).
- hIGFBP ⁇ 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, hIGFBP ⁇ 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 hyd roxyapatite (12).
- 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:
- Plasmids containing the acidic peptide sequences derived from hMEPE, hStatherin, hMGP, hOPN, hBSP2, hIGFBP ⁇ 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 hIGFBP ⁇ 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 Notl.
- 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 GBS specific sequences. pGEX- 4T-3 vectors were transformed into AP401 protease minus E. coli bacteria strain (/on::mini tetR ara- Mac-pro nalA argEam rifR thi ⁇ [F' pro AB laclq Z M15 ). [00105] Protein production and purification, and pharmacodistribution of the iodinated fusion protein were performed as described in Example 1.
- 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.
- 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) 3 ⁇ , 707-13) using the following oligonucleotide primers: [00110] ⁇ '-
- 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 Figure 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 Figure 12 showing the specific activity of this construct) but may be dispensed with.
- the pCDNA3-RSV-D 10 sPHEX-NEO vector was transfected in LLC-PK1 cells (Porcine Kidney cells; ATCC No. CRL-1392) using the Lipofectarhine-PlusTM 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 DiosPHEX 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 immunobloting using a monoclonal antibody raised against a recombinant human PHEX fragment (K121-E294) as described previously (Ruchon AF et al. J. Bone Miner. Res. (2000) 1 ⁇ , 1440-14 ⁇ 0).
- a G-418 resistant clone expressing 1 to 2 mg of DIOsPHEX per litre was used for protein production.
- Cells were seeded in Cellstack-10TM (Corning) at a density of 7 X 10 7 in 1.75 litres of media (199 media, 6% FBS, 1 mM NaPyruvate, Penicillin 1x10 5 U/litre, Streptomycin 100 mg/litre and 1% G-418.
- D 10 sPHEX expression was increased by incubating the cells in 1.75 litre of DMEM + 10 mM sodium butyrate for four days at 37°C and ⁇ % CO 2 prior to harvest of the spent medium.
- 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 NaCI step in the SP buffer. D ⁇ 0 sPHEX was then eluted with a 280 mM NaCI step (Fig. 4A). Fractions were analyzed by SDS-PAGE and with the PHEX enzymatic activity assay.
- DIOsPHEX was eluted by using NaCI gradient. Purity was determined to be above 90%. DiosPHEX was concentrated and dialyzed against 1 mM sodium P04 pH 7.4, 160 mM NaCI using Centriprep- ⁇ OTM cartridges. Dialyzed sample was filtered in a sterile environment on 0.22 ⁇ m membrane. Purified D-
- 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 3 ⁇
- 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.
- DiosPHEX 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.
- Each mouse was anesthetized with vaporized Isoflurane (2%) and DiosPHEX, or sPHEX were injected as an intravenous bolus through the subclavian vein. The dose was ⁇ 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 +1 ⁇ (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.
- ALP Alkaline phosphatase
- Recombinant purified proteins were labelled with fluorescein- isothiocyanate (FITC, Molecular Probes F143). Reaction was carried out by adding proteins to 10 mM sodium phosphate, ⁇ O mM NaCI 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-10TM column (Pharmacia) prior to dialysis in the binding buffer (1 mM sodium phosphate 1 ⁇ 0 mM NaCI, pH 7.4).
- FITC fluorescein- isothiocyanate
- D ⁇ -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).
- ALP tissue non-specific alkaline phosphatase
- An aliquot representing 1/20 th of the RT step was used directly in a PCR reaction with ALP specific oligos (forward ⁇ '-gataaagcaggtcttggggtgcacc-3' (SEQ ID NO: *); reverse ⁇ '-gttggcatctgtcacgggcttgtgg-3' (SEQ ID NO: *)) and the Expand High Fidelity Enzyme KitTM (Roche).
- the resulting ALP specific product (1644 bp) was separated on and purified from an agarose gel (1%) using the Qiaquick Gel Extraction KitTM (QIAGEN).
- the ALP cDNA was then ligated into the pCR4- blunt-TOPOTM vector (Invitrogen), transformed into Top10TM bacteria (Invitrogen), and a positive clone identified by colony PCR. The identity of the cDNA was verified by automated DNA sequencing.
- ALP secreted forms of ALP having the GPI anchor signal removed were constructed by PCR using Expand High Fidelity Enzyme KitTM. They comprised residues 1- ⁇ 02 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 ( ⁇ '-tggafccaccatgatttcaccattcttagtac-3' (SEQ ID NO: 40)) covered the initiator methionine (underlined) and included a BamHI site (italicized).
- the reverse primers (sALP: ⁇ '- tfcfagactacgagctggcaggagcacagtggccg-3' (SEQ ID NO: 41); sALP-D ⁇ 0 5'- tfcfagactagtcgtcatcatcgtcatcatcgtcgtcatccgagctggcaggagcacagtggccg-3' (SEQ ID NO: 42)) contained a stop codon (underlined) and an Xbal site (italicized).
- the PCR products were digested with BamHI and Xbal and cloned into the pCDNA3.1-RSV that had been pre-digested with the same enzymes. Plasmid DNA were sequenced.
- Enzymatic activity of sALP and sALP-D ⁇ o was assayed using 4- methylumbelliferyl phosphate (MUP, Molecular Probes, M842 ⁇ ) as a fluorigenic substrate according to Gee KR et al. (Anal. Biochem. 273, 41-48 (1999))
- MUP 4- methylumbelliferyl phosphate
- 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 GeminiTM (Molecular Devices) plate reader every minute for 30 minutes at 4 ⁇ 0 nm upon excitation at 360 nm. Emission wavelength cut-off was set at 43 ⁇ nm.
- ALP initial speed rate was estimated by linear regression fitting (with r 2 equal or greater than 0.98).
- each construct (pCDNA3-RSV-sALP-NEO and pCDNA3-RSV-sALP-D ⁇ o-NEO) was transiently transfected in HEK-293S cells (Human Embryonic Kidney cells; ATCC No. CRL-1392) using the Lipofectamine-Plus liposome transfection kitTM (Invitrogen).
- HEK-293S cells were also mock ransfected 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 ⁇ min at 4°C to remove dead cells and debris.
- the supernatants were assayed for sALP or sALP-Dio enzymatic activity and expression using the ALP fluorescent enzymatic assay and Western blotting respectively.
- 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 1X Laemmli sample buffer with DTT and boiled for ⁇ min.
- 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 ⁇ % dried milk) and a rabbit anti-mouse IgG coupled to horseradish peroxidase (Sigma) (1 :12000 in PBST with ⁇ % dried milk).
- the signal was developed with the Western Lightning Chemiluminescence Reagent PlusTM (PerkinElmer).
- HEK293 after transient transfection was very high and of similar magnitude for pCDNA3-RSV-sALP-NEO (sALP) and pCDNA3-RSV-sALP-D ⁇ 0 -NEO (sALP- D-io) (Figure 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 3 ⁇ -times greater than that measured in the cell extracts thus attesting to the secretory nature of sALP and sALP-D 10 .
- HEK293 cells constitutivelv secreting sALP and sALP-Dm [00133] To induce the stable expression of the sALP and sALP-D ⁇ 0 proteins, the pCDNA3-RSV-sALP-NEO and pCDNA3-RSV-sALP-D ⁇ 0 -NEO vectors was transfected separately in HEK-293S cells using the Lipofectamine- Plus liposome transfection kitTM (Invitrogen). Transfected cells were selected by adding 800 ⁇ g/ml G418 (Life Technologies) to the medium.
- a pool of G-418 resistant cells were analyzed for sALP or sALP-D 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.
- 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.
- the 96 wells plate was shaken for 10 seconds every minute for the duration of the assay.
- 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-Dio respectively. A binding difference of more than 13 times in favour of sALP-Dio 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.
- Such fusion protein can be targeted directly to bones where the accumulation of PPi inhibits skeletal mineralization.
- UMR106 cells were grown to confluence. They were then cultured for a further 7 days in media containing 10mM ⁇ -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-io-sALP produced from HEK293, human embryonic kidney cells. Mineralization was assessed by 45 Ca uptake. Parameters used for this experiment are presented in table 2 below.
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DE602005027461T DE602005027461D1 (en) | 2004-04-21 | 2005-04-21 | CONJUGATES FOR INTRODUCTION TO BONE AND METHOD FOR THEIR USE IN THE INTRODUCTION OF PROTEINS FOR BONE |
DK05739065.0T DK1759001T3 (en) | 2004-04-21 | 2005-04-21 | Conjugate for Bone Delivery and Method of Preparation by Targeting Proteins to the Bone |
PL18173111T PL3404102T3 (en) | 2004-04-21 | 2005-04-21 | Bone delivery conjugates and method of using same to target proteins to bone |
EP05739065A EP1759001B1 (en) | 2004-04-21 | 2005-04-21 | Bone delivery conjugates and method of using same to target proteins to bone |
PL05739065T PL1759001T3 (en) | 2004-04-21 | 2005-04-21 | Bone delivery conjugates and method of using same to target proteins to bone |
AT05739065T ATE505551T1 (en) | 2004-04-21 | 2005-04-21 | CONJUGATES FOR BONE DELIVERY AND METHODS OF USE THEREOF IN ADDING PROTEINS TO BONE |
PL11000196T PL2348114T3 (en) | 2004-04-21 | 2005-04-21 | Bone delivery conjugates and method of using same to target proteins to bone |
JP2007508698A JP4874954B2 (en) | 2004-04-21 | 2005-04-21 | Bone delivery complex and methods of use for targeting bone to proteins |
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Cited By (29)
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JP2008501307A (en) * | 2004-06-10 | 2008-01-24 | 俊治 戸松 | Protein added with short-chain peptides consisting of acidic amino acids |
WO2008033488A2 (en) * | 2006-09-15 | 2008-03-20 | University Of Kansas Medical Center | Polypeptides for bone mineralization |
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