Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/50—Fusion polypeptide containing protease site
• • 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)
  • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
  • C12Y301/04001—Phosphodiesterase I (3.1.4.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
  • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
  • C12Y306/01009—Nucleotide diphosphatase (3.6.1.9), i.e. nucleotide-pyrophosphatase

Definitions

• ENPP human ectonucleotide pyrophosphatase
• ENPP1-ENPP7 seven extracellular, glycosylated proteins
• ENPPs are cell-surface enzymes, with the exception of ENPP2, which is exported to the plasma membrane but is cleaved by furin and released into the extracellular fluid.
• ENPP enzymes have high degrees of sequence and structural homology, but exhibit a diverse substrate specificity encompassing nucleotides to lipids.
• ENPP1 (also known as PC-1) is a type 2 extracellular membrane-bound glycoprotein located on the mineral-depositing matrix vesicles of osteoblasts and chondrocytes, and hydrolyzes extracellular nucleotides (principally ATP) into adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi).
• PPi functions as a potent inhibitor of ectopic tissue mineralization by binding to nascent hydroxyapatite (HA) crystals, thereby preventing the future growth of these crystals.
• ENPP1 generates PPi via hydrolysis of nucleotide triphosphates (NTPs), Progressive Ankylosis Protein (ANK) transports intracellular PPi into the extracellular space, and Tissue Non-specific Alkaline Phosphatase (TNAP) removes PPi via direct hydrolysis of PPi into Pi.
• NTPs nucleotide triphosphates
• ANK Progressive Ankylosis Protein
• TNAP Tissue Non-specific Alkaline Phosphatase
• Ectopic tissue mineralization is associated with numerous human diseases, including chronic joint disease and acutely fatal neonatal syndromes. To prevent unwanted tissue calcification, factors that promote and inhibit tissue mineralization must be kept in tight balance.
• the balance of extracellular inorganic pyrophosphate (PPi) and phosphate (Pi) is an important regulator of ectopic tissue mineralization.
• the activity of the three extracellular enzymes -TNAP, ANK, and ENPP1 - tightly control the concentration of Pi and PPi in mammals at 1-3 mM and 2-3 mM respectively.
• PPi is a regulator of biomineralization, inhibiting the formation of basic calcium phosphate from amorphous calcium phosphate.
• ENPP1 polypeptides have been shown to be effective in treating certain diseases of ectopic tissue calcification.
• ENPPl-Fc has been shown to reduce generalized arterial calcifications in a mouse model for GACI (generalized arterial calcification of infants), which is a severe disease occurring in infants and involving extensive arterial calcification
• polypeptides that can be used to treat certain calcification or ossification diseases in vivo.
• Such polypeptides should have in vivo half-lives that allow for convenient and effective dosing of the polypeptides to the subject in need thereof.
• the present invention fulfills this need.
• the present disclosure provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to a Fc region of an immunoglobulin, wherein the polypeptide fusion comprises at least one point mutation as described herein.
• the present disclosure further provides an ENPP1 mutant polypeptide comprising at least one point mutation as described elsewhere herein.
• the present disclosure further provides a polypeptide fusion or mutant polypeptide, either of which is expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2, 6-sialyltransferase (ST6GAL1).
• ST6GAL1 human ST6 beta-galactoside alpha-2, 6-sialyltransferase
• the present disclosure further provides a polypeptide fusion and/or mutant polypeptide, either of which is grown in a cell culture supplemented with sialic acid and/or N-acetylmannosamine (l,3,4-0-Bu3ManNAc).
• the present disclosure further provides a method of reducing and/or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure.
• the present disclosure further provides a method of reducing and/or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure.
• the present disclosure further provides a method of reducing and/or preventing progression of ectopic calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure.
• the present disclosure further provides a method of treating, reversing, and/or preventing progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure.
• OPLL posterior longitudinal ligament
• the present disclosure further provides a method of treating, reverting, and/or preventing progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure.
• the present disclosure further provides a method of reducing and/or preventing progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, aging related hardening of arteries, idiopathic infantile arterial calcification (IIAC), Generalized Arterial Calcification of Infancy (GACI), and calcification of atherosclerotic plaques in a subject diagnosed with the at least one disease, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure.
• CKD chronic kidney disease
• ESRD end stage renal disease
• CUA calcific uremic arteriolopathy
• OPLL ossification of the posterior longitudinal ligament
• OPLL hypophosphatemic ricke
• the present disclosure further provides a method of reducing and/or preventing progression of aging related hardening of arteries in a subject in need thereof the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure.
• the present disclosure further provides a method of raising pyrophosphate (PPi) levels in a subject having PPi level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure, whereby upon the administration the level of the PPi in the subject is elevated to a normal level of at least 2mM and is maintained at approximately the same level.
• PPi pyrophosphate
• the present disclosure further provides a method of reducing and/or preventing the progression of pathological calcification or ossification in a subject having pyrophosphate (PPi) level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure, whereby pathological calcification or ossification in the subject is reduced and/or progression of pathological calcification or ossification in the subject is prevented.
• PPi pyrophosphate
• the present disclosure further provides a method of treating ENPP1 deficiency manifested by a reduction of extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion and/or the mutant polypeptide of the disclosure, whereby the level of the PPi in the subject is elevated.
• PPi extracellular pyrophosphate
• FIG. 1 illustrates an ENPP1 polypeptide contemplated within the disclosure (SEQ ID NO:7).
• Point mutations are identified with reference to SEQ ID NO:7, which may also be referred to as the“parent compound,”“parent polypeptide,” or“Construct #770.”
• the labelling scheme identifies the amino acid number and residue with reference to the numbering scheme illustrated in SEQ ID NO:7 followed by the amino acid that has been substituted for the residue in SEQ ID NO:7.
• mutation C25N refers to the substitution of an asparagine (Asn or N) for a cysteine (Cys or C) at position 25 of SEQ ID NO: 7.
• FIG. 2A illustrates the domain structure of the parent Construct # 770.
• the 2 somatomedin B domains, catalytic domain, and endonuclease domain of human ENPP1 was fused N-terminally with the signal sequence of human ENPP7 and c-terminally with the Fc domain of human IgGl.
• FIG. 2B illustrates pharmacokinetic analysis of the parent Construct # 770. After an initial raise in plasma activity 17 hours after subcutaneous injection there is a steep drop off of plasma activity and a calculated half-life of 34 hours.
• FIG. 2C illustrates a non-limiting effect of additional N-glycosylation consensus sequences engineered into the parent Construct # 770.
• FIG. 2D illustrates mass spectroscopy of the digested peptide fragment
• FIG. 2E illustrates the finding that Michaelis-Menten kinetic assays indicate that the velocity of the enzyme at varying substrate concentrations is nearly identical at two different enzyme concentrations for the two I256T containing clones (Clone 17 in yellow, and Clone 19 in red) compared to the parent Construct # 770 in black.
• FIG. 4 illustrates in vivo pharmacokinetic data for Construct #981, as measured using the /iNP-TMP assay to record enzyme activity in plasma samples of a mouse following subcutaneous injection of the construct.
• the half-life was estimated to be around 122 hours based on the single subcutaneous bolus injection into 5 mice. Separate experiments to arrive at the half life are described elsewhere herein.
• FIG. 5 illustrates selected in vivo pharmacokinetic data for Construct #1014,
• FIG. 7 A illustrates certain domains of human ENPP1 with known loss of function mutations that result in the human disease“Generalized Arterial Calcification of Infancy” (GACI).
• GACI Human disease“Generalized Arterial Calcification of Infancy”
• glycosylation sites are not introduced near regions with known loss of function mutations that result in GACI (illustrated in FIG. 7 A).
• FIG. 7B illustrates the crystal structure of ENPP1, with residues where known loss of function mutations resulting in GACI are highlighted (and marked with *). The residue in
• (B) is located in the catalytic domain and corresponds to T238A.
• calcium atom As in FIG. 6: calcium atom
• FIGs. 8A-8D illustrate selected results from high throughput T P-/ NP (thymidine monophosphate-p-nitrophenyl) assays of ENPPl polypeptides for phosphodiesterase activity.
• This is a high throughput assay designed be the inventors to rapidly screen glycosylation isoforms introduced into Construct #770.
• the figure illustrates designing and executing a high throughput screen that is capable of rapid assessment of biological efficacy of mutants forms of the parent polypeptide - Construct #770.
• Construct numbers in (#) represent the original WT clone before mutations were introduced.
• Construct numbers in (*) show clones with possible gain of function mutations.
• FIG. 9 A is ribbon diagram illustrating the Fc domain of human IgGl. This domain is fused onto the C-terminal portion of ENPPl to increase efficacy. Mutations in Fc domain were introduced to enhance pH-dependent recycling by FcRn.
• A) sites that abrogate binding of acidic dependence.
• B) sites that enhance binding.
• FIG. 9B illustrates mutations in the Fc domain of human IgGl known to enhance pH dependent recycling by FcRn.
• FIG. 10 comprises a graph and a table illustrating the effect of glycosylation in PK (in terms of half-life, hours) and bioavailability of ENPPl polypeptides.
• the PK for all the mutations were comparable to that of Construct # CC07 (770B), except the I256T mutation of Construct #922.
• This mutation located in the insertion loop of the catalytic domain was modeled after the equivalent glycosylation site in ENPP3.
• Construct #951 showed similar PK value to that of Construct # CC07, but Construct #951 grown in cell lines stably transfected with ST6GAL1 (Construct #951-ST) showed improved PK and bioavailability. Bioavailability was improved for Construct #922, which contains the I256T mutation.
• Construct #930 had similar half-life, but lower bioavailability, than Construct # CC07. In contrast, Construct #1020 had higher bioavailability than Construct # CC07.
• PK and bioavailability data are presented in the table, determined as illustrated in FIGs. 4, 5, and 13 and calculated using Equation 1.
• FIG. 11 comprises a graph and a table illustrating the effect of glycosylation and H1064K/N1065F Fc mutations in half-life (PK, in hours) and bioavailability (AUC) of ENPP1 polypeptides. All H1064/N1065-containing Constructs showed improved half-life and AUC values over Construct #770B. Note that Constructs #1048 and #1051 correspond to the same cDNA in two distinct clones, illustrating the reproducibility of the PK/AUC analysis provided herein. Construct #1064 was also grown in cell lines stably transfected with ST6GAL1 (Construct #1064-ST).
• Construct #1057 was also grown in cell lines stably transfected with ST6GAL1 (“-ST”)(Construct #1057-ST) and grown in cell lines stably transfected with ST6GAL1 and supplemented with l,3,4-0-Bu3-ManNAc (“-A”) (Construct #1057-ST-A).
• Construct #1089 is identical to Construct #1014 but for an added mutation to eliminate a potential trypsin cleavage site.
• Construct #1014 was also grown in cell lines stably transfected with ST6GAL1 but in this case there was no improvement in PK and bioavailability. PK and bioavailability data are presented in the table, determined as illustrated in FIGs. 4, 5, and 13 and calculated using Equation 1.
• FIG. 12 comprises a graph and a table illustrating the effect of glycosylation and M883Y/S885T/T887E Fc mutation in PK (in terms of half-life, hours) and bioavailability of ENPP1 polypeptides.
• Construct #1030 has a lower AUC than other Constructs possibly due to the S766N mutation.
• Constructs #981, #1028, and #1101 showed an increase in both PK and AUC values when grown in cell lines stably transfected with ST6GAL1.
• Construct #1101 has improved PK and AUC values.
• PK and bioavailability data are presented in the table, determined as illustrated in FIGs. 4, 5, and 13 and calculated using Equation 1.
• FIG. 13 comprises a set of graphs illustrating the effect of expressing the constructs in CHO cells stably transfected with human a-2,6-ST to produce recombinant biologies with terminal sialic acid residues possessing both alpha-2,3 and alpha-2,6 linkages. These cells are referred to as ST6GAL1 cells or ST cells (denoted as“-ST”). This figure also illustrates the effect of growing constructs in ST6GAL1 cells in the presence of sialic acid, or a high flux precursor of sialic acid known as l,3,4-0-Bu3-ManNAc (denoted as“-A”). PK and bioavailability data are presented in the table, determined as illustrated in FIGs. 4, 5, and 13 and calculated using Equation 1.
• FIG. 14A illustrates pharmacokinetic analysis of clones containing the Fc-HR mutation (Clones 9, 10, 11, 12 and 15) and the Fc-MST mutation (Clones 8, 13, 14, 16, and 17) compared with the parent clone 770 and the I256T containing Clone 7.
• the area under the curve (left y-axis) is represented by the bars and the half-life in hours (right y-axis) is represented by the line.
• Clone 7 has only a modest increase in half-life compared with Clones 16 and 17 (line) it has an almost equivocal AUC (bars) as a result of its greater initial activity after injection.
• FIG. 14B illustrates biological availability as represented by the slope of the area under the curve.
• 14C illustrates AUC (bars, left axis) and half-life in hours (line, right axis) for clones grown in unmodified CHO cells or CHO cells over expressing a-2,6-sialytransferase (a-2,6-ST), or CHO cells over-expressing a-2,6-ST combined with l,3,4-0 ⁇ Bu 3 Man Ac supplementation.
• a-2,6-ST Clones 1, 2, 9, 10, 14, 15, 17 and 18
• Clones 9 (3 left arrows) has an increase in AUC and half-life with both CHO cells over-expressing a-2,6-ST, and in CHO over-expressing ⁇ x-2,6-ST combined with 1 ,3 ,4-0-Bu 3 ManNAc
• FIG. 14D illustrates the finding that Anion-Exchange Chromatography with Pulsed
• FIG. 14E illustrates AUC pharmacokinetic analysis for parent Construct #770 grown in CHO cell alone (labelled 770) as compared to Clone 9 grown in CHO cells alone (labelled 9), or CHO cell over-expressing a-2,6-ST (labelled 9(ST)), or CHO cells over-expressing a-2,6-ST combined with l,3,4-0-Bu 3 ManNAc supplementation (labelled 9(ST)A).
• Clone 9 contains an Fc-HN mutation that provides a mild increase in half- life and AUC over parent clone 770. However, when Clone 9 is grown m CHO cells over- expressing a-2,6-ST or the combination of a-2,6-ST and supplementation with
• FIG. 15A illustrates AUC pharmacokinetic analysis for Fc-MST containing Clone 14- ST compared with Fc-MST/I256T containing Clones 17-ST, 19-ST, and 19-ST-A.
• the AUC for Fc-MST containing clones can be further enhanced by the I256T mutation and increased even more when grown in the presence of l,3,4-0-Bu 3 ManNAc precursor.
• FIG. 15B illustrates AUC pharmacokinetics of the parent Clone 770 compared with Clone 19-ST-A.
• FIG. 15C illustrates the finding that MALDl-TOF/TOF analysis for N-glycan profiling revealed that the % glycans containing sialic acid is higher in Clone 19-ST-A (99.214) compared to parent Clone 770 (78.4%) when calculated based on the structures that contains at least one galactose for transfer of sialic acid.
• FIG. 15C illustrates the finding that MALDl-TOF/TOF analysis for N-glycan profiling revealed that the % glycans containing sialic acid is higher in Clone 19-ST-A (99.214) compared to parent Clone 770 (78.4%) when calculated based on the structures that contains at least one galactose for transfer of sialic acid.
• 15D illustrates the pharmacodynamic effect after a single dose at 0.3 mg/kg of either the parent Clone 770 (red squares) or the optimized ENPPl-Fc Clone 19-ST (red circles), as measured by the generation of PPi (left y-axis) in Enppl asj/asj mice.
• Physiological levels of PPi in normal mice (shaded grey) is between 1.5 and 2.5um PPi while Enppl asj/asj mice have nearly undetectable amounts.
• a single dose of Clone 770 restores physiological level of PPi that returns baseline before 89 hours, while Clone 19-ST can maintain near physiological levels out to 263 hours.
• the error bars associated with PPi generation appear much larger than the error bars associate with % activity of the enzyme (right y-axis) (compare red circle vs black circle).
• FIG. 16A-16B illustrate domains of ENPP1 and selected point mutations introduced into the parent polypeptide (SEQ ID NO:7).
• the figure identifies specific point mutations introduced into SEQ ID NO: 7.
• Constructs that have been stably transfected into CHO cells stably transfected with human a-2,6-ST are referred with an“ST”.
• PK and bioavailability data are presented in the table, determined as illustrated in FIGs. 4, 5, and 13 and calculated using Equation 1.
• FIG. 17 illustrates bioavailability (as Area Under Curve, or AUC) for certain
• FIG. 18 illustrates bioavailability (as Area Under Curve, or AUC) for certain
• FIG. 19A comprises an image illustrating stable CHO cell sub-clones co-transfected with plasmid cDNA for both ENPPl-Fc and hST6GALl where screened for expression of alpha-2, 6-sialyltransferase by immunofluorescence of paraformaldehyde fixed cells using a rabbit anti-hST6GAll antibody (R&D Systems cat# AF5924) followed by donkey polyclonal Goat IgG Alexa Fluor594 (Abeam Abl50140). Characteristic Golgi localization was observed with those cell clones that were also positive by Western blot with the same antibody.
• FIG. 19B illustrates a representative western blot using the same primary antibody as FIG.
• 19A demonstrates a single band at approximately 48kD indicating expression of alpha-2, 6-sialyltransferase at varying intensities for some of the CHO cell sub-clones used in this study.
• the first lane is CHO cell lysate untransfected as a negative control.
• the next 3 lanes are 3 unique sub-clones of the unmodified parent plasmid 770 labeled A, B, and C.
• the other lanes are a selection of sub-clones used in the paper for Clones 1, 2, 10, 14, and 18.
• The“X” represents sub-clones of constructs that were not used further herein.
• the present disclosure relates, in one aspect, to the discovery that certain ENPPl-Fc polypeptides having improved in vivo half-lives as compared to the ENPPl-Fc polypeptides known in the art.
• glycosylation was promoted to shield the ENPPl-Fc polypeptides from degradation. This was achieved by introducing additional N-glycan consensus sequences onto the exterior surface of the predicted tertiary structure, guided by three-dimensional models of ENPP1.
• pH-dependent FcRn-mediated cellular recycling was increased by mutating the Fc domain to enhance the affinity of the fusion protein for the neonatal receptor (FcRn).
• sialyation of the fusion protein was enhanced by expressing ENPPl-Fc in CHO cell lines stably transfected with human ST6 beta-galactoside alpha-2, 6-sialyltransferase (also known as ST6GAL1).
• sialic acid capping was enhanced by
• N-acetylmannosamine also known as l,3,4-0-Bu 3 ManNAc
• l,3,4-0-Bu 3 ManNAc N-acetylmannosamine
• enhancing protein sialyation by expressing the biologic in CHO cells stably transfected with human alpha-2, 6-sialyltransferase substantially improved ENPPl-Fc bioavailability (C max ) when dosed subcutaneously.
• increasing the pH-dependent FcRn-mediated cellular recycling by manipulating the Fc domain led to improvements of in vivo biologic half-life.
• combining CHO cells stably transfected with human a-2, 6-sialyltransferase and growing the cells in N-acetylmannosamine led to dramatic increases half-life and/or biologic exposure (AUC).
• AUC half-life and/or biologic exposure
• combining two or more methods described herein into a single construct led to dramatic increases in half-life and/or biologic exposure (AUC).
• the polypeptides of the disclosure are more highly glycosylated than other ENPPl-Fc polypeptides in the art.
• the polypeptides of the disclosure have higher affinity for the neonatal orphan receptor (FcRn) than other ENPPl-Fc polypeptides in the art.
• the polypeptides of the disclosure have higher in vivo half-lives than other ENPPl-Fc polypeptides in the art.
• the kinetic properties of the parent polypeptide (Construct #770) are altered such that the changes represent a“gain of function” alteration in the enzymatic rate constants.
• the kinetic properties of the parent polypeptide are not significantly altered by certain site-directed mutagenesis, and thus the resulting mutant enzyme has enzymatic rate constants that are substantively identical to the parent polypeptide.
• certain point mutations in the parent polypeptide lead to introduction of a glycan at the mutated residue, increasing biologic exposure for the mutant polypeptide.
• the increase in biologic exposure for the mutant polypeptide is due to increased biologic absorption and/or circulation of the mutant polypeptide.
• any of the ENPP1 mutant polypeptides described herein retain ENPP1 catalytic activity as compared to a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO:7. In certain embodiments, any of the ENPP1 mutant polypeptides described herein retain at least about 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9%7, 98%, 99%, 99.5%, 99.8%, or 100%) of the catalytic activity of a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO: 7. In certain embodiments, any one the ENPP1 mutant polypeptides described herein has greater catalytic activity than a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO: 7.
• any of the ENPP1 mutant polypeptides described herein has improved pharmacokinetic and/or bioavailability properties in a mammal as compared to a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides have a circulating half-life in a mammal that is at least 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9%7, 98%, 99%, 99.5%, 99.8%, 100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or greater than 500%) than the circulating half-life of a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO: 7.
• any one the ENPP1 mutant polypeptides described herein has a greater AUC than that of a soluble ENPP1 polypeptide comprising or consisting of amino acids 23-849 of SEQ ID NO:7.
• the in vivo half-life of an ENPPl-Fc polypeptide of the disclosure is at least about 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times higher than the ENPP-1 polypeptides described in the art.
• the polypeptides of the disclosure are administered to the subject at a lower dose and/or at a lower frequency than other ENPPl-Fc polypeptides in the art.
• the polypeptides of the disclosure are administered to the subject once a month, twice a month, three times a month, and/or four times a month.
• the lower frequency administration of the polypeptides of the disclosure results in better patient compliance and/or increased efficacy as compared with other ENPPl-Fc polypeptides in the art.
• an ENPPl-Fc polypeptide of the disclosure can be used to raise pyrophosphate (PPi) levels in a subject having PPi level lower than normal level (which is around 2mM).
• PPi pyrophosphate
• an ENPPl-Fc polypeptide of the disclosure can be used to reduce or prevent progression of pathological calcification or ossification in a subject having PPi levels lower than normal level.
• an ENPPl-Fc polypeptide of the disclosure can be used to treat ENPP1 deficiency manifested by a reduction of extracellular PPi concentration in a subject.
• the steady state level of plasma PPi achieved after administration of a first dosage of a construct of the disclosure is maintained for a time period of at least 2 days, at least 4 days, at least a week or at least a month.
• a second dosage of a construct of the disclosure is
• ENPP1 polypeptides, mutants, or mutant fragments thereof have been previously disclosed in International PCT Application Publications No. WO 2012/125182, WO 2014/126965, WO 2016/187408, and WO 2018/027024, all of which are incorporated by reference in their entireties herein.
• values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
• a range of“about 0.1 % to about 5%” or“about 0.1 % to 5%” should be interpreted to include not just about 0.1 % to about 5%, but also the individual values ( e.g ., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1 % to 0.5%, 1.1 % to 2.2%, 3.3% to 4.4%) within the indicated range.
• an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.
• the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
• the terms“a,”“an,” or“the” are used to include one or more than one unless the context clearly dictates otherwise.
• the term“or” is used to refer to a nonexclusive“or” unless otherwise indicated.
• the statement“at least one of A and B” or“at least one of A or B” has the same meaning as“A, B, or A and B.”
• Protein symbols are disclosed in non-italicized capital letters.
• ⁇ NRR refer to the protein.
• human ENPP1 is referred to as ‘hENPPl’
• mouse ENPP1 is referred to as‘mENPPl’.
• Human gene symbols are disclosed in italicized capital letters.
• the human gene corresponding to the protein hENPPl is ENPP1.
• Mouse gene symbols are disclosed with the first letter in upper case and the remaining letters in lower case; further, the mouse gene symbol is italicized.
• the mouse gene that makes the protein mEnppl is Enppl. Notations about gene mutations are shown as uppercase text.
• “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, in certain embodiments ⁇ 5%, in certain embodiments ⁇ 1%, in certain embodiments ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
• a disease or disorder is“alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
• alteration refers to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation) or conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations.
• antibody refers to an immunoglobulin molecule that is able to specifically bind to a specific epitope on an antigen.
• Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
• The“ATP hydrolytic activity” of ENPP1 can be determined by using an ATP cleavage assay.
• ENPP1 readily hydrolyzes ATP into AMP and PPi.
• the steady-state Michaelis-Menten enzymatic constants of ENPP1 are determined using ATP as a substrate.
• ENPP1 can be demonstrated to cleave ATP by HPLC analysis of the enzymatic reaction, and the identity of the substrates and products of the reaction are confirmed by using ATP, AMP, and ADP standards.
• the ATP substrate degrades over time in the presence of ENPP1, with the accumulation of the enzymatic product AMP.
• the initial rate velocities for ENPP1 are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants.
• AUC refers to the area under the plasma drug
• concentration-time curve correlates with actual body exposure to drug after administration of a dose of the drug.
• the AUC is expressed in mg*h/L.
• the AUC can be used to measure bioavailability of a drug, which is the fraction of unchanged drug that is absorbed intact and reaches the site of action, or the systemic circulation following administration by any route.
• AUC can be calculated used Linear Trapezoidal method or Logarithmic Trapezoidal method.
• the Linear Trapezoidal method uses linear interpolation between data points to calculate the AUC. This method is required by the OGD and FDA, and is the standard for bioequivalence trials. For a given time interval (ti - 1 2 ), the AUC can be calculated as follows:
• Ci and C2 are the average concentration over the time interval (ti and t2).
• the Logarithmic Trapezoidal method uses logarithmic interpolation between data points to calculate the AUC. This method is more accurate when concentrations are decreasing because drug elimination is exponential (which makes it linear on a logarithmic scale). For a given time interval (ti - 1 2 ), the AUC can be calculated as follows (assuming that Ci > C2):
• bioavailability refers to the extent and rate at which the active moiety (protein or drug or metabolite) enters systemic circulation, thereby accessing the site of action, or the systemic circulation following administration by any route.
• Bioavailability of an active moiety is largely determined by the properties of the dosage form, which depend partly on its design and manufacture. Differences in bioavailability among formulations of a given drug or protein can have clinical significance; thus, knowing whether drug formulations are equivalent is essential.
• the most reliable measure of a drug’s or protein’s bioavailability is area under the plasma concentration-time curve (AUC). AUC is directly proportional to the total amount of unchanged drug or therapeutic protein that reaches systemic circulation. Drug or therapeutic protein may be considered bioequivalent in extent and rate of absorption if their plasma concentration curves are essentially superimposable.
• AUC plasma concentration-time curve
• bioavailability is defined as unity.
• bioavailability is often less than unity. Incomplete bioavailability may be due to a number of factors that can be subdivided into categories of dosage form effects, membrane effects, and site of administration effect.
• Half- life and AUC provide information about the bioavailability of a drug or biologic.
• the terms“conservative variation” or“conservative substitution” as used herein refers to the replacement of an amino acid residue by another, biologically similar residue. Conservative variations or substitutions are not likely to change the shape of the peptide chain. Examples of conservative variations, or substitutions, include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
• a“construct” of the disclosure refers to a fusion polypeptide comprising an ENPP1 polypeptide, or a fragment or site directed mutant thereof.
• A“disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
• A“disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
• the terms“effective amount,”“pharmaceutically effective amount” and“therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
• ENPP ectonucleotide pyrophosphatase/ phosphodiesterase
• ENPP1 protein or“ENPPl polypeptide” refers to ectonucleotide pyrophosphatase/phosphodiesterase- 1 protein encoded by the ENPPl gene.
• the encoded protein is a type II transmembrane glycoprotein and cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars.
• ENPP1 protein has a
• transmembrane domain and soluble extracellular domain The extracellular domain is further subdivided into somatomedin B domain, catalytic domain, and the nuclease domain.
• the sequence and structure of wild-type ENPP1 is described in detail in PCT Application Publication No. WO 2014/126965 to Braddock, et al, which is incorporated herein in its entirety by reference.
• the term“human ENPP1” refers to the human ENPP1 sequence as described in NCBI accession NP_006199.
• the term“soluble human ENPP1” refers to the polypeptide corresponding to residues 96 to 925 of NCBI accession NP_006199.
• the term“enzymatically active” with respect to ENPP1 is defined as being capable of binding and hydrolyzing ATP into AMP and PPi and/or AP3a into ATP.
• ENPPl precursor protein refers to ENPP1 with its signal peptide sequence at the ENPPl N-terminus. Upon proteolysis, the signal sequence is cleaved from ENPPl to provide the ENPPl protein.
• Signal peptide sequences useful within the disclosure include, but are not limited to, ENPPl signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and/or ENPP5 signal peptide sequence.
• ENPPl-Fc refers to ENPPl recombinantly fused and/or chemically conjugated (including both covalent and non-covalent conjugations) to an FcR binding domain of an IgG molecule (preferably, a human IgG).
• an IgG molecule preferably, a human IgG.
• the C -terminus of ENPPl is fused or conjugated to the N-terminus of the FcR binding domain.
• Fc refers to a human IgG (immunoglobulin) Fc domain.
• IgGl immunoglobulin
• IgG2 immunoglobulin-2
• IgG3 immunoglobulin-4
• IgG4 immunoglobulin-4
• the“Fc region” is the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule.
• the Fc region comprises the C-terminal half of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and the binding sites for complement and Fc receptors, including the FcRn receptor.
• the Fc fragment contains the entire second constant domain CH2 (residues 231-340 of human IgGl, according to the Rabat numbering system) and the third constant domain CH3 (residues 341- 447).
• IgG hinge-Fc region or“hinge-Fc fragment” refers to a region of an IgG molecule consisting of the Fc region (residues 231 -447) and a hinge region (residues 216- 230) extending from the N-terminus of the Fc region.
• the term“constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site.
• the constant domain contains the CHI, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.
• Fc receptors refer to proteins found on the surface of certain cells (including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells) that contribute to the protective functions of the immune system. Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. Immunoglobulin Fc receptors (FcRs) are expressed on all hematopoietic cells and play crucial roles in antibody-mediated immune responses.
• Fc receptors have been described for all classes of immunoglobulins: FcyR and neonatal FcR (FcRn) for IgG, FcsR for IgE, FcaR for IgA, Fc5R for IgD and FcpR for IgM.
• Fc receptors structurally belong to the immunoglobulin superfamily, except for FcRn and FcsRII, which are structurally related to class I Major Histocompatibility antigens and C-type lectins, respectively ⁇ Fc Receptors, Neil A. Fangera, et al., in Encyclopedia of Immunology (2 nd Edition), 1998).
• FcRn Receptor refers to the neonatal Fc receptor (FcRn), also known as the Brambell receptor, which is a protein that in humans is encoded by the FCGRT gene.
• FcRn specifically binds the Fc domain of an antibody.
• FcRn extends the half-life of IgG and serum albumin by reducing lysosomal degradation in endothelial cells. IgG, serum albumin, and other serum proteins are continuously internalized through pinocytosis. Generally, serum proteins are transported from the endosomes to the lysosome, where they are degraded.
• FcRn-mediated transcytosis of IgG across epithelial cells is possible because FcRn binds IgG at acidic pH ( ⁇ 6.5) but not at neutral or higher pH. IgG and serum albumin are bound by FcRn at the slightly acidic pH ( ⁇ 6.5), and recycled to the cell surface where they are released at the neutral pH (>7.0) of blood. In this way IgG and serum albumin avoid lysosomal degradation.
• the Fc portion of an IgG molecule is located in the constant region of the heavy chain, notably in the CH2 domain.
• the Fc region binds to an Fc receptor (FcRn), which is a surface receptor of a B cell and also proteins of the complement system.
• FcRn Fc receptor
• the binding of the Fc region of an IgG molecule to an FcRn activates the cell bearing the receptor and thus activates the immune system.
• the Fc residues critical to the mouse Fc-mouse FcRn and human Fc-human FcRn interactions have been identified (Dall’Acqua et al., 2002, J.
• An FcRn binding domain comprises the CH2 domain (or a FcRn binding portion thereol) of an IgG molecule.
• the term“fragment,” as applied to a nucleic acid refers to a subsequence of a larger nucleic acid.
• A“fragment” of a nucleic acid can be at least about 15, 50-100, 100-500, 500-1000, 1000-1500 nucleotides, 1500-2500, or 2500 nucleotides (and any integer value in between).
• the term“fragment,” as applied to a protein or peptide refers to a subsequence of a larger protein or peptide, and can be at least about 20,
• “functional equivalent” or“functional derivative” denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of sequences of ENPPl-Fc constructs shown herein.
• a functional derivative or equivalent may be a natural derivative or is prepared synthetically.
• the functionally-equivalent polypeptides of the disclosure can also be polypeptides identified using one or more techniques of structural and or sequence alignment known in art.
• Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved.
• the substituting amino acid desirably has chemico-physical properties which are similar to that of the substituted amino acid. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like. Typically, greater than 30% identity between two polypeptides is considered to be an indication of functional equivalence.
• functionally equivalent polypeptides of the disclosure have a degree of sequence identity with the ENPPl-Fc constructs of greater than 80%.
• More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98% or 99%, respectively.
• Method for determining whether a functional equivalent or functional derivative has the same or similar or higher biological activity than the ENPPl-Fc construct can be determined by using the Enzymology assays involving ATP cleavage described in WO2016/187408.
• Gene transfer and“gene delivery” refer to methods or systems for reliably inserting a particular nucleic acid sequence into targeted cells.
• An“inducible” promoter is a nucleotide sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer that corresponds to the promoter is present in the cell.
• the term“in vivo half-life” for a protein and/or polypeptide contemplated within the disclosure refers to the time required for half the quantity administered in the animal to be cleared from the circulation and/or other tissues in the animal.
• the curve is usually biphasic with a rapid a-phase (which represents an equilibration of the administered molecules between the intra- and extra-vascular space and which is, in part, determined by the size of molecules), and a longer b-phase (which represents the catabolism of the molecules in the intravascular space).
• the term in vivo half-life” in practice corresponds to the half-life of the molecules in the b-phase.
• “Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the nucleic acid, peptide, and/or compound of the disclosure in the kit for identifying or alleviating or treating the various diseases or disorders recited herein.
• isolated means altered or removed from the natural state.
• a nucleic acid or a polypeptide naturally present in a living animal is not“isolated,” but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is“isolated.”
• An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
• An“isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, /. e.. a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs.
• the term also applies to nucleic acids that have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell.
• the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
• An“oligonucleotide” or“polynucleotide” is a nucleic acid ranging from at least 2, in certain embodiments at least 8, 15 or 25 nucleotides in length, but may be up to 50, 100,
• operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
• a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
• a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
• operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
• the term“patient,”“individual” or“subject” refers to a human.
• the term“pharmaceutical composition” or“composition” refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier.
• the pharmaceutical composition facilitates administration of the compound to a patient.
• Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical administration.
• the term“pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
• the term“pharmaceutically acceptable carrier” means a
• pharmaceutically acceptable material such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function.
• Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient.
• materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives.
• “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the patient.
• “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the disclosure.
• Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
• pharmaceutically acceptable salt refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.
• PPi levels refers to the amount of pyrophosphate present in plasma of animals.
• animals include rat, mouse, cat, dog, human, cow and horse. It is necessary to measure PPi in plasma rather than serum because of release from platelets.
• UPPG uridine-diphosphoglucose
• normal PPi levels in healthy subjects range from about lpm to about 3 mM, in some cases between 1-2 pM.
• Subjects with defective ENPP1 expression tend to exhibit low PPi levels ranging from at least 10% below normal levels, at least 20% below normal levels, at least 30% below normal levels, at least 40% below normal levels, at least 50% below normal levels, at least 60% below normal levels, at least 70% below normal levels, at least 80% below normal levels, and any combinations thereof.
• the PPi levels in blood plasma are found to be less than 1 pM and in some cases are below detech on levels.
• the plasma PPi levels of subjects afflicted with diseases of pathological calcification or ossification are below 0.5 pM (Arterioscler Thromb Vase Biol. 2014, 34(9): 1985-9; Braddock et al., 2015, Nat Commun. 6: 10006.)
• polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogues thereof linked via peptide bonds.
• PPi refers to pyrophosphate
• the term“prevent” or“prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.
• promoter as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
• promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/ regulatory sequence.
• this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product.
• the promoter/ regulatory sequence may for example be one that expresses the gene product in a tissue specific manner.
• recombinant polypeptide as used herein is defined as a polypeptide produced by using recombinant DNA methods.
• recombinant DNA as used herein is defined as DNA produced by joining pieces of DNA from different sources.
• sample or“biological sample” as used herein means a biological material isolated from a subject.
• the biological sample may contain any biological material suitable for detecting a mRNA, polypeptide or other marker of a physiologic or pathologic process in a subject, and may comprise fluid, tissue, cellular and/or non-cellular material obtained from the individual.
• signal peptide refers to a sequence of amino acid residues (ranging in length from, for example, 10-30 residues) bound at the amino terminus of a nascent protein of interest during protein translation.
• the signal peptide is recognized by the signal recognition particle (SRP) and cleaved by the signal peptidase following transport at the endoplasmic reticulum. (Lodish, et al., 2000, Molecular Cell Biology, 4 th edition).
• substantially purified refers to being essentially free of other components.
• a substantially purified polypeptide is a polypeptide that has been separated from other components with which it is normally associated in its naturally occurring state.
• Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity and 100% purity.
• A“tissue-specific” promoter is a nucleotide sequence that, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
• under transcriptional control or“operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a
• polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
• transfected or“transformed” or“transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
• a “transfected” or“transformed” or“transduced” cell has been transfected, transformed or transduced with exogenous nucleic acid.
• the cell includes the primary subject cell and its progeny.
• treatment or“treating” is defined as the application or administration of a therapeutic agent, /. e.. a compound useful within the disclosure (alone or in combination with another pharmaceutical agent), to a patient, or application or
• a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder, a symptom of a disease or disorder or the potential to develop a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the potential to develop the disease or disorder.
• Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
• “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical.
• a variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination.
• a variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or may be a variant that is not known to occur naturally.
• Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.
• A“vector” is a composition of matter that comprises an isolated nucleic acid and that may be used to deliver the isolated nucleic acid to the interior of a cell.
• Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
• the term “vector” includes an autonomously replicating plasmid or a virus.
• the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
• examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
• virus is defined as a particle consisting of nucleic acid (RNA or DNA) enclosed in a protein coat, with or without an outer lipid envelope, which is capable of transfecting the cell with its nucleic acid .
• wild-type refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is most frequently observed in a population and is thus arbitrarily designed the“normal” or“wild-type” form of the gene.
• modified or“mutant” refers to a gene or gene product that displays modifications in sequence and/or functional properties (i.e.. altered characteristics) when compared to the wild-type gene or gene product. Naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) when compared to the wild-type gene or gene product.
• 1,3,4-0-B3 ⁇ 4MEIINAO N- acetylmannosamine
• ST6GAL1 ST6 beta-galactoside alpha-2, 6-sialyltransferase.
• ranges throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• the disclosure provides an ENPPl-Fc polypeptide.
• the disclosure contemplates that the polypeptide of the disclosure can have one or more of the mutations described herein.
• the disclosure provides an ENPP1 mutant polypeptide comprising at least one amino acid substitution at position 256 as relating to SEQ ID NO:7.
• the amino acid substitution is the substitution of isoleucine (I) for threonine (T) at position 256 relative to SEQ ID NO:7.
• the amino acid substitution is the substitution of isoleucine (I) for serine (S) at position 256 relative to SEQ ID NO:7.
• the ENPP1 mutant polypeptide comprises the catalytic domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide comprises the endonuclease domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the nuclease domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the transmembrane domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the intracellular domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks both the intracellular domain and the transmembrane domains of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks a signal sequence.
• the ENPP1 mutant polypeptide comprises an amino acid sequence that is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids 23-849 of SEQ ID NO:7.
• the disclosure provides an ENPP1 mutant polypeptide comprising an amino acid sequence that is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids 23-849 of SEQ ID NO:7, wherein the mutant polypeptide comprises an amino acid substitution at position 256 as relating to SEQ ID NO:7.
• the amino acid substitution is I256T.
• the amino acid substitution is I256S.
• the disclosure provides an ENPP1 mutant polypeptide comprising amino acids 23-849 of SEQ ID NO:7 in which no more than ten (10) (e.g., no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than one) amino acid substitution(s) relative to amino acids 23-849 of SEQ ID NO:7 are present.
• the ENPP1 mutant polypeptide comprises an amino acid substitution at position 256 relative to SEQ ID NO:7.
• the amino acid substitution is I256T.
• the amino acid substitution is I256S.
• the ENPP1 polypeptide comprises at least one mutation in the signal sequence region as recited in FIG. 16A and/or FIG. 16B.
• the polypeptide comprises mutation I256T as relating to SEQ ID NO:7.
• the mutation is selected from the group consisting of C25N, K27T, and V29N as relating to SEQ ID NO:7.
• the mutation is C25N as relating to SEQ ID NO:7.
• the mutation is K27T as relating to SEQ ID NO:7.
• the mutation is V29N as relating to SEQ ID NO:7.
• the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N/K27T and V29N as relating to SEQ ID NO:7.
• the ENPP1 polypeptide comprises at least one mutation in the catalytic region as recited in FIG. 16A and/or FIG. 16B.
• the mutation is selected from the group consisting of I256T, K369N, and I371T as relating to SEQ ID NO:7.
• the mutation is I256Y as relating to SEQ ID NO:7.
• the mutation is K369N as relating to SEQ ID NO:7. In certain embodiments, the mutation is I371T as relating to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of I256T and K369N/I371T as relating to SEQ ID NO:7.
• the ENPP1 polypeptide comprises at least one mutation in the endonuclease domain as recited in Table 1, Table 2, Table 3, Table 4, Table 5, FIG. 7A, FIG. 16A, FIG. 16B, FIG. 17, and/or FIG. 18.
• the mutation is selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N as relating to SEQ ID NO:7.
• the mutation is P534N as relating to SEQ ID NO:7.
• the mutation is V536T as relating to SEQ ID NO:7.
• the mutation is R545T as relating to SEQ ID NO:7. In certain embodiments, the mutation is P554L as relating to SEQ ID NO:7. In certain embodiments, the mutation is E592N as relating to SEQ ID NO:7. In certain embodiments, the mutation is R741D as relating to SEQ ID NO:7. In certain embodiments, the mutation is S766N as relating to SEQ ID NO:7. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N as relating to SEQ ID NO:7.
• the ENPP1 polypeptide comprises at least one mutation in the linker region as recited in FIG. 16A and/or FIG. 16B.
• the mutation is selected from the group consisting of E864N and L866T as relating to SEQ ID NO: 7.
• the ENPP1 polypeptide comprises at least the mutation E864N/L866T as relating to SEQ ID NO:7.
• the mutation is E864N as relating to SEQ ID NO:7.
• the mutation is L866T as relating to SEQ ID NO:7.
• the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, wherein the FcRn binding domain comprises any mutation recited in Table 1, Table 2, FIG. 7A, FIG. 16A, FIG. 16B, FIG. 17, and/or FIG. 18.
• the mutation is selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F as relating to SEQ ID NO:7.
• the mutation is M883Y as relating to SEQ ID NO:7.
• the mutation is S885N as relating to SEQ ID NO:7.
• the mutation is S885T as relating to SEQ ID NO:7. In certain embodiments, the mutation is T887E as relating to SEQ ID NO:7. In certain embodiments, the mutation is H1064K as relating to SEQ ID NO:7. In certain embodiments, the mutation is N1065F as relating to SEQ ID NO: 7. In certain embodiments, the FcRn binding domain comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and H1064K/N1065F as relating to SEQ ID NO:7.
• the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N, K27T, V29N, C25N/K27T, I256T, K369N, I371T, K369N/I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N,
• the polypeptide comprises at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F,
• the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, the polypeptide comprising mutations M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, the polypeptide comprising mutations P534N, V536T, M883Y,
• the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, the polypeptide comprising mutations E592N, H1064K, and N1065F as relating to SEQ ID NO:7.
• the polypeptide comprises an ENPP1 mutant polypeptide, wherein the mutant polypeptide comprises an ENPP1 mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N as relating to SEQ ID NO: 7.
• the ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N/V 536T, P554L/R545T, and E592N as relating to SEQ ID NO:7.
• the polypeptide further comprises an FcRn binding domain of an IgG.
• the polypeptide comprises mutations selected from the group consisting of: S885N, S766N, M883Y/S885T/T887E, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and
• P534N/V 536T/M883Y/S885T/T887E as relating to SEQ ID NO:7.
• the polypeptide comprises an S885N mutation in the FcRn binding domain as relating to SEQ ID NO:7.
• the polypeptide comprises an S766N mutation in the ENPP1 mutant polypeptide as relating to SEQ ID NO:7.
• the polypeptide comprises mutations M883Y, S885T, and T887E in the FcRn binding domain as relating to SEQ ID NO: 7.
• the polypeptide comprises mutations P534N and V536T in the ENPP1 mutant polypeptide and mutations H1064K and N1065F in the FcRn binding domain as relating to SEQ ID NO:7.
• the polypeptide comprises mutations P554L and R545T in the ENPP1 mutant polypeptide as relating to SEQ ID NO:7.
• the polypeptide comprises mutation S766N in the ENPP1 mutant polypeptide and mutations H1064K and N1065F in the FcRn binding domain as relating to SEQ ID NO:7.
• the polypeptide comprises mutation E592N in the ENPP1 mutant polypeptide and mutations H1064K and N1065F in the FcRn binding domain as relating to SEQ ID NO:7.
• the polypeptide comprises mutations P534N and V536T in the ENPP1 mutant polypeptide and mutations M883Y, S885T, and T887E in the FcRn binding domain as relating to SEQ ID NO:7. In certain embodiments, the polypeptide comprises mutation I256T as relating to SEQ ID NO:7.
• the polypeptide comprises mutations I256T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• the polypeptide comprises mutations V29N, I256T, P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• the disclosure features an ENPP1 mutant polypeptide comprising an amino acid sequence that is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids 23-849 of SEQ ID NO:7, wherein the mutant polypeptide comprises mutation I256T as relating to SEQ ID NO:7, and further comprises a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N as relating to SEQ ID NO:7.
• any of the mutant polypeptides described herein comprises at least one amino acid substitution selected from the group consisting of S766N,
• P534N/V536T, P554L/R545T, and E592N as relating to SEQ ID NO:7.
• any of the mutant polypeptides described herein comprises the amino acid substitution V29N.
• the mutant polypeptide comprises or consists of the amino acid sequence depicted in SEQ ID NO: 11.
• ENPP1 mutant polypeptide fusions comprising any of the ENPP1 mutant polypeptides described herein and a heterologous protein, such as an FcRn binding domain.
• the heterologous protein is carboxy -terminal to the ENPP1 mutant polypeptide portion of the fusion.
• the heterologous protein is amino-terminal to the ENPP1 mutant polypeptide portion of the fusion.
• the FcRn binding domain is an albumin polypeptide. In certain embodiments, the FcRn binding domain is a Fc portion of an immunoglobulin molecule, such as an IgGl immunoglobulin molecule.
• the FcRn binding domain comprises one more amino acid substitutions relative to a wild type FcRn binding domain.
• the FcRn binding domain is the Fc portion of a human IgGl molecule and comprises the following amino acid substitutions: M883Y, S885T, and T887E (the MST/YTE substitutions), each relative to SEQ ID NO:7.
• a fusion described herein comprises one or more of the following substitutions: S885N, S766N, M883Y/S885T/T887E, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F,
• the ENPP1 mutant polypeptide comprises or consists of the amino acid sequence depicted in SEQ ID NO:l 1.
• the ENPP1 mutant polypeptide comprises or consists of the amino acid sequence depicted in SEQ ID NO: 12.
• any of the fusions described herein comprise: (a) an ENPP1 mutant polypeptide comprising or consisting of the amino acid sequence depicted in SEQ ID NO: 11 or SEQ ID NO: 12, (b) a variant human IgGl Fc region, such as the amino acid sequence depicted in SEQ ID NO: 14, which is carboxy -terminal to the ENPP1 mutant polypeptide; and (c) a linker amino acid sequence separating (a) and (b), wherein the linker sequence is LIN (SEQ ID NO: 8) or GGGGS (SEQ ID NO: 9).
• the heterologous moiety increases or further increases the pharmacokinetic and/or bioavailability of the mutant polypeptides in a mammal.
• the heterologous moiety is an oligomer of ethylene glycol and/or propylene glycol, such as but not limited to polyethylene glycol (PEG) and/or polypropylene glycol (PPG).
• any of the ENPP1 mutant polypeptide fusions or conjugates described herein comprises the S885N mutation as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises the S766N mutation as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptide fusions or conjugates described herein comprises mutations M883Y, S885T, and T887E as relating to SEQ ID NO: 7.
• any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutations P534N, V536T, H1064K, and N1065F as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutations P554L and R545T as relating to SEQ ID NO: 7.
• any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutation S766N, H1064K, and N1065F as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutation E592N, H1064K, and N1065F as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutations P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• the disclosure provides an ENPP1 mutant polypeptide fusion comprising an ENPP1 mutant polypeptide fused to a Fc region of an immunoglobulin, wherein the ENPP1 mutant polypeptide comprises a substitution at position 256 relative to SEQ ID NO:7.
• the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N,
• the Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and H1064K/N1065F as relating to SEQ ID NO:7.
• the ENPP1 mutant polypeptide further comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N as relating to SEQ ID NO:7.
• an ENPP1 mutant polypeptide, or a fusion described herein comprises at least one mutation selected from the group consisting of C25N/K27T and V29N as relating to SEQ ID NO:7.
• an ENPP1 mutant polypeptide described herein further comprises at least one mutation selected from the group consisting of K369N and 137 IT as relating to SEQ ID NO:7.
• an ENPP1 mutant polypeptide, or a fusion comprising such a mutant polypeptide, described herein comprises the mutation K369N/I371T as relating to SEQ ID NO:7.
• an ENPP1 mutant polypeptide, or fusion comprising such a mutant polypeptide, described herein further comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N as relating to SEQ ID NO:7.
• an ENPP1 mutant polypeptide, or a fusion comprising such a mutant polypeptide, described herein comprises at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides or fusions described herein further comprises at least one mutation selected from the group consisting of E864N and L866T as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides or fusions described herein comprises at least the mutation E864N/L866T as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides or fusions described herein comprise at least one mutation selected from the group consisting of C25N, K27T, V29N, C25N/K27T, K369N, 137 IT, K369N/I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N/V536T, P554L/R545T, E592N/R741D, E864N, L866T,
• any of the fusions described herein comprise an Fc region of an IgG, such as IgGl.
• any of the ENPP1 mutant polypeptides described herein, or the fusion proteins comprising such ENPP1 mutant polypeptides comprise at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N, and E592N as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides described herein, or the fusion proteins comprising such ENPP1 mutant polypeptides comprise at least one mutation selected from the group consisting of S766N, P534N/Y536T, P554L/R545T, and E592N as relating to SEQ ID NO:7.
• any of the ENPP1 mutant polypeptides described herein, or the fusion proteins comprising such ENPP1 mutant polypeptides comprise at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H 1064K/N 1065F, and P534N/V536T/M883Y/S885T/T887E as relating to SEQ ID NO: 7.
• any of the fusions described herein comprise mutations I256T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• any of the fusions described herein comprise an ENPP1 polypeptide and a Fc region of an immunoglobulin, the polypeptide fusion comprising mutations I256T, P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• any of the fusions described herein comprise the ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and a Fc region of an immunoglobulin, the polypeptide fusion comprising mutations I256T, E592N, H1064K, and N1065F as relating to SEQ ID NO:7.
• the ENPP1 mutant polypeptide fusions described herein comprise a linker amino acid sequence, for example, between the ENPP1 mutant polypeptide portion of the fusion and the heterologous protein moiety.
• the linker amino acid sequence comprises or consists of SEQ ID NO: 8.
• the disclosure features ENPP1 -containing polypeptides comprising or consisting of the amino acid sequence depicted in SEQ ID NO: 15 or SEQ ID NO: 16, and conjugates thereof.
• the ENPP1 polypeptide lacks a nuclease domain. In other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain. In yet other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain from about residue 524 to about residue 885 relative to SEQ ID NO: l, leaving only the catalytic domain from about residue 186 to about residue 586 relative to SEQ ID NO: 1, which serves to preserve the catalytic activity of the protein.
• the ENPP1 polypeptide is modified with a segment of the extracellular region of ENPP1 containing a peptidase cleavage site after the signal peptide, and between the transmembrane and extracellular domain, as compared to SEQ ID NO: 1.
• the ENPP1 polypeptide is modified with a segment of the extracellular region of ENPP1 containing a furin cleavage site between the transmembrane and extracellular domain, as compared to SEQ ID NO: l. In other embodiments, the ENPP1 polypeptide is not modified with a segment of the extracellular region of ENPP1 containing a furin cleavage site between the transmembrane and extracellular domain, as compared to SEQ ID NO: l.
• the ENPP1 polypeptide is modified with a segment of the extracellular region of ENPP2 containing a signal peptidase cleavage site, as compared to SEQ ID NO: 1. In other embodiments, the ENPP1 polypeptide is not modified with a segment of the extracellular region of ENPP2 containing a signal peptidase cleavage site, as compared to SEQ ID NO: 1.
• the disclosure provides an ENPP1 mutant polypeptide, a ENPP1 -containing polypeptide, or a fusion, which is expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2, 6-sialyltransferase (also known as ST6GAL1).
• the disclosure provides an ENPP1 mutant polypeptide, a ENPP1 -containing polypeptide, or a fusion, which is grown in a cell culture supplemented with sialic acid and/or N-acetylmannosamine (also known as l,3,4-0-Bu3ManNAc).
• compositions comprising any one of the ENPP1 mutant polypeptides, the ENPP1 mutant polypeptide fusions, conjugates, or other polypeptides and proteins described herein and a pharmaceutically acceptable carrier.
• the polypeptide is soluble. In other embodiments, the polypeptide is a recombinant polypeptide. In yet other embodiments, the polypeptide comprises an ENPP1 polypeptide that lacks the ENPP1 transmembrane domain. In yet other embodiments, the polypeptide comprises an ENPP1 polypeptide wherein the ENPP1 transmembrane domain has been removed (and/or truncated) and replaced with the transmembrane domain of another polypeptide, such as, by way of non-limiting example, ENPP2, ENPP5, or ENPP7.
• the polypeptide comprises a signal peptide resulting in the secretion of a precursor of the ENPP1 polypeptide, which undergoes proteolytic processing to yield a polypeptide comprising the ENPP1 polypeptide.
• the signal peptide is selected from the group consisting of signal peptides of ENPP2, ENPP5, and ENPP7.
• the polypeptide comprises an ENPP1 polypeptide comprising transmembrane domains of ENPP1 and another polypeptide, such as, by way of non-limiting example, ENPP2.
• the ENPP1 polypeptide comprises a cleavage product of a precursor ENPP1 polypeptide comprising an ENPP2 transmembrane domain.
• the ENPP2 transmembrane domain comprises residues 12-30 of SEQ ID NO:7, which corresponds to IISLFTFAVGVNICLGFTA.
• the ENPP1 polypeptide is C-terminally fused to the Fc domain of human immunoglobulin 1 (IgGl), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and/or human immunoglobulin 4 (IgG4).
• the ENPP1 polypeptide is N-terminally fused to the Fc domain of human immunoglobulin 1 (IgGl), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and/or human immunoglobulin 4 (IgG4).
• the presence of IgFc domain improves half-life, solubility, reduces immunogenicity, and increases the activity of the ENPP1 polypeptide.
• the ENPP1 polypeptide is C-terminally fused to human serum albumin.
• Human serum albumin may be conjugated to ENPP1 protein through a chemical linker, including but not limited to naturally occurring or engineered disulfide bonds, or by genetic fusion to ENPP1, or a fragment and/or variant thereof.
• the polypeptide is further pegylated (fused with a poly(ethylene glycol) chain).
• the polypeptide has a k cat value for the substrate ATP greater than or equal to about 3.4 ( ⁇ 0.4) s 1 enzyme 1 , wherein the k cat is determined by measuring the rate of hydrolysis of ATP for the polypeptide.
• the polypeptide has a A M value for the substrate ATP less than or equal to about 2 mM, wherein the K M is determined by measuring the rate of hydrolysis of ATP for the polypeptide.
• the polypeptide is formulated as a liquid formulation.
• the disclosure provides a dry product form of a pharmaceutical composition comprising a therapeutic amount of a polypeptide of the disclosure, whereby the dry product is reconstitutable to a solution of the compound in liquid form.
• the disclosure provides a kit comprising at least one polypeptide of the disclosure, or a salt or solvate thereof, and instructions for using the polypeptide within the methods of the disclosure.
• the polypeptide lacks a negatively-charged bone-targeting sequence.
• a polyaspartic acid domain (from about 2 to about 20 or more sequential aspartic acid residues) is a non-limiting example of a negatively-charged bone-targeting sequence.
• the polypeptide has a negatively-charged bone-targeting sequence.
• an ENPP1 polypeptide according to the disclosure includes not only the native human proteins, but also any fragment, derivative, fusion, conjugate or mutant thereof having ATP hydrolytic activity of the native protein.
• the phrase“an ENPP1 polypeptide, mutant, or mutant fragment thereof’ also includes any compound or polypeptide (such as, but not limited to, a fusion protein) comprising an ENPP1 polypeptide, mutant, or mutant fragment thereof. Fusion proteins according to the disclosure are considered biological equivalents of ENPP1, but are intended to provide longer half-life or greater potency due to increased in vivo biologic exposure, as judged by the“area under the curve” (AUC) or increased half-life in pharmacokinetic experiments..
• nucleic acids that encode any one of the ENPP1 mutant polypeptides, ENPP1 -containing polypeptides, or fusions described herein.
• the disclosure further provides vectors, such as expression vectors, that comprise such nucleic acids.
• vectors such as expression vectors, that comprise such nucleic acids.
• a cell, cells, or a plurality of cells e.g., mammalian cells that comprise any one of the nucleic acids, vectors, or expression vectors described herein.
• Also provided are methods for producing a protein e.g., any one of the ENPP1 mutant polypeptides, ENPP1- containing polypeptides, or fusions described herein
• the methods in certain embodiments comprising culturing the cell, cells, or plurality of cells under conditions suitable for expression of the protein by the cell or cells from the nucleic acid, vector, or expression vector.
• the methods can also include purifying the protein from the cell, cells, or plurality of cells, or from the media in which the cell, cells, or plurality of cells were cultured.
• the disclosure provides proteins purified by any such methods.
• the disclosure further provides an autonomously replicating or an integrative mammalian cell vector comprising a recombinant nucleic acid encoding a polypeptide of the disclosure.
• the vector comprises a plasmid or a virus.
• the vector comprises a mammalian cell expression vector.
• the vector further comprises at least one nucleic acid sequence that directs and/or controls expression of the polypeptide .
• the recombinant nucleic acid encodes a polypeptide comprising an ENPP1 polypeptide of the disclosure and to a signal peptide, wherein the polypeptide is proteolytically processed upon secretion from a cell to yield the ENPP1 polypeptide of the disclosure.
• the disclosure provides an isolated host cell comprising a vector of the disclosure.
• the cell is a non-human cell.
• the cell is mammalian.
• the vector of the disclosure comprises a recombinant nucleic acid encoding a polypeptide comprising a ENPP1 polypeptide of the disclosure and a signal peptide.
• the polypeptide is proteolytically processed upon secretion from a cell to yield the ENPP1 polypeptide of the disclosure.
• ENPP1 is prepared as described in US 2015/0359858 Al, which is incorporated herein in its entirety by reference.
• ENPP1 is a transmembrane protein localized to the cell surface with distinct intramembrane domains.
• the transmembrane domain of ENPP1 may be swapped for the transmembrane domain of ENPP2, which results in the accumulation of soluble, recombinant ENPP1 in the extracellular fluid of the baculovirus cultures.
• Signal sequences of any other known proteins may be used to target the extracellular domain of ENPP1 for secretion as well, such as but not limited to the signal sequence of the immunoglobulin kappa and lambda light chain proteins.
• the disclosure should not be construed to be limited to the polypeptides described herein, but also includes polypeptides comprising any enzymatically active truncation of the ENPP1 extracellular domain.
• ENPP1 is made soluble by omitting the transmembrane domain.
• Human ENPP1 (SEQ ID NO: l) was modified to express a soluble, recombinant protein by replacing its transmembrane region (e.g., residues 77-98) with the corresponding subdomain of human ENPP2 (NCBI accession NP_00112433 5, e.g., residues 12-30).
• the modified ENPP1 sequence was cloned into a modified pFastbac FIT vector possessing a TEV protease cleavage site followed by a C-terminus 9-F1IS tag, and cloned and expressed in insect cells, and both proteins were expressed in a baculovirus system as described previously (Albright, et al, 2012, Blood 120:4432-4440; Saunders, et al, 2011, J. Biol. Chem. 18:994-1004; Saunders, et al, 2008, Mol. Cancer Ther. 7:3352-3362), resulting in the accumulation of soluble, recombinant protein in the extracellular fluid.
• a soluble ENPP1 polypeptide including IgG Fc domain or enzymatically /biologically active fragments thereof, are efficacious in treating, reducing, and/or preventing progression of diseases or disorders contemplated herein.
• the soluble ENPP1 polypeptide does not include a bone targeting domain, such as 2-20 consecutive polyaspartic acid residues or 2-20 consecutive poly glutamic acid residues.
• ENPP1 was fused to the Fc domain of IgG (referred to as“NPPl-Fc”) and the fusion protein was expressed in stable CHO cell lines.
• the protein can also be expressed from HEK293 cells, Baculovirus insect cell system or CHO cells or Yeast Pichia expression system using suitable vectors.
• the protein can be produced in either adherent or suspension cells.
• the fusion protein is expressed in CHO cells.
• the nucleic acid sequence encoding ENPP1 constructs are cloned into an appropriate vector for large scale protein production.
• ENPP1 fusion protein including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Kluyveronmyces lactis and Pichia pastoris), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
• bacteria for example E. coli and Bacillus subtilis
• yeasts for example Saccharomyces cerevisiae, Kluyveronmyces lactis and Pichia pastoris
• filamentous fungi for example Aspergillus
• plant cells animal cells and insect cells.
• insect cells for example E. coli and Bacillus subtilis
• the desired protein can be produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid.
• the yeasts can be transformed with a coding sequence for the desired protein in any of the usual ways, for example electroporation. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente, 1990, Methods Enzymol. 194: 182. Successfully transformed cells, i.e., cells that contain a DNA construct of the present disclosure, can be identified by well-known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide.
• Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method, such as that described by Southern, 1975, J. Mol. Biol, 98:503 and/or Berent, et al, 1985, Biotech 3:208.
• a method such as that described by Southern, 1975, J. Mol. Biol, 98:503 and/or Berent, et al, 1985, Biotech 3:208.
• the presence of the protein in the supernatant can be detected using antibodies.
• Useful yeast plasmid vectors include pRS403— 406 and pRS413— 416 and are generally available fronl Strat: 1.gene Cloning Systems, La Jolla, CA, USA
• Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers I-11S3, TRP1, LEU2 and 1JRA3.
• Plasmids pRS413— 416 are Yeast Centromere plasmids (YCps).
• complementary homopolymer tract can be added to the DNA segment to be inserted to the vector DNA.
• the vector and DNA segment are then joined by hydrogen bonding between the complementary homopoly meric tails to form recombinant DNA molecules.
• Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
• the DNA segment generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, which are enzymes that remove protruding, 3'-single-stranded termini with their 3'-5' -exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities.
• the combination of these activities thus generates blunt-ended DNA segments.
• the blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
• an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
• the products of the reaction are DNA segments carrying polymeric linker sequences at their ends.
• These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
• Clones of single, stably transfected cells are then established and screened for high expressing clones of the desired fusion protein.
• Screening of the single cell clones for ENPP1 protein expression can be accomplished in a high-throughput manner in 96 well plates using the synthetic enzymatic substrate pNP-TMP as previously described (Albright, et al, 2015, Nat. Commun. 6: 10006).
• protein production can be accomplished in shaking flasks or bio-reactors are previously described in Albright, et al, 2015, Nat. Commun. 6: 10006.
• ENPP1 Purification of ENPP1 can be accomplished using a combination of standard purification techniques known in the art. Examples of which are described above in production of ENPP1 protein. Following purification, ENPPl-Fc was dialyzed into PBS supplemented with Zn 2+ and Mg 2+ (PBSplus) concentrated to between 5 and 7 mg/ml, and frozen at -80 °C in aliquots of 200-500 pi. Aliquots were thawed immediately prior to use and the specific activity of the solution was adjusted to 31.25 au/ml (or about 0.7 mg/ml depending on the preparation) by dilution in PBSplus.
• PBSplus Zn 2+ and Mg 2+
• nucleic acids encoding the polypeptide(s) useful within the disclosure may be used in gene therapy protocols for the treatment of the diseases or disorders contemplated herein.
• the improved construct encoding the polypeptide(s) can be inserted into the appropriate gene therapy vector and administered to a patient to treat or prevent the diseases or disorder of interest.
• Vectors such as viral vectors
• the vectors have been used in the prior art to introduce genes into a wide variety of different target cells.
• the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide (e.g, a receptor).
• the transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
• the (viral) vector transfects liver cells in vivo with genetic material encoding the polypeptide(s) of the disclosure.
• AAV a parvovirus belonging to the genus Dependovirus
• AAV has several features that make it particularly well suited for gene therapy applications. For example, AAV can infect a wide range of host cells, including non-dividing cells. Furthermore, AAV can infect cells from a variety of species. Importantly, AAV has not been associated with any human or animal disease, and does not appear to alter the physiological properties of the host cell upon integration. Finally, AAV is stable at a wide range of physical and chemical conditions, which lends itself to production, storage, and transportation requirements.
• the AAV genome which is a linear, single-stranded DNA molecule containing approximately 4,700 nucleotides (the AAV-2 genome consists of 4,681 nucleotides, the AAV-4 genome 4,767), generally comprises an internal non-repeating segment flanked on each end by inverted terminal repeats (ITRs).
• ITRs are approximately 145 nucleotides in length (AAV-1 has ITRs of 143 nucleotides) and have multiple functions, including serving as origins of replication, and as packaging signals for the viral genome.
• the internal non-repeated portion of the genome includes two large open reading frames (ORFs), known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode replication and capsid gene products, which allow for the replication, assembly, and packaging of a complete AAV virion. More specifically, a family of at least four viral proteins are expressed from the AAV rep region: Rep 78, Rep 68, Rep 52, and Rep 40, all of which are named for their apparent molecular weights. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.
• AAV is a helper-dependent virus, that is, it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form functionally complete AAV virions.
• a helper virus e.g., adenovirus, herpesvirus, or vaccinia virus
• AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but infectious virions are not produced.
• the helper virus must be of the same species as the host cell.
• human AAV replicates in canine cells that have been co-infected with a canine adenovirus.
• a suitable host cell line can be transfected with an AAV vector containing the heterologous nucleic acid sequence, but lacking the AAV helper function genes, rep and cap.
• the AAV -helper function genes can then be provided on a separate vector.
• only the helper virus genes necessary for AAV production i.e., the accessory function genes
• the AAV helper function genes i.e., rep and cap
• accessory function genes can be provided on one or more vectors. Helper and accessory function gene products can then be expressed in the host cell where they will act in trans on rAAV vectors containing the heterologous nucleic acid sequence.
• the rAAV vector containing the heterologous nucleic acid sequence will then be replicated and packaged as though it were a wild-type (wt) AAV genome, forming a recombinant virion.
• wtAAV wild-type AAV genome
• AAV-1 AAV-1 through AAV-11
• AAV-2 is the most prevalent serotype in human populations; one study estimated that at least 80% of the general population has been infected with wt AAV-2 (Bems and Linden, 1995, Bioessays 17:237-245).
• AAV-3 and AAV-5 are also prevalent in human populations, with infection rates of up to 60% (Georg-Fries, et al, 1984, Virology 134:64-71).
• AAV-1 and AAV -4 are simian isolates, although both serotypes can transduce human cells (Chiorini, et al, 1997, J Virol 71:6823-6833; Chou, et al, 2000, Mol Ther 2:619-623).
• AAV-2 is the best characterized. For instance, AAV-2 has been used in a broad array of in vivo transduction experiments, and has been shown to transduce many different tissue types including: mouse (U.S. Patent Nos.
• AAV-2 vectors have been used to deliver the following genes: the cystic fibrosis transmembrane conductance regulator gene to rabbit lungs (Flotte, et al, 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); Factor NIII gene (Burton, et al, 1999, Proc. Natl. Acad. Sci. USA 96: 12725-12730) and Factor IX gene (Nakai, et al, 1999, J. Virol. 73:5438-5447; Snyder, et al, 1997, Nat. Genet. 16:270-276; U.S. Patent No.
• rAAV-delivered NEGF to mouse myocardium resulted in neovascular formation (Su, et al, 2000, Proc. Natl. Acad. Sci. USA 97: 13801-13806), and expression of rAAV-delivered AADC to the brains of parkinsonian monkeys resulted in the restoration of dopaminergic function.
• AAV vector comprising DNA encoding the protein of interest
• the disclosure should be construed to include AAV vectors comprising DNA encoding the polypeptide(s) of interest. Once armed with the present disclosure, the generation of AAV vectors comprising DNA encoding this/these polypeptide(s)s will be apparent to the skilled artisan.
• the rAAV vector of the disclosure comprises several essential DNA elements.
• these DNA elements include at least two copies of an AAV ITR sequence, a promoter/enhancer element, a transcription termination signal, any necessary 5’ or 3’ untranslated regions which flank DNA encoding the protein of interest or a biologically active fragment thereof.
• the rAAV vector of the disclosure may also include a portion of an intron of the protein on interest.
• the rAAV vector of the disclosure comprises DNA encoding a mutated polypeptide of interest.
• the vector comprises a promoter/regulatory sequence that comprises a promiscuous promoter which is capable of driving expression of a heterologous gene to high levels in many different cell types.
• promoters include, but are not limited to the cytomegalovirus (CMV) immediate early promoter/enhancer sequences, the Rous sarcoma virus promoter/enhancer sequences and the like.
• CMV cytomegalovirus
• the promoter/regulatory sequence in the rAAV vector of the disclosure is the CMV immediate early promoter/enhancer.
• the promoter sequence used to drive expression of the heterologous gene may also be an inducible promoter, for example, but not limited to, a steroid inducible promoter, or may be a tissue specific promoter, such as, but not limited to, the skeletal a-actin promoter which is muscle tissue specific and the muscle creatine kinase promoter/enhancer, and the like.
• the rAAV vector of the disclosure comprises a transcription termination signal. While any transcription termination signal may be included in the vector of the disclosure, in certain embodiments, the transcription termination signal is the SV40 transcription termination signal.
• the rAAV vector of the disclosure comprises isolated DNA encoding the polypeptide of interest, or a biologically active fragment of the polypeptide of interest.
• the disclosure should be construed to include any mammalian sequence of the polypeptide of interest, which is either known or unknown.
• the disclosure should be construed to include genes from mammals other than humans, which polypeptide functions in a substantially similar manner to the human polypeptide.
• the nucleotide sequence comprising the gene encoding the polypeptide of interest is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the gene encoding the polypeptide of interest.
• the disclosure should be construed to include naturally occurring variants or recombinantly derived mutants of wild type protein sequences, which variants or mutants render the polypeptide encoded thereby either as therapeutically effective as full-length polypeptide, or even more therapeutically effective than full-length polypeptide in the gene therapy methods of the disclosure.
• variants which retain the polypeptide’s biological activity.
• variants include proteins or polypeptides which have been or may be modified using recombinant DNA technology, such that the protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to, variants conferring enhanced stability on the protein in plasma and enhanced specific activity of the protein.
• Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function.
• the disclosure is not limited to the specific rAAV vector exemplified in the experimental examples; rather, the disclosure should be construed to include any suitable AAV vector, including, but not limited to, vectors based on AAV-1, AAV-3, AAV-4 and AAV-6, and the like.
• Also included in the disclosure is a method of treating a mammal having a disease or disorder in an amount effective to provide a therapeutic effect.
• the method comprises administering to the mammal an rAAV vector encoding the polypeptide of interest.
• the mammal is a human.
• the number of viral vector genomes/mammal which are administered in a single injection ranges from about 1 c 10 8 to about 5* 10 16 .
• the number of viral vector genomes/mammal which are administered in a single injection is from about 1 xlO 10 to about l x lO 15 ; more preferably, the number of viral vector genomes/mammal which are administered in a single injection is from about 5xl0 10 to about 5x l0 15 ; and, most preferably, the number of viral vector genomes which are administered to the mammal in a single injection is from about 5x l0 n to about 5xl0 14 .
• the method of the disclosure comprises multiple site simultaneous injections, or several multiple site injections comprising injections into different sites over a period of several hours (for example, from about less than one hour to about two or three hours)
• the total number of viral vector genomes administered may be identical, or a fraction thereof or a multiple thereof, to that recited in the single site injection method.
• a composition comprising the virus is injected directly into an organ of the subject (such as, but not limited to, the liver of the subject).
• the rAAV vector may be suspended in a pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8.
• a pharmaceutically acceptable carrier for example, HEPES buffered saline at a pH of about 7.8.
• Other useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
• the rAAV vector of the disclosure may also be provided in the form of a kit, the kit comprising, for example, a freeze-dried preparation of vector in a dried salts formulation, sterile water for suspension of the vector/salts composition and instructions for suspension of the vector and administration of the same to the mammal.
• SEQ ID NO:4 hENPP5 protein export signal sequence
• SEQ ID NO:5 hENPP7 protein export signal sequence
• SEQ ID NO:6 hENPP7 protein export signal sequence
• SEQ ID NO:8 Exemplary Amino Acid Linker Sequence
• SEQ ID NO: 9 Exemplary Amino Acid Linker Sequence
• SEQ ID NO: 10 Exemplary Extracellular Domain of human ENPP1
• SEQ ID NO:ll Exemplary ENPP1 Mutant Polypeptide (substitutions relative to wildtype human ENPP1 are shown in bold/underline)
• SEQ ID NO:12 Exemplary ENPP1 Mutant Polypeptide (substitutions relative to wildtype human ENPP1 are shown in bold/underline)
• SEQ ID NO: 13 Exemplary Human IgGl Fc Region
• SEQ ID NO: 14 Exemplary Variant Human IgGl Fc Region (containing the MST/YTE Substitutions (bold/underline))
• SEQ ID NO: 16 Exemplary ENPP1 -containing Fusion: ENPP1 extracellular domain (SEQ ID NO: 10; italics) fused at its C-terminus to the amino acid sequence LIN (SEQ ID NO: 8; double underlined), which is fused at its C-terminus to a variant human IgG Fc Region (SEQ ID NO: 14; unmodified text)
• SEQ ID NO: 17 Exemplary ENPP1 Mutant Polypeptide Fusion: ENPP1 Mutant
• Polypeptide (SEQ ID NO: 11; italics) fused at its C-terminus to LIN (SEQ ID NO: 8; double underlined), which is fused at its C-terminus to a variant human IgG Fc Region (SEQ ID NO: 14; unmodified text)
• SEQ ID NO: 18 Exemplary ENPP1 Mutant Polypeptide Fusion: ENPP1 Mutant
• the disclosure includes a method of reducing or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of reducing or preventing progression of ectopic calcification of soft tissue, including reducing, ameliorating, or preventing vascular calcification, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of reducing or preventing progression of diseases caused by ENPP1 deficiency.
• ENPP1 deficiency is characterized by reduced levels of ENPP1 activity and or defective expression of ENPP1 levels (compared to that of ENPP1 activity levels or ENPP1 expression levels respectively in normal healthy subjects) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of reducing or preventing progression of diseases caused by lower levels of plasma PPi in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptides of the disclosure to increase the plasma PPi of the subjects to normal (1-3 mM) or above (30-50% higher than) normal levels and then to maintain the plasma PPi at a constant normal or above normal level thereafter.
• the method further comprises administering additional therapeutic effective amounts at intervals of two days, three days, one week or one month in order to maintain the Plasma PPi of the subject at a constant normal or above normal level to reduce or prevent the progression of pathological calcification or ossification.
• the disclosure further includes a method of treating, reversing, or preventing progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• OPLL posterior longitudinal ligament
• the disclosure further includes a method of treating, reverting, or preventing progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of reducing or preventing progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, aging related hardening of arteries, idiopathic infantile arterial calcification (IIAC), Generalized Arterial Calcification of Infancy (GACI), and calcification of atherosclerotic plaques in a subject diagnosed with the at least one disease, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• CKD chronic kidney disease
• ESRD end stage renal disease
• CUA calcific uremic arteriolopathy
• OPLL ossification of the posterior longitudinal ligament
• OPLL hypophosphatemic rickets
• osteoarthritis aging related hard
• the disclosure further includes a method of reducing or preventing progression of aging related hardening of arteries in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of reducing or preventing progression of a disease caused by ENPP1 deficiency (for example, reduced levels of ENPP1 activity and/or defective expression of ENPP1 levels, as compared to that of ENPP1 activity levels or ENPP1 expression levels, respectively, in normal healthy subjects) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• ENPP1 deficiency for example, reduced levels of ENPP1 activity and/or defective expression of ENPP1 levels, as compared to that of ENPP1 activity levels or ENPP1 expression levels, respectively, in normal healthy subjects
• the disclosure further includes a method of reducing or preventing progression of a disease caused by lower than normal levels of plasma PPi in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure to increase and/or sustain the plasma PPi of the subjects to a level that is about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of the normal PPi level (about 1-3 mM).
• the method further comprises further administration of the polypeptide of the disclosure every two days, three days, one week, or one month in order to maintain the plasma PPi levels at a level that is about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of the normal PPi level, thus preventing the progression of pathological calcification or ossification.
• the disclosure further includes a method of treating, reversing, or preventing progression of Pseudoxanthoma Elasticum (PXE) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• PXE Pseudoxanthoma Elasticum
• the disclosure further includes a method of treating, reversing, or preventing progression of calcification of atherosclerotic plaques in vascular arteries in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of treating, reversing, or preventing progression of osteoarthritis in a subject in need thereof, the method comprising
• the disclosure further includes a method of treating, reversing, or preventing progression of hardening of arteries due to progeria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of treating, reversing, or preventing progression of X-linked hypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets (HHRH), hypophosphatemic bone disease (HBD), autosomal dominant hypophosphatemic rickets (ADHR), and/or and autosomal recessive hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• XLH X-linked hypophosphatemic rickets
• HHRH hereditary hypophosphatemic rickets
• HHD hypophosphatemic bone disease
• ADHR autosomal dominant hypophosphatemic rickets
• the disclosure further includes a method of treating, reversing, or preventing progression of age-related osteopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide t of the disclosure.
• the disclosure further includes a method of treating, reversing, or preventing progression of ankylosing spondylitis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of treating, reversing, or preventing progression of strokes in pediatric sickle cell anemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the disclosure.
• the disclosure further includes a method of reducing or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, to thereby reduce or prevent progression of pathological calcification in the subject.
• the disclosure further includes a method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, to thereby reduce or prevent progression of pathological ossification in the subject.
• the disclosure further includes a method of reducing or preventing progression of ectopic calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, to thereby reduce or prevent progression of ectopic calcification of soft tissue in the subject.
• the disclosure further includes a method of treating, reversing, or preventing progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, to thereby reduce, reverse, or prevent ossification of the posterior longitudinal ligament (OPLL) in the subject.
• OPLL posterior longitudinal ligament
• the disclosure further includes a method of treating, reverting, or preventing progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, to thereby reduce, reverse, or prevent progression of hypophosphatemic rickets in the subject.
• the disclosure further includes a method of reducing or preventing progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, aging related hardening of arteries, idiopathic infantile arterial calcification (IIAC),
• CKD chronic kidney disease
• ESRD end stage renal disease
• CUA calcific uremic arteriolopathy
• OPLL ossification of the posterior longitudinal ligament
• OPLL hypophosphatemic rickets
• osteoarthritis aging related hardening of arteries
• IIAC idiopathic infantile arterial calcification
• GCI Generalized Arterial Calcification of Infancy
• calcification of atherosclerotic plaques in a subject diagnosed with the at least one disease comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, to thereby reduce or prevent progression of the disease.
• the disclosure further includes a method of reducing or preventing progression of aging related hardening of arteries in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, to thereby reduce or prevent progression of aging related hardening of arteries in the subject.
• the disclosure further includes a method of raising pyrophosphate (PPi) levels in a subject having PPi level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, whereby upon the administration the level of the PPi in the subject is elevated to a normal level of at least 2mM and is maintained at approximately the same level.
• PPi pyrophosphate
• the disclosure further includes a method of reducing or preventing the progression of pathological calcification or ossification in a subject having pyrophosphate (PPi) level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, whereby pathological calcification or ossification in the subject is reduced or progression of pathological calcification or ossification in the subject is prevented.
• PPi pyrophosphate
• the disclosure further includes a method of treating ENPP1 deficiency manifested by a reduction of extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the ENPP1 mutant polypeptides, fusions, ENPP-1 containing polypeptides, or conjugates described herein, whereby the level of the PPi in the subject is elevated.
• PPi extracellular pyrophosphate
• the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and calcification of atherosclerotic plaques.
• the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.
• OPLL posterior longitudinal ligament
• hypophosphatemic rickets hypophosphatemic rickets
• osteoarthritis osteoarthritis
• the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis.
• the soft tissue is selected from the group consisting of atherosclerotic plaques, muscular arteries, joint, spine, articular cartilage, vertebral disk cartilage, vessels, and connective tissue.
• the soft tissue comprises atherosclerotic plaques.
• the soft tissue comprises muscular arteries.
• the soft tissue is selected from the group consisting of joint and spine.
• the joint is selected from the group consisting of joints of the hands and joints of the feet.
• the soft tissue is selected from the group consisting of articular cartilage and vertebral disk cartilage.
• the soft tissue comprises vessels.
• the soft tissue comprises connective tissue.
• the subject is diagnosed with progeria.
• the ENPP1 mutant polypeptide, fusion, or ENPP1 -containing polypeptide is a secreted product of a ENPP1 precursor protein expressed in a mammalian cell, wherein the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein undergoes proteolytic processing to yield the ENPP1 polypeptide.
• the polypeptide of the disclosure is a secreted product of a ENPP1 precursor protein expressed in a mammalian cell.
• the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein undergoes proteolytic processing to the polypeptide of the disclosure.
• the signal peptide sequence in the ENPP1 precursor protein the signal peptide sequence is conjugated to the ENPP1 polypeptide N-terminus. Upon proteolysis, the signal sequence is cleaved from the ENPP1 precursor protein to provide the ENPP1 polypeptide.
• the signal peptide sequence is selected from the group consisting of ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and ENPP5 signal peptide sequence.
• the polypeptide is administered acutely or chronically to the subject. In other embodiments, the polypeptide is administered locally, regionally, parenterally or systemically to the subject.
• the subject is a mammal. In other embodiments, the mammal is human.
• the ENPP1 mutant polypeptide, the ENPP1 -containing polypeptide, or fusion, or its precursor protein is administered by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal,
• the ENPP1 mutant polypeptide, the ENPP1 -containing polypeptide, or fusion, or its precursor protein is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.
• the ENPP1 mutant polypeptide, the ENPP1 -containing polypeptide, or fusion, or its precursor protein is administered acutely or chronically to the subject.
• the ENPP1 mutant polypeptide, the ENPP1 -containing polypeptide, or fusion, or its precursor protein is administered locally, regionally or systemically to the subject.
• the polypeptide, or its precursor protein is delivered on an encoded vector, wherein the vector encodes the protein and it is transcribed and translated from the vector upon administration of the vector to the subject.
• the disclosure is not limited to treatment of a disease or disorder once it is established.
• the symptoms of the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant pathology from disease or disorder does not have to occur before the present disclosure may provide benefit.
• the present disclosure includes a method for preventing diseases and disorders in a subject, in that a polypeptide of the disclosure, as discussed elsewhere herein, can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the disease or disorder from developing.
• a polypeptide of the disclosure as discussed elsewhere herein, can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the disease or disorder from developing.
• the symptoms of the disease or disorder have not manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant pathology from the disease or disorder does not have to occur before the present disclosure may provide benefit.
• the present disclosure includes methods for preventing or delaying onset, or reducing progression or growth, of a disease or disorder in a subject, in that a polypeptide of the disclosure can be administered to a subject prior to detection of the disease or disorder.
• the polypeptide of the disclosure is administered to a subject with a strong family history of the disease or disorder, thereby preventing or delaying onset or progression of the disease or disorder.
• the prevention of a disease or disorder in a subject encompasses administering to a subject a polypeptide of the disclosure as a preventative measure against the disease or disorder.
• compositions comprising a polypeptide of the disclosure within the methods described herein.
• Such a pharmaceutical composition is in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more
• compositions may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
• the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.
• compositions of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
• the composition may comprise between about 0.1% and about 100% (w/w) active ingredient.
• compositions that are useful in the methods of the disclosure may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration.
• Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.
• the route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
• compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
• preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
• a“unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
• the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.
• the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
• the regimen of administration may affect what constitutes an effective amount. For example, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
• administration of the compound of the disclosure to a subject elevates the subject’s plasma PPi to a level that is close to normal, where a normal level of PPi in mammals is 1-3 mM.
• “Close to normal” refers to 0 to 1.2 pM or 0-40% below or above normal, 30 nM to 0.9 pM or 1-30% below or above normal, 0 to 0.6 pM or 0-20% below or above normal, or 0 to 0.3 mM or 0-10% below or above normal.
• compositions of the present disclosure may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient.
• An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response.
• Dosage is determined based on the biological activity of the therapeutic compound which in turn depends on the half-life and the area under the plasma time of the therapeutic compound curve.
• the polypeptide according to the disclosure is administered at an appropriate time interval of every 2 days, or every 4 days, or every week or every month so as to achieve a continuous level of plasma PPi that is either close to the normal (1-3 mM) level or above (30-50% higher than) normal levels of PPi.
• Therapeutic dosage of the polypeptides of the disclosure may also be determined based on half-life or the rate at which the therapeutic polypeptide is cleared out of the body.
• the polypeptide according to the disclosure is administered at appropriate time intervals of either every 2 days, or every 4 days, every week or every month so as to achieve a constant level of enzymatic activity of ENPP 1.
• an effective dose range for a therapeutic compound of the disclosure is from about 0.01 and 50 mg/kg of body weight/per day.
• the effective dose range for a therapeutic compound of the disclosure is from about 50 ng to 500 ng/kg, preferably 100 ng to 300 ng/kg of body weight.
• One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
• the compound can be administered to a patient as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
• the frequency of the dose is readily apparent to the skilled artisan and depends upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the patient.
• Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• a medical doctor e.g., physician, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
• the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
• compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
• the frequency of administration of the various combination compositions of the disclosure varies from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physical taking all other factors about the patient into account.
• the present disclosure is directed to a packaged
• composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second
• compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g, sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,
• compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like.
• the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
• Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
• parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.
• Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos.
• Controlled- or sustained-release formulations of a pharmaceutical composition of the disclosure may be made using conventional technology.
• the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions.
• Single unit dosage forms suitable for oral administration such as tablets, capsules, gelcaps, and caplets, which are adapted for controlled-release are encompassed by the present disclosure.
• the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
• sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
• the period of time may be as long as a month or more and should be a release that is longer that the same amount of agent administered in bolus form.
• the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds.
• the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
• the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
• delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
• pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
• immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
• short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
• rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
• Human NPP1 (Human: NCBI accession NP 006199) was modified to express soluble, recombinant protein was fused to IgGl by sub cloning into pFUSE-hlgGl -Fcl or pFUSE- mlgGl -Fcl plasmids (InvivoGen, San Diego CA), respectively.
• the constructs were generated from SEQ ID NO:7) using site-directed mutagenesis using commercially available kits. (Q5® Site-Directed Mutagenesis Kit/New England Biolabs). The constructs thus generated were sequenced to verify the nucleic acid sequence and then used for expression of protein.
• FORTICHOTM medium (A1148301, Thermo Fischer) or PEPROGROWTM AF-CHO
• CHO-K1 cells were modified to generate CHO-K1-MOD cells stably expressing human a-2,6-sialytransferase (a-2,6-ST) enzyme. Stable transfections of the ENPPl-Fc constructs were established in CHO Kl-MOD cells, and protein was expressed following the same protocol as described above.
• the cell culture medium of CHO-K1-MOD cells expressing the corresponding constructs were supplemented with sialic acid or a“high-flux” precursor of sialic acid called l,3,4-0-Bu3ManNAc to facilitate higher levels of glycosylate during protein production
• the liquid cultures were centrifuged at 4300 x g for 5 mm and the supernatants were filtered through a 0.2 pm membrane and concentrated via tangential flow using a Pellicon®3 0. 0.11 m 2 Ultracell® 30 D cassette (Millipore, Billerica MA).
• the concentrated supernatant was then purified by a combination of chromatographic techniques in a multi-step process. These techniques are performed sequentially and may include any of the following: affinity chromatography with protein A or protein G, cation-exchange chromatography, anion- exchange chromatography, size exclusion chromatography, hydrophobic exchange chromatography, high-pressure liquid chromatography (HPLC), precipitation steps, extractions steps, lyophilization steps, and/or crystallization steps.
• the steady state hydrolysis of ATP by ENPP1 constructs was determined by HPLC. Briefly, enzyme reactions were started by addition of 10 nM PPi to varying concentrations of ATP in the reaction buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 4.5 nM KC1, 14 mM ZnCfy ImM MgCE. and ImM CaCT. At various time points, 50 pi reaction solution were removed and quenched with an equal volume of 3M formic acid.
• the quenched reaction solution was loaded on a C-18 (5 m t 250 X 4.6 mm) column (Higgins Analytical) equilibrated in 5 mM ammonium acetate (pH 6.0) solution and eluted with a 0% to 20% methanol gradient. Substrate and products were monitored by UV absorbance at 259 nm and quantified according to the integration of their correspondent peaks and standard curves.
• phosphodiesterase activity was analyzed using thymidine 5’-monophosphate p-nitrophenyl ester (/;NP-TMP) (Saunders, et cil, 2008, Mol. Cancer Ther. 7(10):3352-62; Albright, et al., 2015, Nat Commun. 6: 10006).
• the area under the plasma concentration versus time curve also called the area under the curve (AUC) can be used as a means of evaluating the volume of distribution (V), total elimination clearance (CL), and bioavailability (F) for extravascular drug delivery.
• AUC area under the curve
• V volume of distribution
• CL total elimination clearance
• F bioavailability
• the drug half-life (h / 2) is the time it takes for the plasma concentration or the amount of drug or biologic in the body to be reduced by 50%.
• Half-life values for each expressed and purified ENPPl-Fc construct were carried out following protocols described in the prior art and/or herein, such as Equation 1, which allows for determining half-life and
• a ENPPl-Fc construct was subjected to mutations so as to introduce putative additional glycosylation sites and/or increase affinity of the Fc for the neonatal orphan receptor (FcRn). Mutations tested are illustrated elsewhere herein, and specific Constructs of the discussion are illustrated below.
• ENPP1 amenable to hyperglycosylation that would not adversely impact catalytic activity
• a combination of structural modeling, clinical data, and genetic data on ENPP1 in GACI patients was used.
• N-linked glycosylation consensus sequences were identified in ENPP2-7, and sequences that would easily permit the introduction of a glycosylation site via the alteration of a single adjacent residues were evaluated.
• ENPP2-7 was then structurally modeled using standard software to thread the sequences through the mouse Enppl structure (PDB ID code 4GTW). The location of proposed glycosylation sites were compared to sites of known inactivating ENPP1 mutations in GACI (FIGs. 7A-7B) as well as the locations of di-sulfide bonds in the enzyme.
• the additional N-linked glycosylation consensus sequences were then introduced into human ENPPl-Fc (hENPPl-Fc, Construct #770) via site directed mutagenesis.
• the proteins were transiently expressed in CHO cells in 96 well plates and the enzymatic activity of the extracellular supernatant from each clone was screened in triplicate in a high throughput assay using fyNP-TMP as a chromogenic substrate as described in the methods (FIGs. 7A- 7D).
• the rate of pNP-TMP hydrolysis in 10 ENPPl-Fc isoforms was equal to or better than Construct #770 (FIGs. 7A-7D), and these 10 gly coforms were selected for combinatorial optimization with one-another and the IgGl Fc domain as described elsewhere herein.
• FcRn is the main homeostatic regulator of human IgGl Fc serum half-life, and mutations in the Fc domain which enhance the pH dependent interactions of Fc with FcRn extend the circulatory half-life of biologic antibodies. Effects of two Fc mutations reported to enhance pH dependent recycling were examined herein - H433K/N434F, hereafter referred to as HN mutations, and M242Y/S254T/T246E, hereafter referred to as MST mutations (FIGs. 9A-9B). Either of the two variants of the Fc domain were combined randomly with one or more of the 10 ENPPl-Fc gly coforms demonstrating acceptable hydrolysis rates to create 12 additional ENPPl-Fc clones (Table 3).
• Non-human Chinese Hamster Ovary (CHO) cells are widely employed for the production of biologies due to the similarity of the CHO and human glycosylation patterns in recombinantly produced protein. Nevertheless, glycosylation differences between the two exist, most notably, human N-linked glycans contain terminal sialic acid residues with both a-2,3 and a-2,6 linkages while CHO cells contain only a-2,3 linkages.
• a CHO cell line stably expressing human a- 2,6-sialytransferase (a-2,6-ST) was established as a host, and this clone was used for the production of 7 ENPP1 isoforms to compared the effect of a-2,6 linkages on PK and bioavailability in the various Constructs (Table 5; Construct numbers ending in‘-ST’).
• ENPPl-Fc isoforms were purified to homogeneity using an identical purification scheme and the Michaelis-Menton enzymatic rate constants and pharmacokinetic properties were determined as described elsewhere herein. Finally, to quantitate the biological impact of clone optimization, the pharmacodynamic effects of select ENPPl-Fc isoforms were quantitated by determining plasma PPi concentrations at multiple time points following a single subcutaneous dose of each isoform.
• ENPP2 lacks this loop and can accommodate larger lipid substrates in the catalytic pocket; both ENPP1 and ENPP3 have the loop but only ENPP3 has the N-glycan consensus sequences (N- GCS).
• the size of the ENPPl-Fc isoforms in Table 2 were compared by SDS-PAGE gels to determine which sequence variation resulted in increased glycosylation, and showed increases in the molecular weight consistent with the addition of glycosylations. To determine whether the sequence changes in Construct #1020 successfully introduced glycosylations MALDI-TOF was used, which also confirmed the presence of glycosylations at these sites.
• steric hinderances associated with the position of the N-GCS may occur such that the Asn residue is unable to accept the gly can due to specific flanking amino acids. Therefore, until a purified protein is analyzed for gly can content, one cannot be sure whether any Pk effect of a particular N-GCS is due to the shielding phenomenon of a new gly can or that the amino acid change has altered the kinetics of the enzyme or both. For this reason, any effect on PK to be associated with the N-GCS should be verified by gly can analysis.
• Antibodies containing mutations in the Fc domain that enhance their affinity for the FcRn and increase the pH dependent antibody recycling have never been used in therapeutic enzymes fused to the Fc domain.
• Some Fc mutations successfully increased affinity of the Fc domain for the FcRn receptor but resulted in unfavorable PK properties in antibody PK in vivo, while others were shown to enhance PK properties in vivo.
• FcRn is the main homeostatic regulator of human IgGl Fc serum half-life.
• analogous Fc changes enhance the PK properties of enzyme fusion proteins.
• two specific IgGl mutations in Fc previously used in biologic antibodies were investigated - H433K/N434F and M242Y/S254T/T246E. In general, the
• M242Y/S254T/T246E mutations were found to be superior to H433K/N434F at improving the properties of ENPPl-Fc.
• Construct #981 possessing only the
• M242Y/S254T/T246E mutation when compared to Construct #770, increased the half-life by 3.3 fold and the AUC by 5.8 fold.
• constructs with the H433K/N434F mutation in the context of various ENPP1 mutations achieved more modest half-life increases of between 1.2-1.7 fold.
• the Fc MST mutation increased biologic exposure to a greater degree than Fc HN mutations (Table 3, FIGs. 14A-14E).
• adding the MST mutation to the parent isoform increased AUC by 6-fold and half-life by about 2.5 fold, in comparison to the HN mutation which increased AUC by 4.5 fold in the presence of additional glycans (compare Clonel4 with Clones 9-12 in Table 3 and FIGs. 14A-14E).
• specific N-GCS mutations actually decrease bioavailability in the context of specific Fc mutations, i.e., the N-GCS mutation at residue 766 decreases the AUC of MST-Fc containing constructs (compare Clone 8 with Clone 14, Table 3, FIG.
• FIG. 14B To compare the PK effects of the MST Fc mutation with the effect of the additional glycan at position 256 (via the 1256T mutation) the plasma activity of the ENPPl-Fc isoforms was plotting with time (FIG. 14B).
• the figure demonstrates that the Fc mutation enhances PK by increasing the half-life of the biologic in the plasma, as demonstrated by the reduced slope of the activity vs time curve in Clone 14 vs. Clone 7.
• the addition of the I256T glycosylation enhances PK by increasing drug absorption into plasma, /. e.. increasing C maX , demonstrated by the greater maximal activity present in clone 7 (FIG. 14B).
• ENPPl-Fc is the only enzyme in mammals capable of generating plasma PPi, and plasma PPi is therefore a biomarker for predicting the efficacy of ENPP1 enzyme replacement therapy in ENPP1 deficiency.
• Enppl asi " v/ mice were dosed with a subcutaneous dose of 0.3 mg/Kg of either Construct #770 or Clone 19-ST and plasma PPi and enzyme presence were measured in plasma for 263 hours (FIG. 15D).
• Table 2 Effect of additional N-linked glycosylation on pharmacokinetics (PK).
• Table 3 Effect of Fc mutations on pharmacokinetics (PK).
• Table 4 Effect of cell lines and mutations on pharmacokinetics (PK). Those Constructs marked as“-ST” were prepared using a modified CHO cells line stably transfected with human a-2,6-sialytransferase (a-2,6-ST); this enhances the amount of sialyation of the fusion protein when compared with the fusion protein expressed in normal CHO cell lines.
• PK pharmacokinetics
• Table 5 Effect of sialic acid supplementation on pharmacokinetics (PK).
• PK pharmacokinetics
• Those Constructs marked as“-ST” were prepared using a modified CHO cells line stably transfected with human a-2,6-sialytransferase (a-2,6-ST); this enhances the amount of sialyation of the fusion protein when compared with the fusion protein expressed in normal CHO cell lines.
• Those Constructs marked as“-A” were prepared in cells grown in culture media supplemented with
• Embodiment 1 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to a Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises the mutation I256T as relating to SEQ ID NO:7.
• Embodiment 2 provides the polypeptide fusion of Embodiment 1, wherein the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 3 provides the polypeptide fusion of Embodiment 1, wherein the Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and H1064K/N1065F as relating to SEQ ID NO:7.
• Embodiment 4 provides the polypeptide fusion of any of Embodiments 1-3, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N as relating to SEQ ID NO:7.
• Embodiment 5 provides the polypeptide fusion of any of Embodiments 1-4, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N/K27T and V29N as relating to SEQ ID NO:7.
• Embodiment 6 provides the polypeptide fusion of any of Embodiments 1-5, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of K369N, and I371T as relating to SEQ ID NO:7.
• Embodiment 7 provides the polypeptide fusion of any of Embodiments 1-6, wherein the ENPP1 polypeptide comprises the mutation K369N/I371T as relating to SEQ ID NO:7.
• Embodiment 8 provides the polypeptide fusion of any of Embodiments 1-7, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N as relating to SEQ ID NO:7.
• Embodiment 9 provides the polypeptide fusion of any of Embodiments 1-8, wherein the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N as relating to SEQ ID NO:7.
• Embodiment 10 provides the polypeptide fusion of any of Embodiments 1-9, wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of E864N and L866T as relating to SEQ ID NO:7.
• Embodiment 11 provides the polypeptide fusion of any of Embodiments 1-10, wherein the ENPP1 polypeptide comprises at least the mutation E864N/L866T as relating to SEQ ID NO:7.
• Embodiment 12 provides the polypeptide fusion of any of Embodiments 1-11, comprising at least one mutation selected from the group consisting of C25N, K27T, V29N, C25N/K27T, K369N, I371T, K369N/I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N/V536T, P554L/R545T, E592N/R741D, E864N, L866T, E864N/L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y/S885T/T887E, H1064K/N1065F as relating to SEQ ID NO:7.
• Embodiment 13 provides the polypeptide fusion of any of Embodiments 1-12, wherein the Fc region is
• Embodiment 14 provides the polypeptide fusion of any of Embodiments 1-13, comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N, and E592N as relating to SEQ ID NO:7.
• Embodiment 15 provides the polypeptide fusion of any of Embodiments 1-13, comprising at least one mutation selected from the group consisting of S766N,
• P534N/Y536T, P554L/R545T, and E592N as relating to SEQ ID NO:7.
• Embodiment 16 provides the polypeptide fusion of any of Embodiments 1-13, comprising at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and
• P534N/V 536T/M883Y/S885T/T887E as relating to SEQ ID NO:7.
• Embodiment 17 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and a Fc region of an immunoglobulin, the polypeptide fusion comprising mutations I256T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• Embodiment 18 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and a Fc region of an immunoglobulin, the polypeptide fusion comprising mutations I256T, P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• Embodiment 19 provides an ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and a Fc region of an immunoglobulin, the polypeptide fusion comprising mutations I256T, E592N, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 20 provides an ENPP1 mutant polypeptide comprising amino acids 23- 849 of SEQ ID NO:7, wherein the mutant polypeptide comprises mutation I256T and further comprises a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N as relating to SEQ ID NO:7.
• Embodiment 21 provides the mutant polypeptide of Embodiment 20, which comprises the amino acid sequence of SEQ ID NO:7.
• Embodiment 22 provides the mutant polypeptide of any of Embodiments 20-21, which comprises the amino acid sequence of SEQ ID NO:7.
• Embodiment 23 provides the mutant polypeptide of any of Embodiments 20-23, wherein the mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N/V 536T,
• Embodiment 24 provides the mutant polypeptide of any of Embodiments 21-23, comprising mutations selected from the group consisting of: S885N, S766N,
• P534N/V 536T/M883Y/S885T/T887E as relating to SEQ ID NO:7.
• Embodiment 25 provides the mutant polypeptide of any of Embodiments 21-24, comprising a S885N mutation as relating to SEQ ID NO:7.
• Embodiment 26 provides the mutant polypeptide of any of Embodiments 20-25, comprising a S766N mutation as relating to SEQ ID NO:7.
• Embodiment 27 provides the mutant polypeptide of any of Embodiments 21-26, comprising mutations M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• Embodiment 28 provides the mutant polypeptide of any of Embodiments 21-27, comprising mutations P534N, V536T, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 29 provides the mutant polypeptide of any of Embodiments 20-28, comprising mutations P554L and R545T as relating to SEQ ID NO:7.
• Embodiment 30 provides the mutant polypeptide of any of Embodiments 21-29, comprising mutation S766N, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 31 provides the mutant polypeptide of any of Embodiments 21-30, comprising mutation E592N, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 32 provides the mutant polypeptide of any of Embodiments 21-31, comprising mutations P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO: 7.
• Embodiment 33 provides the polypeptide fusion of any of Embodiments 1-19 or the mutant polypeptide of any of Embodiments 20-32, which is expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2, 6-sialyltransferase (also known as ST6GAL1).
• ST6GAL1 human ST6 beta-galactoside alpha-2, 6-sialyltransferase
• Embodiment 34 provides the polypeptide fusion of any of Embodiments 1-19 or the mutant polypeptide of any of Embodiments 20-32, which is grown in a cell culture supplemented with sialic acid and/or N-acetylmannosamine (also known as
• Embodiment 35 provides a method of reducing or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of any of Embodiments 1-19 and 33-34 or the mutant polypeptide of any of Embodiments 20-34.
• Embodiment 36 provides a method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of any of
• Embodiment 37 provides a method of reducing or preventing progression of ectopic calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of any of
• Embodiment 38 provides a method of treating, reversing, or preventing progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of any of Embodiments 1-19 and 33-34 or the mutant polypeptide of any of Embodiments 20-34.
• OPLL posterior longitudinal ligament
• Embodiment 39 provides a method of treating, reverting, or preventing progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of any of
• Embodiment 40 provides a method of reducing or preventing progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, aging related hardening of arteries, idiopathic infantile arterial calcification (IIAC), Generalized Arterial Calcification of Infancy (GACI), and calcification of atherosclerotic plaques in a subject diagnosed with the at least one disease, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of any of Embodiments 1-19 and 33-34 or the mutant polypeptide of any of Embodiments 20-34.
• CKD chronic kidney disease
• ESRD end stage renal disease
• CUA calcific uremic arteriolopathy
• OPLL ossification
• Embodiment 41 provides a method of reducing or preventing progression of aging related hardening of arteries in a subject in need thereof, the method comprising
• Embodiment 42 provides the method of Embodiment 35, wherein the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and calcification of atherosclerotic plaques.
• IIAC idiopathic infantile arterial calcification
• Embodiment 43 provides the method of Embodiment 36, wherein the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.
• OPLL posterior longitudinal ligament
• hypophosphatemic rickets hypophosphatemic rickets
• osteoarthritis ossification of the posterior longitudinal ligament
• Embodiment 44 provides the method of Embodiment 37, wherein the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis.
• Embodiment 45 provides the method of Embodiment 37, wherein the soft tissue is selected from the group consisting of atherosclerotic plaques, muscular arteries, joint, spine, articular cartilage, vertebral disk cartilage, vessels, and connective tissue.
• Embodiment 46 provides a method of raising pyrophosphate (PPi) levels in a subject having PPi level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the polypeptide fusion of any of Embodiments 1-19 and 33-34 or the mutant polypeptide of any of Embodiments 20-34, whereby upon the administration the level of the PPi in the subject is elevated to a normal level of at least 2mM and is maintained at approximately the same level.
• PPi pyrophosphate
• Embodiment 47 provides a method of reducing or preventing the progression of pathological calcification or ossification in a subject having pyrophosphate (PPi) level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of Embodiments 1-19 and 33-34 or the mutant polypeptide of any of Embodiments 20-34, whereby pathological calcification or ossification in the subject is reduced or progression of pathological calcification or ossification in the subject is prevented.
• PPi pyrophosphate
• Embodiment 48 provides a method of treating ENPP1 deficiency manifested by a reduction of extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of any of Embodiments 1-19 and 33-34 or the mutant polypeptide of any of Embodiments 20-34, whereby the level of the PPi in the subject is elevated.
• PPi extracellular pyrophosphate
• Embodiment 49 provides the method of any of Embodiments 35-48, wherein the polypeptide fusion or mutant polypeptide is a secreted product of a ENPP1 precursor protein expressed in a mammalian cell, wherein the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein undergoes proteolytic processing to yield the ENPP1 polypeptide.
• Embodiment 50 provides the method of Embodiment 49, wherein in the ENPP1 precursor protein the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide.
• Embodiment 51 provides the method of any of Embodiments 49-50, wherein the signal peptide sequence is selected from the group consisting of ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and ENPP5 signal peptide sequence.
• Embodiment 52 provides the method of any of Embodiments 35-51, wherein the polypeptide fusion or mutant polypeptide is administered acutely or chronically to the subject.
• Embodiment 53 provides the method of any of Embodiments 35-52, wherein the polypeptide fusion or mutant polypeptide is administered locally, regionally, parenterally, or systemically to the subject.
• Embodiment 54 provides the method of any of Embodiments 35-53, wherein the polypeptide fusion or mutant polypeptide is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal,
• Embodiment 55 provides the method of any of Embodiments 35-54, wherein the polypeptide fusion or mutant polypeptide is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.
• Embodiment 56 provides the method of any of Embodiments 35-55, wherein the subject is a mammal.
• Embodiment 57 provides the method of Embodiment 56, wherein the mammal is human.
• Embodiment 58 provides an ENPP1 mutant polypeptide comprising one or more amino acid substitutions as relating to SEQ ID NO:7, wherein the polypeptide comprises an amino acid substitution at position 256 relative to SEQ ID NO:7.
• Embodiment 59 provides the ENPP1 mutant polypeptide of Embodiment 58, wherein the ENPP1 mutant polypeptide amino acid sequence is at least 90% identical to amino acids 23-849 of SEQ ID NO: 7.
• Embodiment 60 provides an ENPP1 mutant polypeptide comprising amino acids 23- 849 of SEQ ID NO:7, wherein no more than ten (10) amino acid substitutions relative to amino acids 23-849 of SEQ ID NO:7 are present, and wherein the ENPP1 mutant polypeptide comprises an amino acid substitution at position 256 relative to SEQ ID NO:7.
• Embodiment 61 provides the ENPP1 mutant polypeptide of any one of Embodiments 58-60, wherein the amino acid substitution is the substitution of isoleucine (I) for threonine (T) at position 256 relative to SEQ ID NO:7.
• Embodiment 62 provides the ENPP1 mutant polypeptide of any one of Embodiments 58-60, wherein the amino acid substitution is the substitution of isoleucine (I) for serine (S) at position 256 relative to SEQ ID NO:7.
• Embodiment 63 provides an ENPP1 mutant polypeptide comprising an amino acid sequence that is at least 90% identical to amino acids 23-849 of SEQ ID NO:7, wherein the mutant polypeptide comprises mutation I256T as relating to SEQ ID NO:7, and wherein the mutant polypeptide further comprises a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N as relating to SEQ ID NO: 7.
• Embodiment 64 provides the ENPP1 mutant polypeptide of Embodiment 63, wherein the mutant polypeptide comprises at least one amino acid substitution selected from the group consisting of S766N, P534N/V536T, P554L/R545T, and E592N as relating to SEQ ID NO:7.
• Embodiment 65 provides the ENPP1 mutant polypeptide of Embodiment 63, wherein the mutant polypeptide comprises the amino acid substitution V29N.
• Embodiment 66 provides the ENPP1 mutant polypeptide of any one of Embodiments 58-61, wherein the mutant polypeptide comprises the amino acid sequence of SEQ ID NO: l l.
• Embodiment 67 provides an ENPP1 mutant polypeptide fusion comprising the ENPP1 mutant polypeptide of any one of Embodiments 58-66 and a heterologous protein.
• Embodiment 68 provides the ENPP1 mutant polypeptide fusion of Embodiment 67, wherein the heterologous protein is an FcRn binding domain.
• Embodiment 69 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 67-68, wherein the heterologous protein is carboxy -terminal to the ENPP1 mutant polypeptide of the fusion.
• Embodiment 70 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 67-68, wherein the heterologous protein is amino-terminal to the ENPP1 mutant polypeptide of the fusion.
• Embodiment 71 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70, wherein the FcRn binding domain is an albumin polypeptide.
• Embodiment 72 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70, wherein the FcRn binding domain is a Fc portion of an immunoglobulin molecule.
• Embodiment 73 provides the ENPP1 mutant polypeptide fusion of Embodiment 72, wherein the immunoglobulin molecule is an IgGl.
• Embodiment 74 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-73, wherein the FcRn binding domain comprises one more amino acid substitutions relative to a wild type FcRn binding domain.
• Embodiment 75 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-74, wherein the FcRn binding domain is the Fc portion of a human IgGl molecule and comprises the following amino acid substitutions: M883Y,
• Embodiment 76 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-75, wherein the fusion comprises one or more of the following substitutions: S885N, S766N, M883Y/S885T/T887E, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, or
• P534N/V 536T/M883Y/S885T/T887E each as relating to SEQ ID NO:7.
• Embodiment 77 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-76, wherein the fusion comprises the S885N mutation as relating to SEQ ID NO:7.
• Embodiment 78 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-77, wherein the fusion comprises the S766N mutation as relating to SEQ ID NO:7.
• Embodiment 79 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-78, wherein the fusion comprises mutations M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• Embodiment 80 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-79, wherein the fusion comprises mutations P534N, V536T, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 81 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-80, wherein the fusion comprises mutations P554L and R545T as relating to SEQ ID NO:7.
• Embodiment 82 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-81, wherein the fusion comprises mutation S766N, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 83 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-82, wherein the fusion comprises mutation E592N, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 84 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-83, wherein the fusion comprises mutations P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• Embodiment 85 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-84, wherein the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 86 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-85, wherein the Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and
• H1064K/N1065F as relating to SEQ ID NO:7.
• Embodiment 87 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-86 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65, wherein the fusion comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N as relating to SEQ ID NO:7.
• Embodiment 88 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-87 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 85, wherein the fusion or the ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of C25N/K27T and V29N as relating to SEQ ID NO: 7.
• Embodiment 89 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-88 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 87-88, wherein the fusion or the ENPP1 mutant polypeptide comprises one mutation selected from the group consisting of K369N and I371T as relating to SEQ ID NO: 7.
• Embodiment 90 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-89 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 87-89, wherein the fusion or ENPP1 mutant polypeptide comprises the mutation K369N/I371T as relating to SEQ ID NO:7.
• Embodiment 91 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-90 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 87-90, wherein the fusion or ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N as relating to SEQ ID NO:7.
• Embodiment 92 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-91 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 87-91, wherein the fusion or ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N as relating to SEQ ID NO:7.
• Embodiment 93 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-92 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 87-92, wherein the fusion or ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of C25N, K27T, V29N, C25N/K27T, K369N, I371T, K369N/I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N,
• Embodiment 94 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-93 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 87-93, wherein the fusion or ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N, and E592N as relating to SEQ ID NO:7.
• Embodiment 95 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-94 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 87-94, wherein the fusion or ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N/Y 536T, P554L/R545T, and E592N as relating to SEQ ID NO:7.
• Embodiment 96 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-95 or the ENPP1 mutant polypeptide of any one of Embodiments 58-65 and 87-95, wherein the fusion or ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H 1064K/N 1065F, and P534N/V536T/M883Y/S885T/T887E as relating to SEQ ID NO: 7.
• Embodiment 97 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-96, wherein the fusion comprises mutations I256T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• Embodiment 98 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-96, wherein the fusion comprises mutations I256T, P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.
• Embodiment 99 provides the ENPP1 mutant polypeptide fusion of any one of Embodiments 68-70 and 72-96, wherein the fusion comprises mutations I256T, E592N, H1064K, and N1065F as relating to SEQ ID NO:7.
• Embodiment 100 provides the fusion of any one of Embodiments 67-99, comprising a linker amino acid sequence.
• Embodiment 101 provides the fusion of Embodiment 100, wherein the linker amino acid sequence connects the ENPP1 mutant polypeptide portion of the fusion and the heterologous protein.
• Embodiment 102 provides the fusion of any one of Embodiments 100-101, wherein the linker amino acid sequence comprises SEQ ID NO:8 or SEQ ID NO:9.
• Embodiment 103 provides a nucleic acid encoding the ENPP1 mutant polypeptide of any one of Embodiments 58-66 or the fusion of any one of Embodiments 67-102.
• Embodiment 104 provides a vector comprising the nucleic acid of Embodiment 103.
• Embodiment 105 provides an expression vector comprising the nucleic acid of Embodiment 103.
• Embodiment 106 provides a cell or plurality of cells, each comprising the nucleic acid of Embodiment 103, the vector of Embodiment 104, and/or the expression vector of Embodiment 105.
• Embodiment 107 provides the cell or plurality of cells of Embodiment 106, wherein the cell(s) is/are a CHO cell(s) and/or an NS0 cell(s).
• Embodiment 108 provides the cell or plurality of cells of Embodiment 107, wherein the CHO cell is stably transfected with human ST6 beta-galactoside alpha-2, 6- sialyltransferase.
• Embodiment 109 provides a method of producing an ENPP1 mutant polypeptide or fusion, the method comprising culturing the cell or plurality of cells of any one of
• Embodiments 106-108 under conditions suitable for expression of the ENPP1 mutant polypeptide or fusion by the cell or cells.
• Embodiment 110 provides the method of Embodiment 109, wherein the cells are cultured in a medium supplemented with sialic acid and/or N-acetylmannosamine.
• Embodiment 111 provides the method of any one of Embodiments 109-110, further comprising purifying the ENPP1 mutant polypeptide or fusion from the cell, plurality of cells, or the media in which the cell or plurality of cells were cultured.
• Embodiment 112 provides an ENPP1 mutant polypeptide or fusion purified by the method of Embodiment 111.
• Embodiment 113 provides a conjugate comprising (i) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112 and/or the ENPP1 mutant polypeptide fusion of any one of Embodiments 67-102 and 112 and (ii) a heterologous moiety.
• Embodiment 114 provides the conjugate of Embodiment 113, wherein the heterologous moiety is polyethylene glycol.
• Embodiment 115 provides a pharmaceutical composition comprising the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112, the fusion of any one of Embodiments 67-102 and 112, the nucleic acid of Embodiment 103, the vector of Embodiment 104, the expression vector of Embodiment 105, and/or the conjugate of any one of Embodiments 113-114 and a pharmaceutically acceptable carrier.
• Embodiment 116 provides a method of reducing or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of: (a) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112; (b) the fusion of any one of Embodiments 67-102 and 112; (c) the conjugate of any one of Embodiments 113-114; and/or (d) the pharmaceutical composition of Embodiment 115, to thereby reduce or prevent progression of pathological calcification in the subject.
• Embodiment 117 provides a method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of: (a) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112; (b) the fusion of any one of Embodiments 67- 102 and 112; (c) the conjugate of any one of Embodiments 113-114; and/or (d) the pharmaceutical composition of Embodiment 115, to thereby reduce or prevent progression of pathological ossification in the subject.
• Embodiment 118 provides a method of reducing or preventing progression of ectopic calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of: (a) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112; (b) the fusion of any one of Embodiments 67-102 and 112; (c) the conjugate of any one of Embodiments 113-114; and/or (d) the pharmaceutical composition of Embodiment 115, to thereby reduce or prevent progression of ectopic calcification of soft tissue in the subject.
• Embodiment 119 provides a method of treating, reversing, or preventing progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of: (a) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112; (b) the fusion of any one of Embodiments 67-102 and 112; (c) the conjugate of any one of Embodiments 113-114; and/or (d) the pharmaceutical composition of Embodiment 115, to thereby reduce, reverse, or prevent ossification of the posterior longitudinal ligament (OPLL) in the subject.
• Embodiment 120 provides a method of treating, reverting, or preventing progression of hypophosphatemic rickets in a subject in need thereof, the method comprising
• Embodiment 121 provides a method of reducing or preventing progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, aging related hardening of arteries, idiopathic infantile arterial calcification (IIAC), Generalized Arterial Calcification of Infancy (GACI), and calcification of atherosclerotic plaques in a subject diagnosed with the at least one disease, the method comprising administering to the subject a therapeutically effective amount of: (a) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112; (b) the fusion of any one of Embodiments 67-102 and 112; (c) the conjugate of any one of Embodiments 113-114; and
• Embodiment 115 a pharmaceutical composition of Embodiment 115, to thereby reduce or prevent progression of the disease.
• Embodiment 122 provides a method of reducing or preventing progression of aging related hardening of arteries in a subject in need thereof, the method comprising
• Embodiment 123 provides a method of raising pyrophosphate (PPi) levels in a subject having PPi level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of: (a) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112; (b) the fusion of any one of Embodiments 67-102 and 112; (c) the conjugate of any one of Embodiments 113-114; and/or (d) the
• Embodiment 115 whereby upon the administration the level of the PPi in the subject is elevated to a normal level of at least 2mM and is maintained at approximately the same level.
• Embodiment 124 provides a method of reducing or preventing the progression of pathological calcification or ossification in a subject having pyrophosphate (PPi) level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of: (a) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112; (b) the fusion of any one of Embodiments 67-102 and 112; (c) the conjugate of any one of Embodiments 113-114; and/or (d) the pharmaceutical composition of:
• Embodiment 115 whereby pathological calcification or ossification in the subject is reduced or progression of pathological calcification or ossification in the subject is prevented.
• Embodiment 125 provides a method of treating ENPP1 deficiency manifested by a reduction of extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of: (a) the ENPP1 mutant polypeptide of any one of Embodiments 58-66, 87-96, and 112; (b) the fusion of any one of Embodiments 67-102 and 112; (c) the conjugate of any one of Embodiments 113-114; and/or (d) the pharmaceutical composition of Embodiment 115, whereby the level of the PPi in the subject is elevated.
• PPi extracellular pyrophosphate
• Embodiment 126 provides the method of any one of Embodiments 116-125, wherein the mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered acutely or chronically to the subject.
• Embodiment 127 provides the method of any one of Embodiments 116-126, wherein the mutant polypeptide, fusion, conjugate, or pharmaceutical composition is administered locally, regionally, parenterally, or systemically to the subject.
• Embodiment 128 provides the method of any one of Embodiments 116-127, wherein the subject is human.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Microbiology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• General Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Physical Education & Sports Medicine (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Epidemiology (AREA)
• Plant Pathology (AREA)
• Physics & Mathematics (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicinal Preparation (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)

Priority Applications (13)

Applications Claiming Priority (6)

Related Child Applications (2)

Publications (1)

Family

ID=72667412

Family Applications (1)

Country Status (12)

Cited By (2)

Citations (3)

• 2020
  • 2020-04-03 CA CA3136118A patent/CA3136118A1/en active Pending
  • 2020-04-03 BR BR112021020037A patent/BR112021020037A2/pt unknown
  • 2020-04-03 JP JP2021559256A patent/JP2022527557A/ja active Pending
  • 2020-04-03 MX MX2021012197A patent/MX2021012197A/es unknown
  • 2020-04-03 EP EP20783595.0A patent/EP3947660A4/en active Pending
  • 2020-04-03 CN CN202080041580.2A patent/CN113993993A/zh active Pending
  • 2020-04-03 AU AU2020256253A patent/AU2020256253B2/en active Active
  • 2020-04-03 KR KR1020217036178A patent/KR20210149146A/ko active Pending
  • 2020-04-03 US US17/601,387 patent/US20220195402A1/en not_active Abandoned
  • 2020-04-03 WO PCT/US2020/026643 patent/WO2020206302A1/en not_active Ceased
  • 2020-04-06 TW TW109111502A patent/TW202043292A/zh unknown
• 2021
  • 2021-10-04 IL IL286946A patent/IL286946A/en unknown
• 2024
  • 2024-08-26 US US18/815,173 patent/US20250230423A1/en active Pending
  • 2024-09-26 JP JP2024167228A patent/JP2024178422A/ja active Pending
• 2026
  • 2026-01-30 AU AU2026200700A patent/AU2026200700A1/en active Pending

Patent Citations (3)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events