Biopharmaceuticals – Philippe Crine, Guy Boileau, Isabelle Lemire, Thomas P. Loisel, Alexion Holding BV, Alexion Pharmaceuticals Inc
Abstract for “Bone delivery conjugates: Method of using them to target proteins to bones”
“A bone delivery conjugate whose structure is selected from: A) Dn-Y protein-Z; and (B) Z-protein Y-Dn X, wherein X or an amino sequence of at most one amino acids; Y or Y are absent; Z is absent; or Z is an amino sequence of at minimum one amino acid; and Dn is either a poly aspartate whose n=10-16 Methods of using the same compositions and combinations thereof.
Background for “Bone delivery conjugates: Method of using them to target proteins to bones”
“Technological advancements in molecular biology, large-scale protein purification and recombinant proteins have enabled the production of large quantities of proteins that are now used as biopharmaceuticals. Monoclonal antibodies as well as soluble forms TNF-a receptors have been used to treat autoimmune conditions such Crohn’s disease and severe forms of psoriasis (1). Enzyme replacement therapy (ERT) is another example of the use of recombinant proteins. Lysosomal storage disorders can be treated with ERT. This group of genetic disorders involves the impairment of lysosome enzyme function, which can lead to severe somatic and sometimes neuronal pathologies. These patients receive large amounts of normal enzymes in ERT. The infused enzymes are then taken from the circulation via cell surface receptors (mannose-6phosphate receptor) and entered the endocytic path on the way to the lysosome, their place of action. Some attempts to treat genetic disorders with ERT are not successful.
“Hypophosphatasia” is a rare and heritable form of rickets. It can also be called osteomalacia. The more severe forms of the disease are less common at 1 in 100,000 births. The milder forms are more common. Mutations in the gene that codes for the tissue-nonspecific alkalinephosphatase enzyme can cause this inborn metabolic defect. It is biochemically characterized by low serum alkalinephosphatase activity. Deficit in alkaline phosphatase in osteoblasts or chondrocytes can cause skeletal mineralization problems, such as rickets and osteomalacia.
“There are many expressions of hypophosphatasia. It can range from mild to severe, with stillbirths often occurring from an unmineralized bone skeleton to a more mild form that causes only the premature loss of teeth. Hypophosphatasia is an autosomal recessive trait that affects severely affected children and infants. There are four types of hypophosphatasia: perinatal (infantile), childhood (childhood), and adult. Perinatal hypophosphatasia is a condition that occurs during pregnancy. Most affected newborns are only able to survive for a short time. Clinical signs of infantile hypophosphatasia are usually present before six months. Half of all patients will die within one year. Although childhood hypophosphatasia can be severe, most patients will experience skeletal symptoms throughout their lives. Hypophosphatasia in adulthood is characterized by painful recurrent stress fractures and poor healing.
“Osteoblasts, chondrocytes have a high level of tissue-nonspecific alkalinephosphatase that is attached to the cell’s surface. Hypophosphatasia is characterized by the accumulation of extracellular phosphorus-compounds that are believed to be substrates for the enzyme. These include phosphoethanolamine, inorganic pyrophosphate and pyridoxal 5-?-phosphate. PPi, an inhibitor of hydroxyapatite cristal growth, is responsible for impaired skeletal mineralization. Patients suffering from hypophosphatasia should be provided with active enzyme. This will reduce extracellular PPi and increase skeletal mineralization.
Hypophosphatasia is not currently treated medically. Trials for enzyme replacement with intravenous infusions alkalinephosphatase failed. It seems that alkalinephosphatase activity must increase not only in the circulation, but also in the skeleton. Recent bone marrow transplantation confirmed this hypothesis. The poor engraftment meant that the benefits of transplantation were only temporary.
“There is therefore an urgent need for enzyme replacement therapy to provide active enzyme to the patients with hypophosphatasia.”
“Bone-targeted protein could be used not only to treat or prevent hypophosphatasia (loss in function alkaline phosphatase), but also to treat or prevent other genetic diseases characterized with defective enzymatic activities involved in bone metabolism such as X-linked Hypophosphatemic Rickets (XLH), which is a loss of function for the phosphate regulating genes with homology of endopeptidases (PHEX )).”).
“XLH” is the most common of all familial hypophosphatemias. (OMIM 307800 and 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 can cause growth retardation, deformities in the lower extremities, and radiologic and histomorphometric evidence for rickets. The disease is thought to be caused by combined renal defects in tubular vitamin D metabolism and tubular phosphate-reabsorption. It also has a functional disorder of bone and teeth. XLH is caused by inactivating mutations within the PHEX gene. This is a member the zinc metallopeptidase type II integral membrane glycoproteins. These mutations block the expression of the functional PHEX enzyme on osteoblast cells. The treatment of XLH sufferers is limited to oral inorganic phosphate supplementation (Pi) in four to five divided doses daily, with co-administration of 1,25OH(2D to offset the inadvertent synthesis of 1,25OH(2D). Patients may not comply with treatment due to gastrointestinal intolerances and diarrhea caused by high doses. The phosphate load can lead to secondary hyperparathyroidism, which may require parathyroidectomy. On the other hand, excess 1,25(OH),2D could cause hypercalciuria, hypercalcemia, and nephrocalcinosis.
“Useful ERT would seek to replace defective PHEX enzymes in XLH patients by a functional enzyme obtained using recombinant genetic technology. The normal PHEX enzyme has been anchored in the osteoblast plasma membrane with a hydrophobic protein. Therefore, it is impossible to produce and purify sufficient amounts of PHEX in sufficient quantities for pharmaceutical use. A soluble form (or sPHEX), of recombinant PHEX was created in cell cultures and purified. It is then prepared for intravenous (IV), administration (WO 00/50580). sPHEX was then injected into Hyp mice to create a model mouse for XLH. No. 10/362,259. There were improvements in several bone-related serum parameters, including a decrease in serum alkalinephosphatase. These experiments were successful. However, it was thought that therapeutic sPHEX could be more effective if the recombinant proteins were modified to increase their binding to bone minerals.
“There are therefore many ways to target proteins and bone matrix successfully.”
Biphosphonates have been shown to be highly affinity bound to hydroxyapatite, and can therefore be used to target small molecules ((4) and proteins ((5)) to bone. This strategy involves chemical modifications to the proteins and can cause interference with protein activity.
Conjugate small molecules to bone with acidic peptides like poly-Asp(6). This strategy was created after it became apparent that many proteins synthesized from osteoblasts, bone-forming cells, bind to bone matrix via sequences rich in acidic amino acids residues (Asp or Glu). This is what happened with osteopontin (7 and bone sialoprotein (two non-collagenous proteins). Acidic peptides (E2-10, D2-10) were developed to target small molecules (e.g. Methotrexate and FITC were used to target small molecules (i.e. biotin, Fmoc. To target small molecules (e.g. FITC, Fmoc and estradiol) were used to hydroxyapatite intravenously. E6 also conferred to BSA, IgG and hemoglobin the ability to bind to hydroxyapatite, in vitro. Chemically linking the acidic sequences was used in all cases.
“The invention sought to address these and other needs.”
“The present description refers a number of documents, whose content is herein incorporated in their entirety by reference.”
“The invention shows that large complex molecules, such as proteins, can be fused to acidic peptides in order to effectively target bone in vivo.”
“Accordingly to a particular embodiment of the invention, a bone delivery compound is provided having a structure selected among the following: A) Dn-Y protein-Z; and (B) Z-protein Y-Dn X. In which X is absent; or is an amino acid sequence of at most one amino acids; Y is absent; or Z is an amino acid sequence containing at least one of those amino acids; and Dn is a n=10-16 poly aspartate. A bone delivery conjugate with homology to endopeptidases (sPHEX) is another embodiment of the invention. The structure of the conjugate in another specific embodiment is: XDn-Y -sPHEXZ-Z. The sPHEX can be selected from the following sequence in another embodiment of the invention: X-Dn-Y-sPHEXZ. 10; 47- 749 of FIG. 10; 47 to 749 of FIG. 10; 48 to 749 of FIG. 10; 49 to 749 of FIG. 10; 50 to 749 of FIG. 10; 51 to 749 of FIG. 10; 52 to 749 of FIG. 10; 53 to 749 of FIG. 10. In one specific embodiment, n is 10 for these bone delivery conjugates. An additional specific embodiment of this bone-delivery conjugate has n at 11. An additional specific embodiment of this bone-delivery conjugate is n = 12. An alternative embodiment of this bone delivery conjugate is n = 13. An additional specific embodiment of this bone-delivery conjugate is n = 14. An additional specific embodiment of this bone-delivery conjugate is n = 15. In another specific embodiment of this bone-delivery conjugate, n = 16. The sPHEX is a specific embodiment of this invention and consists of the sequences of amino acids 46 through 749 in FIG. 10, and n=10.
“In an alternative embodiment of the invention, the protein contained in the conjugate is a soluble alkalinephosphatase (sALP). The structure of the conjugate in another specific embodiment is Z-sALPX-DnY. FIG. 16A shows another specific embodiment of sALP. It is encoded using the sequence shown in FIG. 16A. 16A. 16B. 16B. An additional specific embodiment of this bone-delivery conjugate has n at 11. An additional specific embodiment of this bone-delivery conjugate is n = 12. An alternative embodiment of this bone delivery conjugate is n = 13. An additional specific embodiment of this bone-delivery conjugate is n = 14. An additional specific embodiment of this bone-delivery conjugate is n = 15. An additional specific embodiment of this bone-delivery conjugate is n=16. In another embodiment, n is 10.
“There is also provided a sequence-recombinant vector.” A recombinant host cells is also available that contain said vector.”
“There is also provided an isolate sPHEX-polypeptide consisting of a sequence chosen from the following group: amino acids 54-749 as shown in FIG. 10; the amino acids 53-749. As set forth in FIG. 10; amino acids 52-749. As set forth in FIG. 10; amino acids 52 to 749 as shown in FIG. 10; amino acids 51 to 749 as shown in FIG. 10; amino acids 49-749. As set forth in FIG. 10; amino acids 49 to 749 as shown in FIG. 10; the FIG. 10; and amino acid 46 to 749, as shown in FIG. 10.”
“There is also provided a composition for bone delivery that includes a bone delivery compound of the invention and a pharmaceutically acceptable carrier.”
“There is also a method for delivering protein to bone tissue in a mammal. This involves administering to the mammal a sufficient amount of a bone delivery compound as described in the present invention.”
“There is also a method for delivering sPHEX into bone tissue of a mammal. This involves administering to the mammal a dose of the bone delivery conjugate according to the present invention.”
“There is also a method for delivering ALP to bone tissue in a mammal that has a need of it. This involves administering to the mammal a dose of the bone delivery conjugate according to the present invention.”
“The present invention also provides a method for treating a condition or disease that is related to a bone defect. This involves administering to a mammal who has a need, a conjugate of this invention in a pharmaceutically acceptable carrier. “X-linked hypophosphatemic Rickets (XLH) is the specific condition.
“There is also a method for treating a condition or disease that results in a bone defect. This involves administering a conjugate according to the invention to a mammal who has a need. The conjugate is in a pharmaceutically acceptable container. Hypophosphatasia is a condition that can be described in specific embodiments.
“There is also a method for screening peptides to use in a bone delivery peptide-peptide conjugate. It involves the following steps: fusing the candidate protein to a reporter to form a protein peptide conjugate; contacting conjugate with bone tissue and mineral phase of bones; wherein the candidate protein is selected when the reporter protein’s presence on bone tissue is greater when it is bound with the candidate protein than when it’s not.”
“Accordingly to a particular embodiment of the invention, there is provided a bone-delivery conjugate of a protein fused with a peptide selected in the group consisting deca-aspartate(D10) to Hexadeca?aspartate(D16).
“In certain embodiments of conjugates according to the present invention, the sPHEX fused at its Nterminal to D10. Another specific embodiment of the sPHEX fused at its N terminal to D11. Another specific embodiment of the sPHEX fused at its N terminal to D12. Another specific embodiment of the sPHEX fused at its N terminal to D13. Another specific embodiment of the sPHEX fused at its N terminal to D14. Another specific embodiment of the sPHEX fused at its N terminal to D15. Another specific embodiment of the sPHEX fused at its N terminal to D16
“According to specific embodiments, conjugates of this invention, the sALP fused at its Cterminal to D10. Another specific embodiment of the sALP fused at its Cterminal to D10. Another specific embodiment of the sALP fused at its Cterminal to D12. Another specific embodiment of the sALP fused at its Cterminal to D13. Another specific embodiment fuses the sALP at its C-terminal, D14. Another specific embodiment fuses the sALP at its C-terminal to reach D15. Another specific embodiment of the sALP fused is at its C-terminal to D16.
It is understood that any functionally soluble protein can be used in the conjugate according to the invention. While results are shown for conjugates containing one specific sPHEX and sALP according to the present invention, it is also understood that other functional sPHEXes or sALPs may be used.
“sPHEX”
“SPHEX” is any biologically active fragment or mutein of PHEX that is soluble in water. For optimal production of sPHEX, skilled artisans may create expression constructs that are not described in this article. Skilled artisans can also design fragments of DNA encoding biologically active fragments or muteins of naturally occurring PHEX that have the same or similar biological activity as the full-length enzyme.
A large number of expression vectors can be created and tested to express a PHEX cDNA in order to create a recombinant source of sPHEX. A transient transfection experiment as well as stable transfections may reveal an expression construct that is particularly effective in expressing a particular level of expression.
“Any sPHEX containing at least a native PHEX ectodomain section starting with the cysteine position 54 in the sequence shown at FIG. The present invention includes 10
According to the specific embodiments, the conjugates are any sPHEX that contains this 54-749 Native fragment, more preferable the native 52-749 Fragment, more preferred the native 50-749 Native fragment, more preferably the native 49-749 Native fragment, more desirable the native 47-749 Native fragment, more most preferably, the native 46-749 Native fragment, and more preferably, the native 48-749 Native fragment, more preferable the native 47-749, more preferably, the native 47-749, more preferably, and more preferably, together with a Poly-aspartate from the group consisting D10 to D10 to D16 fusions immediately upstream of this segment, D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10, D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D16, to this fragment.
“The conjugate can optionally contain one or more additional amino acid 1) upstream of the poly-aspartate, and/or 2) between it and the native fragment/functional equivalent. These amino acids may be any amino acid. They may be chosen independently of the group that includes cysteine, proline, and tryptophan, namely amino acids that are known to cause disulfide bonds formation or changes to conformation.
These amino acids could be found in the conjugate if, for example, the cloning technique used to create it introduces them at these locations.
“Amino acids upstream of the polyaspartate can be selected using specific cloning strategies to provide a substrate for specific enzymes in the secretory pathway (e.g. The host cell’s furin (or signal peptidase), will be used to cleave the recombinant PHEXs produced into a secreted bone-targeting sPHEX. An appropriate computer algorithm, such as the one described by Bendtsen and al., can predict the likelihood that a sequence is cleaved in host cells by the signal proteinase. (J Mol Biol. 2004 Jul. 16; 340(4):783-95) and available on the Web at www.cbs.dtu.dk/services/SignalP/ which takes into account parameters including the following: the amino acids at position ?3 and ?1 from the cleavage site by the signal peptidase desirably have small and non charged side chains. Ala, Ser and Gly, Cys and Thr are preferred. Occasionally Gln and Pro are also acceptable. Likewise, those at position ‘3 should be Ala, Ser and Gly, Cys Thr, Ile Leu, Val. Aimable amino acids at position?6 or?4 near the cleavage site can induce the formation of beta-turns (such as Pro) residues.
The present invention also includes conjugates that contain additional amino acids. These may be chosen based on the cloning strategy used in order to create a cleavable PHEX. The cleavable PHEX described in Examples 3 and 4, respectively, contains additional amino acids between the polyaspartate sequence and the native ectodomain. The present invention also includes a conjugate that contains the secPHEX described in co-pending application number. 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. More particularly, FIG. FIG. 12 shows the schematic structure of secPHEXs, which contain additional amino acids upstream from the native 46-749 PHEX fragment. No. Constructs no. 1 through 5 and 3 to 5 could be fused into a poly-aspartate, and used as conjugates according to the present invention. Construct no. Construct no. 4 is a conjugate according to the invention. It includes a D10 polyaspartate as well as a native fragment of ectodomain.
“The conjugates of this invention also include sPHEXs that contain deletions at their Cterminal which are not detrimental to their enzymematic activity.”
“Moreover, the present invention includes conjugates in which the poly-aspartate would attach at the C-terminal to the native PHEX fragment.”
“sALP”
ALP is a membrane-bound protein that is bound to a glycolipid at its C-terminal. After removing a hydrophobic Cterminal end, a glycolipid anchor (GPI), is added to the translational process. This serves as both a transitional membrane anchor and a signal for the addition the GPI. The sALP in Example 6 is composed of an ALP in which the first amino acid from the hydrophobic Cterminal sequence, namely, alanine is replaced with a stop codon. This soluble ALP contains all the amino acids of ALP’s native, and active, anchored form.
“The sALP conjugates according specific embodiments are thus any sALP and a poly-aspartate selected within the group consisting D10 to D16 fused directly downstream of this fragment.”
“The conjugate may optionally contain one or more amino acids either upstream of the poly-aspartate or between the polyaspartate’s native sALP fragment, functional equivalent, or both. Exogenous amino acid may be introduced in these places, for example, when bone targeting conjugate is cloned. The exogenous amino acid should not be added to the transamination site. Ikezawa (Biol Pharm.) describes how to predict the likelihood that a sequence designed will be cleaved in the host cell’s transaminase. Bull. 2002, 25(4) 409-417).”
“The conjugates of this invention also include sALPs that contain deletions at their Nterminal which are not detrimental to their enzyme activity.”
“Furthermore the present invention includes conjugates in which the poly-aspartate would attach at the N-terminal or biologically active fragment of the native ALP-anchored fragment.”
“Recombinant protein” is a term that refers to a protein encoded by a genetically manipulated nucleic acid. “Recombinant protein” is a term that refers to a protein encoded using a genetically modified nucleic acid. It can be placed in a prokaryotic, eukaryotic host cells. The nucleic acids are usually placed in a vector such as a virus or plasmid, depending on the cell’s needs. E. coli was used in the Examples to express the conjugates of this invention. However, a person of ordinary skill will be able to recognize that recombinant proteins can also be produced using other hosts according to routine methods in the art. Maniatis et al. provide examples of representative methods. Cold Springs Harbor Laboratory (1989). ?Recombinant cleavable protein? Recombinant cleavable protein is a term that can be used to describe a recombinant molecule of protein. It may also refer to an enzyme that can cleave the protein to make a secreted/soluble form.
“The term ‘ectodomain fragment?” When used in relation to PHEX, it is meant to mean that PHEX’s fragment is not found within the cellular membrane in its native form.
“Bone tissue” is a term that refers to bone tissue. “Bone tissue” is used herein to describe the tissue that has been synthesized by osteoblasts. It is composed of an organic matrix containing a lot of collagen and mineralized with the deposition of hydroxapatite crystals.
The bone delivery conjugates of this invention contain fusion proteins that are effective in providing a sufficient amount of fusion protein to bone defects. The fusion protein can be administered in standard procedures, such as intravenous injection, in the form of a pharmaceutical formulation in any pharmaceutically acceptable carrier.
“Pharmaceutically acceptable carrier” is a term that refers to a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” is used herein when parenteral administration is chosen as the method of administration. It refers to pharmaceutically acceptable sterile non-aqueous and aqueous solvents, suspensions, or emulsions. Non-aqueous solvents include propylene glycol and polyethylene glycol. Water, water-alcohol solutions, physiological saline, buffered medical parenteral vehicle including sodium chloride, Ringer?s dextrose solution and dextrose plus potassium chloride solution; fluid and nutrient replenishers, electrolyte replenishers, fluids, and Ringer?s solution containing Lactose or fixed oils are all examples of aqueous solvents.
“Effective amount” is a term that refers to the minimum amount of a pharmaceutical composition that should be administered to a mammal in order for it to have measurable therapeutic effects. “Effective amount” is a term that refers to the minimum amount of a pharmaceutical compound that should be given to a mammal to achieve significant therapeutic effects. Many factors will affect the dosages, including the method of administration. The amount of protein in a single dose is usually sufficient to prevent, delay or treat bone-related undesired conditions without causing significant toxic effects. The present invention contains a certain amount of fusion protein that will significantly reduce the clinical symptoms.
The effective amount can be administered daily, weekly, monthly, or in fractions. A pharmaceutical composition of the invention is typically administered in a dose of 0.001 mg to 500 mg per kilogram of body weight per day (e.g. 10 mg, 50mg, 100mg, 250 mg or 250 mg). You can give the dosage in a single or multiple doses. In some cases, the effective dose is the amount that is given to target bone. It can range from 1 mg up to 25 grams of conjugate per day to 50 mg up to 10 grams per day to approximately 1 gram per day. About 50 mg to 10 grams per week of conjugate targeted at bone. 50 mg to 10 grams per week. 100 mg to 5 grams every other day. 1 gram of conjugate targeted to bones once per week.
These are only guidelines. The actual dose must be selected and adjusted by the attending physician according to the clinical factors specific to each patient. Methods of the art will determine the optimal daily dose. Other clinically relevant factors will also be considered. Patients may also be taking medication for other conditions or diseases. While the protein to deliver to bone may be continued while the patient is receiving the medication, it is best to start with low doses in order to see if side effects occur.
“Other objects and advantages, as well as features, of the present invention, will be more evident if you read the following non-restrictive description, which is given only by reference to the accompanying illustrations.
Summary for “Bone delivery conjugates: Method of using them to target proteins to bones”
“Technological advancements in molecular biology, large-scale protein purification and recombinant proteins have enabled the production of large quantities of proteins that are now used as biopharmaceuticals. Monoclonal antibodies as well as soluble forms TNF-a receptors have been used to treat autoimmune conditions such Crohn’s disease and severe forms of psoriasis (1). Enzyme replacement therapy (ERT) is another example of the use of recombinant proteins. Lysosomal storage disorders can be treated with ERT. This group of genetic disorders involves the impairment of lysosome enzyme function, which can lead to severe somatic and sometimes neuronal pathologies. These patients receive large amounts of normal enzymes in ERT. The infused enzymes are then taken from the circulation via cell surface receptors (mannose-6phosphate receptor) and entered the endocytic path on the way to the lysosome, their place of action. Some attempts to treat genetic disorders with ERT are not successful.
“Hypophosphatasia” is a rare and heritable form of rickets. It can also be called osteomalacia. The more severe forms of the disease are less common at 1 in 100,000 births. The milder forms are more common. Mutations in the gene that codes for the tissue-nonspecific alkalinephosphatase enzyme can cause this inborn metabolic defect. It is biochemically characterized by low serum alkalinephosphatase activity. Deficit in alkaline phosphatase in osteoblasts or chondrocytes can cause skeletal mineralization problems, such as rickets and osteomalacia.
“There are many expressions of hypophosphatasia. It can range from mild to severe, with stillbirths often occurring from an unmineralized bone skeleton to a more mild form that causes only the premature loss of teeth. Hypophosphatasia is an autosomal recessive trait that affects severely affected children and infants. There are four types of hypophosphatasia: perinatal (infantile), childhood (childhood), and adult. Perinatal hypophosphatasia is a condition that occurs during pregnancy. Most affected newborns are only able to survive for a short time. Clinical signs of infantile hypophosphatasia are usually present before six months. Half of all patients will die within one year. Although childhood hypophosphatasia can be severe, most patients will experience skeletal symptoms throughout their lives. Hypophosphatasia in adulthood is characterized by painful recurrent stress fractures and poor healing.
“Osteoblasts, chondrocytes have a high level of tissue-nonspecific alkalinephosphatase that is attached to the cell’s surface. Hypophosphatasia is characterized by the accumulation of extracellular phosphorus-compounds that are believed to be substrates for the enzyme. These include phosphoethanolamine, inorganic pyrophosphate and pyridoxal 5-?-phosphate. PPi, an inhibitor of hydroxyapatite cristal growth, is responsible for impaired skeletal mineralization. Patients suffering from hypophosphatasia should be provided with active enzyme. This will reduce extracellular PPi and increase skeletal mineralization.
Hypophosphatasia is not currently treated medically. Trials for enzyme replacement with intravenous infusions alkalinephosphatase failed. It seems that alkalinephosphatase activity must increase not only in the circulation, but also in the skeleton. Recent bone marrow transplantation confirmed this hypothesis. The poor engraftment meant that the benefits of transplantation were only temporary.
“There is therefore an urgent need for enzyme replacement therapy to provide active enzyme to the patients with hypophosphatasia.”
“Bone-targeted protein could be used not only to treat or prevent hypophosphatasia (loss in function alkaline phosphatase), but also to treat or prevent other genetic diseases characterized with defective enzymatic activities involved in bone metabolism such as X-linked Hypophosphatemic Rickets (XLH), which is a loss of function for the phosphate regulating genes with homology of endopeptidases (PHEX )).”).
“XLH” is the most common of all familial hypophosphatemias. (OMIM 307800 and 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 can cause growth retardation, deformities in the lower extremities, and radiologic and histomorphometric evidence for rickets. The disease is thought to be caused by combined renal defects in tubular vitamin D metabolism and tubular phosphate-reabsorption. It also has a functional disorder of bone and teeth. XLH is caused by inactivating mutations within the PHEX gene. This is a member the zinc metallopeptidase type II integral membrane glycoproteins. These mutations block the expression of the functional PHEX enzyme on osteoblast cells. The treatment of XLH sufferers is limited to oral inorganic phosphate supplementation (Pi) in four to five divided doses daily, with co-administration of 1,25OH(2D to offset the inadvertent synthesis of 1,25OH(2D). Patients may not comply with treatment due to gastrointestinal intolerances and diarrhea caused by high doses. The phosphate load can lead to secondary hyperparathyroidism, which may require parathyroidectomy. On the other hand, excess 1,25(OH),2D could cause hypercalciuria, hypercalcemia, and nephrocalcinosis.
“Useful ERT would seek to replace defective PHEX enzymes in XLH patients by a functional enzyme obtained using recombinant genetic technology. The normal PHEX enzyme has been anchored in the osteoblast plasma membrane with a hydrophobic protein. Therefore, it is impossible to produce and purify sufficient amounts of PHEX in sufficient quantities for pharmaceutical use. A soluble form (or sPHEX), of recombinant PHEX was created in cell cultures and purified. It is then prepared for intravenous (IV), administration (WO 00/50580). sPHEX was then injected into Hyp mice to create a model mouse for XLH. No. 10/362,259. There were improvements in several bone-related serum parameters, including a decrease in serum alkalinephosphatase. These experiments were successful. However, it was thought that therapeutic sPHEX could be more effective if the recombinant proteins were modified to increase their binding to bone minerals.
“There are therefore many ways to target proteins and bone matrix successfully.”
Biphosphonates have been shown to be highly affinity bound to hydroxyapatite, and can therefore be used to target small molecules ((4) and proteins ((5)) to bone. This strategy involves chemical modifications to the proteins and can cause interference with protein activity.
Conjugate small molecules to bone with acidic peptides like poly-Asp(6). This strategy was created after it became apparent that many proteins synthesized from osteoblasts, bone-forming cells, bind to bone matrix via sequences rich in acidic amino acids residues (Asp or Glu). This is what happened with osteopontin (7 and bone sialoprotein (two non-collagenous proteins). Acidic peptides (E2-10, D2-10) were developed to target small molecules (e.g. Methotrexate and FITC were used to target small molecules (i.e. biotin, Fmoc. To target small molecules (e.g. FITC, Fmoc and estradiol) were used to hydroxyapatite intravenously. E6 also conferred to BSA, IgG and hemoglobin the ability to bind to hydroxyapatite, in vitro. Chemically linking the acidic sequences was used in all cases.
“The invention sought to address these and other needs.”
“The present description refers a number of documents, whose content is herein incorporated in their entirety by reference.”
“The invention shows that large complex molecules, such as proteins, can be fused to acidic peptides in order to effectively target bone in vivo.”
“Accordingly to a particular embodiment of the invention, a bone delivery compound is provided having a structure selected among the following: A) Dn-Y protein-Z; and (B) Z-protein Y-Dn X. In which X is absent; or is an amino acid sequence of at most one amino acids; Y is absent; or Z is an amino acid sequence containing at least one of those amino acids; and Dn is a n=10-16 poly aspartate. A bone delivery conjugate with homology to endopeptidases (sPHEX) is another embodiment of the invention. The structure of the conjugate in another specific embodiment is: XDn-Y -sPHEXZ-Z. The sPHEX can be selected from the following sequence in another embodiment of the invention: X-Dn-Y-sPHEXZ. 10; 47- 749 of FIG. 10; 47 to 749 of FIG. 10; 48 to 749 of FIG. 10; 49 to 749 of FIG. 10; 50 to 749 of FIG. 10; 51 to 749 of FIG. 10; 52 to 749 of FIG. 10; 53 to 749 of FIG. 10. In one specific embodiment, n is 10 for these bone delivery conjugates. An additional specific embodiment of this bone-delivery conjugate has n at 11. An additional specific embodiment of this bone-delivery conjugate is n = 12. An alternative embodiment of this bone delivery conjugate is n = 13. An additional specific embodiment of this bone-delivery conjugate is n = 14. An additional specific embodiment of this bone-delivery conjugate is n = 15. In another specific embodiment of this bone-delivery conjugate, n = 16. The sPHEX is a specific embodiment of this invention and consists of the sequences of amino acids 46 through 749 in FIG. 10, and n=10.
“In an alternative embodiment of the invention, the protein contained in the conjugate is a soluble alkalinephosphatase (sALP). The structure of the conjugate in another specific embodiment is Z-sALPX-DnY. FIG. 16A shows another specific embodiment of sALP. It is encoded using the sequence shown in FIG. 16A. 16A. 16B. 16B. An additional specific embodiment of this bone-delivery conjugate has n at 11. An additional specific embodiment of this bone-delivery conjugate is n = 12. An alternative embodiment of this bone delivery conjugate is n = 13. An additional specific embodiment of this bone-delivery conjugate is n = 14. An additional specific embodiment of this bone-delivery conjugate is n = 15. An additional specific embodiment of this bone-delivery conjugate is n=16. In another embodiment, n is 10.
“There is also provided a sequence-recombinant vector.” A recombinant host cells is also available that contain said vector.”
“There is also provided an isolate sPHEX-polypeptide consisting of a sequence chosen from the following group: amino acids 54-749 as shown in FIG. 10; the amino acids 53-749. As set forth in FIG. 10; amino acids 52-749. As set forth in FIG. 10; amino acids 52 to 749 as shown in FIG. 10; amino acids 51 to 749 as shown in FIG. 10; amino acids 49-749. As set forth in FIG. 10; amino acids 49 to 749 as shown in FIG. 10; the FIG. 10; and amino acid 46 to 749, as shown in FIG. 10.”
“There is also provided a composition for bone delivery that includes a bone delivery compound of the invention and a pharmaceutically acceptable carrier.”
“There is also a method for delivering protein to bone tissue in a mammal. This involves administering to the mammal a sufficient amount of a bone delivery compound as described in the present invention.”
“There is also a method for delivering sPHEX into bone tissue of a mammal. This involves administering to the mammal a dose of the bone delivery conjugate according to the present invention.”
“There is also a method for delivering ALP to bone tissue in a mammal that has a need of it. This involves administering to the mammal a dose of the bone delivery conjugate according to the present invention.”
“The present invention also provides a method for treating a condition or disease that is related to a bone defect. This involves administering to a mammal who has a need, a conjugate of this invention in a pharmaceutically acceptable carrier. “X-linked hypophosphatemic Rickets (XLH) is the specific condition.
“There is also a method for treating a condition or disease that results in a bone defect. This involves administering a conjugate according to the invention to a mammal who has a need. The conjugate is in a pharmaceutically acceptable container. Hypophosphatasia is a condition that can be described in specific embodiments.
“There is also a method for screening peptides to use in a bone delivery peptide-peptide conjugate. It involves the following steps: fusing the candidate protein to a reporter to form a protein peptide conjugate; contacting conjugate with bone tissue and mineral phase of bones; wherein the candidate protein is selected when the reporter protein’s presence on bone tissue is greater when it is bound with the candidate protein than when it’s not.”
“Accordingly to a particular embodiment of the invention, there is provided a bone-delivery conjugate of a protein fused with a peptide selected in the group consisting deca-aspartate(D10) to Hexadeca?aspartate(D16).
“In certain embodiments of conjugates according to the present invention, the sPHEX fused at its Nterminal to D10. Another specific embodiment of the sPHEX fused at its N terminal to D11. Another specific embodiment of the sPHEX fused at its N terminal to D12. Another specific embodiment of the sPHEX fused at its N terminal to D13. Another specific embodiment of the sPHEX fused at its N terminal to D14. Another specific embodiment of the sPHEX fused at its N terminal to D15. Another specific embodiment of the sPHEX fused at its N terminal to D16
“According to specific embodiments, conjugates of this invention, the sALP fused at its Cterminal to D10. Another specific embodiment of the sALP fused at its Cterminal to D10. Another specific embodiment of the sALP fused at its Cterminal to D12. Another specific embodiment of the sALP fused at its Cterminal to D13. Another specific embodiment fuses the sALP at its C-terminal, D14. Another specific embodiment fuses the sALP at its C-terminal to reach D15. Another specific embodiment of the sALP fused is at its C-terminal to D16.
It is understood that any functionally soluble protein can be used in the conjugate according to the invention. While results are shown for conjugates containing one specific sPHEX and sALP according to the present invention, it is also understood that other functional sPHEXes or sALPs may be used.
“sPHEX”
“SPHEX” is any biologically active fragment or mutein of PHEX that is soluble in water. For optimal production of sPHEX, skilled artisans may create expression constructs that are not described in this article. Skilled artisans can also design fragments of DNA encoding biologically active fragments or muteins of naturally occurring PHEX that have the same or similar biological activity as the full-length enzyme.
A large number of expression vectors can be created and tested to express a PHEX cDNA in order to create a recombinant source of sPHEX. A transient transfection experiment as well as stable transfections may reveal an expression construct that is particularly effective in expressing a particular level of expression.
“Any sPHEX containing at least a native PHEX ectodomain section starting with the cysteine position 54 in the sequence shown at FIG. The present invention includes 10
According to the specific embodiments, the conjugates are any sPHEX that contains this 54-749 Native fragment, more preferable the native 52-749 Fragment, more preferred the native 50-749 Native fragment, more preferably the native 49-749 Native fragment, more desirable the native 47-749 Native fragment, more most preferably, the native 46-749 Native fragment, and more preferably, the native 48-749 Native fragment, more preferable the native 47-749, more preferably, the native 47-749, more preferably, and more preferably, together with a Poly-aspartate from the group consisting D10 to D10 to D16 fusions immediately upstream of this segment, D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10, D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D10 to D16, to this fragment.
“The conjugate can optionally contain one or more additional amino acid 1) upstream of the poly-aspartate, and/or 2) between it and the native fragment/functional equivalent. These amino acids may be any amino acid. They may be chosen independently of the group that includes cysteine, proline, and tryptophan, namely amino acids that are known to cause disulfide bonds formation or changes to conformation.
These amino acids could be found in the conjugate if, for example, the cloning technique used to create it introduces them at these locations.
“Amino acids upstream of the polyaspartate can be selected using specific cloning strategies to provide a substrate for specific enzymes in the secretory pathway (e.g. The host cell’s furin (or signal peptidase), will be used to cleave the recombinant PHEXs produced into a secreted bone-targeting sPHEX. An appropriate computer algorithm, such as the one described by Bendtsen and al., can predict the likelihood that a sequence is cleaved in host cells by the signal proteinase. (J Mol Biol. 2004 Jul. 16; 340(4):783-95) and available on the Web at www.cbs.dtu.dk/services/SignalP/ which takes into account parameters including the following: the amino acids at position ?3 and ?1 from the cleavage site by the signal peptidase desirably have small and non charged side chains. Ala, Ser and Gly, Cys and Thr are preferred. Occasionally Gln and Pro are also acceptable. Likewise, those at position ‘3 should be Ala, Ser and Gly, Cys Thr, Ile Leu, Val. Aimable amino acids at position?6 or?4 near the cleavage site can induce the formation of beta-turns (such as Pro) residues.
The present invention also includes conjugates that contain additional amino acids. These may be chosen based on the cloning strategy used in order to create a cleavable PHEX. The cleavable PHEX described in Examples 3 and 4, respectively, contains additional amino acids between the polyaspartate sequence and the native ectodomain. The present invention also includes a conjugate that contains the secPHEX described in co-pending application number. 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. More particularly, FIG. FIG. 12 shows the schematic structure of secPHEXs, which contain additional amino acids upstream from the native 46-749 PHEX fragment. No. Constructs no. 1 through 5 and 3 to 5 could be fused into a poly-aspartate, and used as conjugates according to the present invention. Construct no. Construct no. 4 is a conjugate according to the invention. It includes a D10 polyaspartate as well as a native fragment of ectodomain.
“The conjugates of this invention also include sPHEXs that contain deletions at their Cterminal which are not detrimental to their enzymematic activity.”
“Moreover, the present invention includes conjugates in which the poly-aspartate would attach at the C-terminal to the native PHEX fragment.”
“sALP”
ALP is a membrane-bound protein that is bound to a glycolipid at its C-terminal. After removing a hydrophobic Cterminal end, a glycolipid anchor (GPI), is added to the translational process. This serves as both a transitional membrane anchor and a signal for the addition the GPI. The sALP in Example 6 is composed of an ALP in which the first amino acid from the hydrophobic Cterminal sequence, namely, alanine is replaced with a stop codon. This soluble ALP contains all the amino acids of ALP’s native, and active, anchored form.
“The sALP conjugates according specific embodiments are thus any sALP and a poly-aspartate selected within the group consisting D10 to D16 fused directly downstream of this fragment.”
“The conjugate may optionally contain one or more amino acids either upstream of the poly-aspartate or between the polyaspartate’s native sALP fragment, functional equivalent, or both. Exogenous amino acid may be introduced in these places, for example, when bone targeting conjugate is cloned. The exogenous amino acid should not be added to the transamination site. Ikezawa (Biol Pharm.) describes how to predict the likelihood that a sequence designed will be cleaved in the host cell’s transaminase. Bull. 2002, 25(4) 409-417).”
“The conjugates of this invention also include sALPs that contain deletions at their Nterminal which are not detrimental to their enzyme activity.”
“Furthermore the present invention includes conjugates in which the poly-aspartate would attach at the N-terminal or biologically active fragment of the native ALP-anchored fragment.”
“Recombinant protein” is a term that refers to a protein encoded by a genetically manipulated nucleic acid. “Recombinant protein” is a term that refers to a protein encoded using a genetically modified nucleic acid. It can be placed in a prokaryotic, eukaryotic host cells. The nucleic acids are usually placed in a vector such as a virus or plasmid, depending on the cell’s needs. E. coli was used in the Examples to express the conjugates of this invention. However, a person of ordinary skill will be able to recognize that recombinant proteins can also be produced using other hosts according to routine methods in the art. Maniatis et al. provide examples of representative methods. Cold Springs Harbor Laboratory (1989). ?Recombinant cleavable protein? Recombinant cleavable protein is a term that can be used to describe a recombinant molecule of protein. It may also refer to an enzyme that can cleave the protein to make a secreted/soluble form.
“The term ‘ectodomain fragment?” When used in relation to PHEX, it is meant to mean that PHEX’s fragment is not found within the cellular membrane in its native form.
“Bone tissue” is a term that refers to bone tissue. “Bone tissue” is used herein to describe the tissue that has been synthesized by osteoblasts. It is composed of an organic matrix containing a lot of collagen and mineralized with the deposition of hydroxapatite crystals.
The bone delivery conjugates of this invention contain fusion proteins that are effective in providing a sufficient amount of fusion protein to bone defects. The fusion protein can be administered in standard procedures, such as intravenous injection, in the form of a pharmaceutical formulation in any pharmaceutically acceptable carrier.
“Pharmaceutically acceptable carrier” is a term that refers to a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” is used herein when parenteral administration is chosen as the method of administration. It refers to pharmaceutically acceptable sterile non-aqueous and aqueous solvents, suspensions, or emulsions. Non-aqueous solvents include propylene glycol and polyethylene glycol. Water, water-alcohol solutions, physiological saline, buffered medical parenteral vehicle including sodium chloride, Ringer?s dextrose solution and dextrose plus potassium chloride solution; fluid and nutrient replenishers, electrolyte replenishers, fluids, and Ringer?s solution containing Lactose or fixed oils are all examples of aqueous solvents.
“Effective amount” is a term that refers to the minimum amount of a pharmaceutical composition that should be administered to a mammal in order for it to have measurable therapeutic effects. “Effective amount” is a term that refers to the minimum amount of a pharmaceutical compound that should be given to a mammal to achieve significant therapeutic effects. Many factors will affect the dosages, including the method of administration. The amount of protein in a single dose is usually sufficient to prevent, delay or treat bone-related undesired conditions without causing significant toxic effects. The present invention contains a certain amount of fusion protein that will significantly reduce the clinical symptoms.
The effective amount can be administered daily, weekly, monthly, or in fractions. A pharmaceutical composition of the invention is typically administered in a dose of 0.001 mg to 500 mg per kilogram of body weight per day (e.g. 10 mg, 50mg, 100mg, 250 mg or 250 mg). You can give the dosage in a single or multiple doses. In some cases, the effective dose is the amount that is given to target bone. It can range from 1 mg up to 25 grams of conjugate per day to 50 mg up to 10 grams per day to approximately 1 gram per day. About 50 mg to 10 grams per week of conjugate targeted at bone. 50 mg to 10 grams per week. 100 mg to 5 grams every other day. 1 gram of conjugate targeted to bones once per week.
These are only guidelines. The actual dose must be selected and adjusted by the attending physician according to the clinical factors specific to each patient. Methods of the art will determine the optimal daily dose. Other clinically relevant factors will also be considered. Patients may also be taking medication for other conditions or diseases. While the protein to deliver to bone may be continued while the patient is receiving the medication, it is best to start with low doses in order to see if side effects occur.
“Other objects and advantages, as well as features, of the present invention, will be more evident if you read the following non-restrictive description, which is given only by reference to the accompanying illustrations.
Click here to view the patent on Google Patents.