Compositions for treating pathological calcification conditions and methods of using the same

Abstract

To provide compositions and methods for treating diseases or disorders associated with pathological calcification or pathological ossification.SOLUTION: Provided are a compound having a structure of protein-Z-domain-X-Y, wherein the protein is a specific sequence derived from ectonucleotide pyrophosphate / phosphodiesterase-1 or a mutant thereof, the domain is a human IgGFc domain or a human serum albumin protein and a fragment thereof, X and Z are polypeptides having a specific amino acid sequence of 20 or less, and Y is a compound consisting of a specific amino acid sequence, and a composition and a treatment method for treating systemic arterial calcification of infancy (GACI), idiopathic arterial calcification of infancy (IIAC), and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 163,500, filed May 19, 2015, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Generalized arterial calcification of infancy (GACI) is an extremely rare neonatal disorder characterized by widespread arterial calcification in large and medium-sized vessels during infancy, resulting in cardiovascular collapse and death during the neonatal period. Clinical manifestations of the disease include heart failure, respiratory distress, hypertension, cyanosis, and cardiac hypertrophy. The prognosis is severe, with older reports of a 6-month mortality rate of 85%, but more recently, intensive bisphosphonate therapy has reduced mortality to 55% at 6 months. This apparent progress has been limited by the severe skeletal toxicity associated with long-term etridonate use in patients with GACI. Due to limited available data, it is difficult to ascertain whether bisphosphonate treatment truly provides protection or whether poor response reflects the natural history of the disease and the ineffectiveness of bisphosphonates, even when initiated early, in preventing death in some patients.

[0003] The overall incidence of GACI is rare, with 200 cases reported in the medical literature, representing a disease prevalence of 1 in 391,000. Although the disease was first described by Bryant and White in 1901, it was not until 2000 that Rutsch and colleagues noticed that serum PPi levels and ENPP1 enzyme activity were significantly decreased in patients with GACI. ENPP1 (also known as NPP1 or PC-1) is a member of the ectonucleotide pyrophosphatase / phosphodiesterase (ENPP or NPP) enzyme family, characterized by phosphodiesterase activity. ENPP1 is a type II extracellular membrane-bound glycoprotein located on mineral-depositing matrix vesicles in osteoblasts and chondrocytes, as well as on the vascular surface of brain capillaries. ENPP1 catalyzes the degradation of extracellular ATP into AMP and PPi. PPi likely inhibits ectopic tissue mineralization by occupying some of the Pi sites on the surfaces of nascent or growing hydroxyapatite (HA) crystals, thereby creating irregularities that slow or terminate crystal growth. Inactivating mutations in ENPP1 account for 75% of GACI patients, and a significantly larger proportion of the remaining patients are due to inactivating mutations in the ATP-dependent membrane transporter MRP6, encoded by the abcc6 gene. The abcc6 mutation has been linked to a reduced extracellular concentration of nucleoside triphosphates, thereby limiting the metabolism of ATP to extracellular PPi by ENPP1.

[0004] The kidneys are essential for maintaining normal bone and mineral metabolism, including phosphate excretion. Patients with renal failure are unable to properly regulate serum mineral balance and tend to retain phosphate absorbed from various dietary sources. High serum phosphate levels are associated with excessive secretion of parathyroid hormone and a tendency for soft tissues, including blood vessels, to calcify.

[0005] In patients with renal failure, excessive removal of phosphate and pyrophosphate ions can occur during hemodialysis or peritoneal dialysis. Depletion of these anions from tissues and plasma leads to disorders of bone and mineral metabolism, including osteomalacia and soft tissue calcification and bone disease. Pyrophosphate deficiency can be a risk factor for calciphylaxis, an inflammatory vasculitis in which calcium deposits in small blood vessels in the skin, leading to gangrene of the skin and underlying tissues and causing severe chronic pain. Calciphylaxis can require amputation of the affected limb; the condition has no effective treatment and is generally fatal. Left untreated, ectopic calcification causes increased morbidity and mortality. It is important to regulate the amount of pyrophosphate in the system and prevent the development of calciphylaxis in patients.

[0006] As revealed by the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKFK / DOQI), 19.5 million U.S. adults had chronic kidney disease (CKD) in 2003, and 13.6 million had stages 2 to 5 of CKD. Adverse outcomes of chronic kidney disease can often be prevented or delayed by early detection and treatment.

[0007] The incidence of end-stage renal disease (ESRD) is increasing at an alarming rate. In 2000, over 90,000 people developed end-stage renal disease in the United States. The patient population undergoing dialysis or requiring a transplant rose from 380,000 in 2003 to 651,000 patients in 2010. Over $18 billion is already spent annually in the United States treating ESRD patients, representing a significant burden to the healthcare system.

[0008] Calcific uremic arteriopathy (CUA) is a potentially fatal disease observed in chronic kidney disease (CKD) patients undergoing dialysis. Calcification of small arteries leads to tissue and skin ischemia, infarction, and thrombosis, resulting in a patient mortality rate approaching 80%. Currently, there are 450,000 dialysis patients in the United States at risk for CUA, and there is no FDA-approved treatment for the disease. CUA shares hallmarks similar to GACI and other calcific disorders, including low levels of PPi and high levels of fibroblast growth factor 23 (i.e., FGF23). In ESRD patients requiring dialysis, this calcification process is more rapid, and life expectancy is 5–6 years.

[0009] Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by elastic fiber calcification in the skin, arteries, and retina, resulting in skin lesions with concomitant laxity and loss of elasticity, arterial insufficiency, cardiovascular disease, and retinal hemorrhages leading to macular degeneration. Mutations associated with PXE are also located in the abcc6 gene. While cutaneous manifestations are among the most common features of PXE, ocular and cardiovascular manifestations contribute to the disease's pathology. Generally, characteristic skin lesions (yellowish papules and plaques, as well as laxity and loss of elasticity, typically found on the face, neck, axillae, antecubital fossae, popliteal fossae, groin, and periumbilical region) are the initial signs of PXE and result from the accumulation of abnormally calcified elastic fibers in the central dermis. Skin lesions are usually detected during childhood or adolescence and progress slowly and often unpredictably. A diagnosis of PXE can be confirmed by a skin biopsy showing fragmented elastic fiber calcification in the central and lower dermis.

[0010] Common cardiovascular complications of PXE are due to the presence of abnormally calcified elastic fibers in the internal elastic lamina of medium-sized arteries. The broad phenotype includes premature atherosclerotic changes, intimal fibroplasia leading to angina or intermittent claudication, or both, premature myocardial infarction, and hypertension. Restrictive cardiomyopathy may also occur due to fibrous thickening of the endocardium and atrioventricular valves. Approximately 10% of patients with PXE also develop gastrointestinal bleeding and central nervous system complications (e.g., stroke and dementia) as a result of systemic arterial wall calcification. Additionally, patients with PXE may have renovascular hypertension and atrial septal aneurysms.

[0011] Low serum phosphate levels are called hypophosphatemia, whereas high serum phosphate levels are called hyperphosphatemia. Hypophosphatemia is often due to renal phosphate wasting and is caused by numerous genetic disorders, including X-linked hypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets with hypercalciuria (HHRH), hypophosphatemic bone disease (HBD), and autosomal dominant hypophosphatemic rickets (ADHR). Although the precise molecular mechanisms by which adequate serum phosphate levels are maintained are not fully understood, maintaining serum phosphate levels is important for alleviating the symptoms of the aforementioned diseases.

[0012] Thus, there is a need in the art for novel compositions and methods for treating diseases and disorders associated with pathological calcification and / or pathological ossification. Such compositions and methods should not unnecessarily disrupt other physiological processes. The present invention fulfills this need. Summary of the Invention

[0013] The present invention provides a compound of formula (I) or a salt or solvate thereof. Furthermore, the present invention provides a method for treating or preventing a disease or disorder associated with pathological calcification or pathological ossification in a subject in need thereof. Furthermore, the present invention provides a method for reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in infants suffering from at least one disease or disorder selected from the group consisting of GACI and PXE.

[0014] In certain embodiments, the compound of formula (I) is protein-Z-domain-XY (I), wherein (I) the protein is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:24, and the domain is selected from the group consisting of human IgG Fc domain (also referred to as Fc), human serum albumin protein (also referred to as ALB), and fragments thereof, and X and Z are independently absent or a polypeptide comprising 1 to 20 amino acids, and Y is absent or TIFF2026015456000001.tif26158, wherein m is an integer of 1 to 15, and n is an integer of 1 to 10.

[0015] In certain embodiments, the domain is Fc or a fragment thereof, hi other embodiments, the domain is ALB or a fragment thereof.

[0016] In certain embodiments, Y is absent and the compound lacks a negatively charged bone-targeting sequence.

[0017] In certain embodiments, the protein has a mutation relative to SEQ ID NO:1 at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536. In other embodiments, the nuclease domain of the protein or variant thereof is absent. In still other embodiments, the nuclease domain from about residue 524 to about residue 885 of SEQ ID NO:1 is absent. In yet other embodiments, the protein or variant thereof may or may not have a segment of the extracellular domain of NPP2 containing a furin or signal peptide cleavage site introduced by a substitution.

[0018] In certain embodiments, the domain is Fc or a fragment thereof, and the protein-Z-domain is (SEQ ID NO:15)-Z-(Fc or a fragment thereof), (SEQ ID NO:17)-Z-(Fc or a fragment thereof), (SEQ ID NO:19)-Z-(Fc or a fragment thereof), (SEQ ID NO:24)-Z-(Fc or a fragment thereof), or a variant thereof, and Compared to NO:1, the variants include those containing at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536.

[0019] In certain embodiments, the protein-Z-domain comprises SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, (SEQ ID NO:24)-Z-(SEQ ID NO:26), or a variant thereof, which comprises at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1.

[0020] In certain embodiments, the domain is ALB or a fragment thereof, and the protein-Z-domain is (SEQ ID NO:15)-Z-(ALB or a fragment thereof), (SEQ ID NO:17)-Z-(ALB or a fragment thereof), (SEQ ID NO:19)-Z-(ALB or a fragment thereof), (SEQ ID NO:24)-Z-(ALB or a fragment thereof), or a variant thereof, and Compared to NO:1, the variants include those containing at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536.

[0021] In certain embodiments, the protein-Z-domain comprises SEQ ID NO:21, (SEQ ID N:17)-Z-(SEQ ID NO:27), SEQ ID NO:22, SEQ ID NO:25, or a variant thereof, which comprises at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1.

[0022] In certain embodiments, the compound has a pH of about 3.4 (±0.4) s -1 enzyme -1 Greater than or equal to k cat has a value k cat is determined by measuring the rate of ATP hydrolysis of the compound.

[0023] In certain embodiments, the compound has a K of less than or equal to about 2 μM. M has a value of K M is determined by measuring the rate of ATP hydrolysis of the compound.

[0024] In certain embodiments, the NPP1 polypeptide is a cleavage product of a precursor NPP1 polypeptide that includes the ectonucleotide pyrophosphate / phosphodiesterase-2 (NPP2) transmembrane domain.

[0025] In certain embodiments, the NPP2 transmembrane domain is residues 12-30 of NCBI Accession No. NP_001124335 (SEQ ID NO:2), which corresponds to SEQ ID NO:23.

[0026] In certain embodiments, the methods comprise administering to the subject a therapeutically effective amount of at least one compound of the present invention.

[0027] In certain embodiments, the disease comprises at least one selected from the group consisting of generalized arterial calcification of infancy (GACI), idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

[0028] In certain embodiments, the disease comprises at least one selected from the group consisting of PXE, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, arteriosclerosis that occurs with aging, calciphylaxis due to end-stage renal disease, and progeria.

[0029] In certain embodiments, Y is absent and the compound lacks a negatively charged bone-targeting sequence.

[0030] In certain embodiments, the method comprises administering to the infant a therapeutically effective amount of a predetermined polypeptide comprising an ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide and an IgG Fc domain, wherein the predetermined polypeptide lacks a polyaspartic acid domain, and wherein administration of the predetermined polypeptide increases extracellular pyrophosphate (PPi) levels in the infant.

[0031] In certain embodiments, the method comprises administering to the infant a therapeutically effective amount of a predetermined polypeptide comprising an ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide and an ALB, wherein the predetermined polypeptide lacks a polyaspartic acid domain, and wherein administration of the predetermined polypeptide increases extracellular pyrophosphate (PPi) concentrations in the infant.

[0032] In certain embodiments, the administration is at least one selected from the group consisting of inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal (intranasal), and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration. In other embodiments, the administration is subcutaneous.

[0033] In certain embodiments, administration restores the infant's extracellular pyrophosphate concentration to levels within the range found in infants not afflicted with GACI and / or PXE.

[0034] In certain embodiments, the infant exhibits and / or has been diagnosed with "failure to thrive" prior to administration. [The present invention 1001] Formula (I): Protein-Z-Domain-XY (I) or a salt thereof, In (I), The protein is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:24; The domain is selected from the group consisting of a human IgG Fc domain (Fc), a human serum albumin protein (ALB), and fragments thereof; X and Z are, independently, null or a polypeptide containing 1 to 20 amino acids; and Y is absent or TIFF2026015456000002.tif33156, wherein m is an integer of 1 to 15 and n is an integer of 1 to 10. The compound or a salt thereof. [The present invention 1002] 1001. The compound of the present invention, wherein said domain is Fc or a fragment thereof. [The present invention 1003] 1001. The compound of the present invention, wherein said domain is ALB or a fragment thereof. [The present invention 1004] 1001. The compound of claim 10, wherein Y is absent and said compound lacks a negatively charged bone-targeting sequence. [The present invention 1005] 1001. The compound of claim 1001, wherein the protein has a mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 compared to SEQ ID NO:1. [The present invention 1006] 1001. The compound of the present invention, wherein the nuclease domain of said protein or a variant thereof is absent. [The present invention 1007] 1001. The compound of the present invention, wherein the nuclease domain at about residue 524 to about residue 885 relative to SEQ ID NO:1 is absent in said protein or variant thereof. [The present invention 1008] 1001. A compound of the present invention, wherein a segment of the extracellular domain of NPP2 containing a furin or signal peptide cleavage site is or is not introduced into said protein or a mutant thereof by substitution. [The present invention 1009] The domain is Fc or a fragment thereof, and the protein-Z-domain is (SEQ ID NO: 15)-Z-(Fc or a fragment thereof), (SEQ ID NO: 17)-Z-(Fc or a fragment thereof), (SEQ ID NO: 19)-Z-(Fc or a fragment thereof), (SEQ ID NO: 24)-Z-(Fc or a fragment thereof), or variants thereof, a variant comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 relative to NO:1; 1001 compounds of the present invention, including: [The present invention 1010] the protein-Z-domain SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, (SEQ ID NO:24)-Z-(SEQ ID NO:26), or variants thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 compared to SEQ ID NO:1. The compound of the present invention 1009, comprising: [The present invention 1011] The domain is ALB or a fragment thereof, and the protein-Z-domain is (SEQ ID NO:15)-Z-(ALB or a fragment thereof), (SEQ ID NO:17)-Z-(ALB or a fragment thereof), (SEQ ID NO:19)-Z-(ALB or a fragment thereof), (SEQ ID NO:24)-Z-(ALB or a fragment thereof), or variants thereof, a variant comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 relative to NO:1; 1001 compounds of the present invention, including: [The present invention 1012] the protein-Z-domain SEQ ID NO:21, (SEQ ID NO:17)-Z-(SEQ ID NO:27), SEQ ID NO:22, SEQ ID NO:25, or variants thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 compared to SEQ ID NO:1. The compound of the present invention comprises: [The present invention 1013] The compound has a viscosity of 3.4 (±0.4) s -1 enzyme -1 More than k cat k has a value catis determined by measuring the ATP hydrolysis rate of the compound. [The present invention 1014] The compound has a K M having a value of K M is determined by measuring the ATP hydrolysis rate of the compound. [The present invention 1015] A method for treating or preventing a disease or disorder associated with pathological calcification or pathological ossification in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of at least one compound of any one of present inventions 1001 to 1014, wherein the disease comprises at least one selected from the group consisting of generalized arterial calcification of infancy (GACI), idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaque. [The present invention 1016] The method of claim 1015, wherein Y is absent and said compound lacks a negatively charged bone-targeting sequence. [The present invention 1017] A method for treating or preventing a disease or disorder associated with pathological calcification or pathological ossification in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of at least one compound of present inventions 1001 to 1014, wherein the disease comprises at least one selected from the group consisting of PXE, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, arteriosclerosis that occurs with aging, calciphylaxis due to end-stage renal disease, and progeria. [The present invention 1018] The method of claim 1017, wherein Y is absent and said compound lacks a negatively charged bone-targeting sequence. [The present invention 1019] A method for reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in an infant suffering from at least one disease or disorder selected from the group consisting of GACI and PXE, comprising the step of administering to the infant a therapeutically effective amount of a predetermined polypeptide comprising an ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide and Fc, wherein the predetermined polypeptide lacks a polyaspartic acid domain, and administration of the predetermined polypeptide increases extracellular pyrophosphate (PPi) concentration in the infant, thereby reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in the infant. [The present invention 1020] 1019. The method of claim 1019, wherein said administration is subcutaneous administration. [The present invention 1021] The method of claim 1019, wherein said administering restores the infant's extracellular pyrophosphate concentration to a level within the range found in infants not afflicted with GACI and / or PXE. [The present invention 1022] The method of claim 1019, wherein said infant exhibits "failure to thrive" and / or has been diagnosed with "failure to thrive" prior to said administration. [The present invention 1023] 1019. The method of claim 1019, wherein said NPP1 polypeptide is a cleavage product of a precursor NPP1 polypeptide containing an ectonucleotide pyrophosphate / phosphodiesterase-2 (NPP2) transmembrane domain. [The present invention 1024] 1019. The method of the present invention, wherein the NPP2 transmembrane domain is residues 12 to 30 of NCBI Accession No. NP_001124335 (SEQ ID NO:2), which corresponds to SEQ ID NO:23. [The present invention 1025] A method for reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in an infant suffering from at least one disease or disorder selected from the group consisting of GACI and PXE, comprising the step of administering to the infant a therapeutically effective amount of a predetermined polypeptide comprising an NPP1 polypeptide and an ALB, wherein the predetermined polypeptide lacks a polyaspartic acid domain, and said administration increases the extracellular PPi concentration in the infant, thereby reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in the infant. [The present invention 1026] 1026. The method of claim 1025, wherein said administering comprises subcutaneous administration. [The present invention 1027] The method of claim 1025, wherein said administering restores extracellular pyrophosphate concentrations in said infant to levels within the range found in infants not afflicted with GACI and / or PXE. [The present invention 1028] The method of claim 1025, wherein said infant exhibits "failure to thrive" and / or has been diagnosed with "failure to thrive" prior to said administration. [Brief explanation of the drawings]

[0035] The following detailed description of illustrative embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, specific embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0036] [Figure 1A]Figures 1A-1G include a set of images and graphs illustrating the natural history study. Figure 1A: Mean daily body weights of ENPP1-asj / asj and ENPP1-WT sibling pairs fed an acceleration diet. Daily body weights of ENPP1-WT (cyan blue squares) and ENPP1-asj / asj mice (green circles) fed an acceleration diet for 70 days. The ENPP1-asj / asj cohort exhibits growth failure at day 26, where their body weights diverge from those of ENPP1-WT. Mortality events are marked with red arrows. [Figure 1B] Figure 1B: Survival curve, natural history study. The median survival time for ENPP1-asj / asj was 58 days. No deaths were observed in the ENPP1-WT cohort. [Figure 1C] Figure 1C: Representative micro-CT. [Figure 1D] Representative histology, natural history study. Some asj / asj animals showed marked, clearly visible cardiac and aortic calcification (E). As shown by aortic alizarin red staining, all aortas from ENPP1-asj / asj mice had near-circumferential calcification extending throughout the vessel wall. [Figure 1E] See legend to Figure 1D. [Figure 1F] Figure 1F: Histology of the left ventricle of an asj / asj mouse (40x magnification). Extensive calcification surrounded by scar tissue reveals the presence of a repeated, old, healed myocardial infarction. [Figure 1G] Figure 1G: asj / asj mouse, septal histology (100X). More typically, asj mice exhibit small calcified lesions with surrounding scar tissue, as seen here in the myocardial septum, also diagnostic of a previous myocardial infarction. [Figure 2A]Figures 2A-2E include a set of images and graphs illustrating the metabolic pathways, as well as the design, stability, and kinetic properties, of therapeutic proteins of the invention. Figure 2A: Schematic representation of the metabolic pathways of purinergic metabolism associated with ectopic calcification. ENPP1 converts extracellular ATP to AMP and PPi, TNAP converts PPi to Pi, and CD73 converts AMP to adenosine and Pi. The abcc6 gene encodes MRP6, a membrane transporter that increases the extracellular concentration of ATP. Loss-of-function mutations in TNAP cause familial hypophosphatasia. Loss-of-function mutations in ENPP1 cause GACI, loss-of-function mutations in MRP6 cause PXE, and loss-of-function mutations in CD73 cause a disease of arterial and joint calcification called "ACDC." [Figure 2B] Figure 2B: Design of ENPP1 protein therapeutics. To produce a soluble recombinant protein, a segment containing the furin cleavage site in the extracellular region of NPP2 was introduced into ENPP1 by substitution, as previously described, and the C-terminus of this protein was fused to the Fc domain of human immunoglobulin 1 (IgG1). [Figure 2C] Figure 2C: Stability of ENPP1 therapeutics. ENPP1-Fc Ap3A activity was found to be stable to freeze-thaw cycles in PBS after storage at -80°C. Figures 2D-2E: Steady-state kinetics of hENPP1-Fc. [Figure 2D] Figure 2D: Time course of AMP formation measured by HPLC analysis after addition of 10 nM hNPP1-Fc to (bottom to top) 1.0 μM, 2.0 μM, 7.8 μM, 125 μM, and 250 μM ATP. The smooth curve through the data is a fit obtained by nonlinear kinetic time course analysis. The inset shows the low [ATP] time course in panel A: (bottom to top) 1.0 μM, 2.0 μM, and 7.8 μM ATP. The 1.0 μM ATP time course shows that ATP was completely depleted after 1 min, and therefore, a rate could not be determined. [Figure 2E]Figure 2E: Initial rate of ATP hydrolysis per enzyme as a function of ATP concentration. The initial rates after 7.8 μM were essentially the same, with k(average) = 3.4 (±0.4) s-1 enzyme-1. The initial rate at 2.0 μM ATP concentration was approximately half the k value, and therefore, a K of approximately 2 μM was estimated for ATP hydrolysis by the hNPP1-Fc protein. [Figure 3-1] Figures 3A-3D contain a set of images and graphs illustrating the proof-of-concept study. Figure 3A: Daily animal weights. Average daily body weights of ENPP1-WT and ENPP1-asj / asj sibling pairs dosed with vehicle (one daily PBS injection supplemented with weekly GK1.5) compared with ENPP1-asj / asj sibling pairs dosed daily with mouse ENPP1-Fc (mENPPI-Fc) 500 au / kg dissolved in PBS (mENPPI-Fc) and weekly with GK1.5 immunosuppression. Dosing and weight measurements began on day 14. Deaths in the ENPP1-asj / asj + vehicle cohort are indicated by red arrows on the day of death. No deaths were observed in the ENPP1-WT + vehicle or ENPP1-asj / asj + ENPP1-Fc cohorts. [Figure 3-2] Figure 3B: Survival curve, proof-of-concept study. Figure 3C: Left ventricular histology, (40x, H&E). Untreated asj / asj mice showed large foci of calcification and microinfarcts in the free wall. Figure 3D: Left ventricular histology, (40x, H&E), treated asj / asj mice. None of the treated ENPP1-asj mice showed abnormal left ventricular histology. [Figure 4-1] Figures 4A-4G include a set of images illustrating representative histology and proof-of-concept studies. Figures 4A-4B: Aorta (40x, Alizarin Red). Untreated ENPP1-asj mice (Figure 4A) showed nearly circumferential aortic calcification, whereas treated ENPP1-asj mice (Figure 4B) showed no aortic calcification. Figure 4C: Untreated ENPP1-asj / asj mice, right ventricle (40x, H&E). Two untreated ENPP1-asj mice had large confluent myocardial infarctions in the free wall of the right ventricle. [Figure 4-2]Figure 4D: Treated ENPP1-asj / asj mouse, right ventricle (40x, H&E). All treated ENPP1-asj mice showed normal right ventricular myocardium. Figure 4E: Untreated ENPP1-asj / asj mouse, coronary artery (100x, H&E). All untreated ENPP1-asj / asj mice had coronary artery calcification, and most showed pericardial calcification of the coronary arteries surrounded by scar tissue, diagnostic of ischemia and myocardial infarction. [Figure 4-3] Figure 4F: Untreated ENPP1-asj / asj mouse, myocardial septum (100x, H&E). Nearly all animals (77%) showed intracardiac calcification surrounded by scar tissue, as evidenced in the myocardial septum of this animal. Figure 4G: Phenotypic comparison between treated and untreated ENPP1-asj / asj mice. There is a significant difference in size between treated and untreated animals, as well as in their mobility and health. The best results can be seen in the video provided in the Supplementary Data. [Figure 5-1] Figures 5A-5F include a set of images and graphs illustrating biomarkers of disease response. Figure 5A: Postmortem high-resolution micro-CT scans revealed extensive calcification in the heart, coronary arteries, and ascending and descending aorta in untreated ENPP1-asj / asj mice, but no calcification in these organs in treated ENPP1-asj / asj cohorts or ENPP1-WT mice. Figure 5B: Plasma [PPi] in ENPP1-WT and treated and untreated ENPP1-asj / asj animals revealed that treatment with ENPP1-FC increased [PPi] in ENPP1-asj / asj mice above WT levels, significantly exceeding the nearly undetectable levels present in untreated ENPP1-asj / asj mice. [Figure 5-2]Figures 5C-5D: Percentage uptake of injected mPYP in the heads of WT and asj / asj animals. Percentage uptake of mPYP in the heads of animals in the natural history study was recorded weekly in WT and asj / asj animals fed the acceleration diet. This demonstrated that mPYP uptake remained fairly constant over the 80 days after birth but differed significantly between the two experimental groups. Figure 5D: In the natural history study, mean mPYP uptake in the heads of WT animals was approximately 15% of the injected dose over the 80 days, whereas mPYP uptake in asj / asj animals was approximately 20% (p<0.001, two-tailed Student's t-test). [Figure 5-3] Figures 5E-5F: Percentage of 99mPYP uptake in the skulls of WT, treated asj / asj, and untreated asj / asj mice relative to the injected dose. In the experimental groups, 99mPYP uptake was recorded midway through the study (days 30-35, Figure 5E) or at the end of the study (days 50-65, Figure 5F). The percent uptake in the skulls of WT and treated asj / asj animals was approximately 15%, whereas the untreated ENPP1-asj / asj cohort was 20% or higher. The difference between treated ENPP1-asj and untreated ENPP1-asj mice was statistically significant (p<0.001, two-tailed Student's t-test), whereas the difference between WT and treated ENPP1-asj mice was not. [Figure 6]Panels a-h, inclusive, illustrate certain non-limiting constructs of NPP1 fusion proteins. X and Y are optional peptides in some embodiments. Z is an optional linker connecting the Fc domain or HSA domain to the C-terminus of the NPP1 protein. In Figure 6, the N- and C-terminal regions of the NPP1 protein are represented by N and C. Panels a-d illustrate fusion proteins containing the NPP2 transmembrane domain (marked with "*") and the NPP1 transmembrane domain (marked with "**") together with the NPP1 enzymatic domain. The NPP1 enzymatic domain begins with the PSCAKE amino acid sequence and ends with the QED amino acid sequence. Panels e-h illustrate fusion proteins containing the NPP2 signal peptide (marked with "*") and the NPP1 transmembrane domain (marked with "**") together with the NPP1 enzymatic domain. [Figure 7] 1 is a graph showing measurements of plasma PPi levels in mice treated with ENPP1-Fc as described in Example 1. [Figure 8] FIG. 1 is a schematic diagram of the plasmid used to express SEQ ID NO:22. [Figure 9] FIG. 1 is a schematic diagram of the plasmid used to express SEQ ID NO:25. [Figure 10] 1 is an image illustrating silver stained images from purified human and mouse NPP1-Fc constructs. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention The present invention relates to the discovery that certain NPP1-containing polypeptides, their variants, or variant fragments are useful for treating diseases and disorders associated with plasma pyrophosphate imbalance, pathological calcification, and / or pathological ossification. Diseases and disorders associated with pathological calcification and / or pathological ossification that can be treated by the compositions and methods of the present invention include, but are not limited to, generalized arterial calcification of infancy (GACI), chronic kidney disease (CKD), end-stage renal disease (ESRD), idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, calcification of atherosclerotic plaques, pseudoxanthoma elasticum (PXE), hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, arterial stiffness that occurs with aging, calciphylaxis (e.g., due to end-stage renal disease), and progeria.

[0038] Such diseases are the result of a myriad of causes—some are genetic mutations, others are complications resulting from diabetes, heart failure, or extensive dialysis—but in certain aspects, such diseases share symptoms of plasma pyrophosphate imbalance and / or widespread calcification.

[0039] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0040] As used herein, each of the following terms has the meaning associated with it in this section.

[0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0042] As used herein, "about," when referring to a measurable value, e.g., amount, duration, etc., is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, and in certain embodiments ±0.1% from the specified value, where such variations are appropriate for practicing the disclosed methods.

[0043] The term "abnormal," when used in the context of an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from an organism, tissue, cell, or component thereof that exhibits the "normal" (expected) respective characteristic. A characteristic that is normal or expected for one cell or tissue type may be abnormal for a different cell or tissue type.

[0044] As used herein, the term "ALB" refers to human serum albumin protein.

[0045] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such symptoms are experienced by a patient, or both, are reduced.

[0046] As used herein, the terms "alteration," "deletion," "variation," or "mutation" refer to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation), or conformation of the polypeptide encoded by the gene. Mutations encompassed by the present invention may be any mutation in a gene in a cell that results in the enhancement or disruption of the function, activity, expression, or conformation of the encoded polypeptide, including the complete absence of expression of the encoded protein, and may include, for example, missense and nonsense mutations, insertions, deletions, frameshifts, and premature terminations. Mutations encompassed by the present invention may, but are not limited to, alter the splicing of mRNA (splice site mutations) or cause a shift in the reading frame (frameshifts).

[0047] The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Typically, an amino acid sequence variant has at least about 70% homology, at least about 80% homology, at least about 90% homology, or at least about 95% homology to the native polypeptide. An amino acid sequence variant has substitutions, deletions, and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence.

[0048] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. An antibody can be an intact immunoglobulin derived from natural sources or from recombinant sources, or an immunoreactive portion of an intact immunoglobulin. Antibodies in the present invention may exist in a variety of formats, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)2, as well as single-chain antibodies (scFv), heavy-chain antibodies, e.g., camelid antibodies, synthetic antibodies, chimeric antibodies, and humanized antibodies (Harlow, et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow, et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston, et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird, et al., 1988, Science 242:423-426).

[0049] As used herein, the term "Ap3P" refers to adenosine-(5')-triphospho-(5')-adenosine or a salt thereof.

[0050] As used herein, the terms "child" and "infant" are used interchangeably.

[0051] As used herein, the term "coding sequence" refers to a sequence of a nucleic acid or its complementary strand, or a portion thereof, that can be transcribed and / or translated to produce mRNA and / or a polypeptide, or a fragment thereof. Coding sequences include exons in genomic DNA or premature primary RNA transcripts, which are spliced ​​together by the cell's biochemical machinery to produce mature mRNA. The antisense strand is the complementary strand of such a nucleic acid, from which the coding sequence can be deduced. In contrast, the term "non-coding sequence" as used herein refers to a sequence of a nucleic acid or its complementary strand, or a portion thereof, that is not translated into amino acids in vivo or with which tRNAs do not interact or attempt to place amino acids. Non-coding sequences include intron sequences in genomic DNA or premature primary RNA transcripts and sequences associated with genes, such as promoters, enhancers, and silencers.

[0052] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., nucleotide sequences) related by the base-pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity can be "partial," in which only some of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that depend on binding between nucleic acids.

[0053] As used herein, the term "conservative variation" or "conservative substitution" refers to the replacement of an amino acid residue with another biologically similar residue.Conservative variation or conservative substitution is unlikely to change the shape of a peptide chain.Examples of conservative variation or conservative substitution include the replacement of a hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue, or the replacement of a polar residue with another polar residue, such as the replacement of lysine with arginine, the replacement of aspartic acid with glutamic acid, or the replacement of asparagine with glutamine.

[0054] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis, and unless the disease improves, the animal's health will continue to deteriorate.

[0055] 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 health is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily result in a further deterioration of the animal's health.

[0056] As used herein, the term "domain" refers to a portion of a molecule or structure that shares common physicochemical characteristics, such as, but not limited to, hydrophobic, polar, globular, and helical domains or properties. Specific examples of binding domains include, but are not limited to, DNA-binding domains and ATP-binding domains.

[0057] As used herein, the terms "effective amount," "pharmaceutically effective amount," and "therapeutically effective amount" refer to a nontoxic but sufficient amount of an agent to produce a desired biological result. This result may be a 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 can be determined by one of skill in the art using routine experimentation.

[0058] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, e.g., a gene, cDNA, or mRNA, to serve as a template in biological processes for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and resulting biological properties. Thus, a gene encodes a protein if the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0059] As used herein, the term "Fc" refers to the human IgG Fc domain.

[0060] As used herein, the term "failure to thrive" refers to a child or infant whose current weight or rate of weight gain is less than the weight or rate of weight gain of other children of similar age and sex. Circumstances in which a child or infant exhibits "failure to thrive" can be identified by consulting a medical professional and / or by comparing the child's or infant's weight or weight gain rate to known average weight or weight gain rate data for their age.

[0061] As used herein, the term "fragment," when applied to a nucleic acid, refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid may be at least about 15 nucleotides in length, e.g., at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides; at least about 1000 nucleotides to about 1500 nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value therebetween). As used herein, the term "fragment," when applied to a protein or peptide, refers to a subsequence of a larger protein or peptide. A "fragment" of a protein or peptide may be at least about 20 amino acids in length, e.g., at least about 50 amino acids in length; at least about 100 amino acids in length; at least about 200 amino acids in length; at least about 300 amino acids in length; or at least about 400 amino acids in length (and any integer value therebetween).

[0062] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both compared molecules is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions × 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC are 50% homologous. Generally, comparisons are performed when the two sequences are aligned to maximize homology.

[0063] As used herein, "immunoassay" refers to any binding assay that uses an antibody capable of specifically binding to a target molecule to detect and quantitate the target molecule.

[0064] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions, such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. IgM is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin with unknown antibody function but may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon allergen exposure.

[0065] "Instructional materials," as that term is used herein, include publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the nucleic acids, peptides, and / or compounds of the invention in the kit for identifying, alleviating, or treating the various diseases or disorders listed herein. Optionally, or alternatively, the educational materials may describe one or more methods of identifying or alleviating a disease or disorder in a subject's cells or tissues. The educational materials of the kit may, for example, be affixed to a container that holds the nucleic acids, polypeptides, and / or compounds of the invention, or may be shipped together with the container that holds the nucleic acids, polypeptides, and / or compositions. Alternatively, the educational materials may be shipped separately from the container, with the intention that the educational materials and compounds will be used cooperatively by the recipient.

[0066] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, for example, it may exist in a non-native environment such as a host cell.

[0067] "Isolated nucleic acid" refers to a nucleic acid segment or fragment that has been separated from adjacent sequences in nature, e.g., a DNA fragment that has been removed from sequences that normally flank the DNA fragment, e.g., sequences that flank the DNA fragment in the genome in which the DNA fragment naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany them, e.g., RNA or DNA or proteins that naturally accompany them in a cell. Thus, the term includes recombinant DNA that is incorporated into, for example, a vector, a self-replicating plasmid or virus, or the genomic DNA of a prokaryote or eukaryote, or exists as a separate molecule independent of other sequences (e.g., as a cDNA or genomic fragment or cDNA fragment generated by PCR or restriction enzyme digestion). The term also includes recombinant DNA that is part of a hybrid gene that encodes an additional polypeptide sequence.

[0068] As used herein, the term "NPP" or "ENPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0069] "Nucleic acid" refers to polynucleotides, including polyribonucleotides and polydeoxyribonucleotides. Nucleic acids according to the present invention may contain any polymer or oligomer of pyrimidine bases, preferably cytosine, thymine, and uracil, and purine bases, preferably guanine. See Albert L. Lehninger, Principles of Biochemistry, 793-800 (Worth Pub. 1982), incorporated herein in its entirety for all purposes. Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variant thereof, such as methylated, hydroxymethyl, or glycosylated forms of these bases. These polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from natural sources or produced artificially or synthetically. Furthermore, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states.

[0070] An "oligonucleotide" or "polynucleotide" is a nucleic acid at least 2 nucleotides in length, and in certain embodiments at least 8, 15, or 25 nucleotides in length, but may be up to 50, 100, 1000, or 5000 nucleotides in length, and may be a compound that specifically hybridizes to a polynucleotide. Polynucleotides include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or mimetics thereof, that may be isolated from natural sources, recombinantly produced, or artificially synthesized. A further example of a polynucleotide of the present invention may be a peptide nucleic acid (PNA) (see U.S. Pat. No. 6,156,501, incorporated herein by reference in its entirety). The present invention also encompasses situations where there is unusual base pairing, such as Hoogsteen base pairing, which has been identified in certain tRNA molecules and is postulated to exist in triple helices. "Polynucleotide" and "oligonucleotide" are used interchangeably herein. When a nucleotide sequence is represented herein as a DNA sequence (e.g., A, T, G, and C), it is understood that this also includes the corresponding RNA sequence (e.g., A, U, G, C) in which "U" is substituted for "T."

[0071] As used herein, the terms "patient," "individual," or "subject" refer to a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. In certain embodiments, the patient, individual, or subject is human.

[0072] As used herein, the term "prevent" or "prevention" means that if nothing has happened, there will be no onset of the disorder or disease, or if the disorder or disease has already occurred, there will be no further onset of the disorder or disease. It is also contemplated that some or all of the symptoms associated with the disorder or disease can be prevented.

[0073] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful in the present invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. Multiple techniques for administering compounds exist in the art, including, but not limited to, intravenous administration, oral administration, aerosol administration, inhalation administration, rectal administration, vaginal administration, transdermal administration, intranasal administration, buccal administration, sublingual administration, parenteral administration, intrathecal administration, intragastric administration, ocular administration, pulmonary administration, and topical administration.

[0074] As used herein, the term "pharmaceutically acceptable" refers to a relatively non-toxic material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound, i.e., the material can be administered to an individual without causing undesired biological effects or interacting adversely with any of the components of the composition in which it is contained.

[0075] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, involved in carrying or transporting a compound useful in the present invention into or to a patient so that the compound can perform its intended function. Typically, such constructs are carried or transported from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful in the present invention, and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcohol; phosphate buffer; and other non-toxic, compatible substances used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like, that are compatible with the activity of the compounds useful in the present invention and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. "Pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compounds useful in the present invention.Other additional ingredients that may be included in pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0076] As used herein, the term "pharmaceutically acceptable salts" refers to salts 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. Suitable pharmaceutically acceptable acid addition salts may be prepared from inorganic acids or organic acids. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid (including monohydrogen phosphate and dihydrogen phosphate). Suitable organic acids may be selected from the aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxyl-containing, and sulfonic acid classes of organic acids. Examples include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include, for example, metal salts including alkali metal salts, alkaline earth metal salts, and transition metal salts, such as calcium salts, magnesium salts, potassium salts, sodium salts, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. All of these salts can be prepared from the corresponding compound, for example, by reacting the compound with the appropriate acid or base.

[0077] As used herein, "polynucleotide" includes cDNA, RNA, DNA / RNA hybrids, antisense RNA, ribozymes, genomic DNA, synthetic, and mixed polymers, both sense and antisense strands, which may be chemically or biochemically modified to contain non-natural, or derivatized, synthetic, or semi-synthetic nucleotide bases. Alterations of wild-type or synthetic genes, including, but not limited to, deletion, insertion, or substitution of one or more nucleotides, or fusion with other polynucleotide sequences, are also contemplated.

[0078] As used herein, the term "polypeptide" refers to a polymeric compound composed of amino acid residues linked via peptide bonds, their related natural structural variants, and synthetic, non-natural analogs. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. As used herein, the term "protein" typically refers to a large polypeptide. As used herein, the term "peptide" typically refers to a short polypeptide. Conventional notation is used herein to represent polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus, and the right-hand end of a polypeptide sequence is the carboxyl-terminus.

[0079] As used herein, amino acids are represented by their full name, their corresponding three-letter code, or their corresponding one-letter code, as follows: aspartic acid (Asp / D); glutamic acid (Glu / E); lysine (Lys / K); arginine (Arg / R); histidine (His / H); tyrosine (Tyr / Y); cysteine ​​(Cys / C); asparagine (Asn / N); glutamine (Gln / Q); serine (Ser / S); threonine (Thr / T); glycine (Gly / G); alanine (Ala / A); valine (Val / V); leucine (Leu / L); isoleucine (Ile / I); methionine (Met / M); proline (Pro / P); phenylalanine (Phe / F); tryptophan (Trp / W).

[0080] As used herein, "sample" or "biological sample" refers to biological material isolated from a subject. A biological sample may contain any biological material suitable for detecting mRNA, polypeptides, or other markers of physiological or pathological processes in a subject, and may include fluids, tissues, cellular material, and / or non-cellular material obtained from an individual.

[0081] As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more other species. However, such cross-species reactivity does not, in and of itself, cause the antibody to be classified as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in and of itself, cause the antibody to be classified as specific. In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with another chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody may recognize and bind to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0082] As used herein, "substantially purified" refers to being essentially free of other components. For example, a substantially purified polypeptide is one that has been separated from other components with which it is normally associated in the natural state.

[0083] The terms "therapy" or "treating" as used herein are defined as the application or administration of a therapeutic agent, i.e., a compound useful in the present invention (alone or in combination with another pharmaceutical agent), to a patient having a disease or disorder, symptoms of a disease or disorder, or likelihood of developing a disease or disorder, or the application or administration of a therapeutic agent to isolated tissues or cell lines derived from a patient (e.g., in the case of diagnostic or ex vivo applications), for the purpose of curing, curing, alleviating, alleviating, altering, palliating, ameliorating, improving, or affecting the disease or disorder, symptoms of the disease or disorder, or the likelihood of developing the disease or disorder. Such therapy may be individualized and modified based on knowledge gained from the field of pharmacogenomics.

[0084] As used herein, the term "wild-type" refers to a gene or gene product isolated from a natural source. A wild-type gene is one that is most frequently observed in a population and is therefore arbitrarily designated as the "normal" or "wild" form of the gene. In contrast, the term "altered" or "mutant" refers to a gene or gene product that exhibits altered 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. They are identified by the fact that they have altered characteristics (including altered nucleic acid sequence) when compared to the wild-type gene or gene product.

[0085] Ranges: Throughout this application, various aspects of the invention may be presented in the form of ranges. It should be understood that the description in range form is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0086] explanation ENPP1 is the main source of extracellular PPi in the body. Despite the multiple genetic etiologies and multifactorial nature of GACI's onset, progression, and severity, our results demonstrate that disruption of extracellular purine metabolism by NPP1 explains the pathological sequelae and mortality associated with GACI, and that enzyme replacement therapy with ENPP1 is a tractable therapeutic approach. This was demonstrated using the ENPP1-asj mouse model of GACI fed an "acceleration diet."

[0087] Ectopic tissue calcification disorders range from extremely rare conditions, such as GACI, to near-universal conditions in the aging population, such as arteriosclerosis and osteoarthritis. The genetic etiology of human GACI suggests that lethal arterial calcification results from impaired extracellular purine metabolism due to either loss-of-function mutations in ENPP1 or an upstream reduction in nucleotide triphosphates metabolized by ENPP1 to extracellular PPi. As demonstrated herein, subcutaneous supplementation with untargeted ENPP1 or untargeted ENPP1-Fc increases extracellular PPi concentrations sufficiently to abolish mortality and cardiac and arterial calcification in an animal model of GACI. These results suggest that untargeted enzyme replacement therapy may be effective in treating GACI and other diseases that cause uncontrolled vascular calcification.

[0088] The present results are surprising in light of previous studies treating hereditary hypophosphatasia (HPP), which argued that a bone-targeting motif was required for efficacy. HPP is a rickets-like disease with hypo / absent bone mineralization, and treatment with recombinant TNAP evoked the need for bone targeting to achieve clinical efficacy (Millan, et al., 2008, J. Bone Mineral Res. 23:777-787; Whyte, et al., 2012, New Engl. J. Med. 366:904-913). Clinical trials attempting to treat HPP using serum enriched with untargeted TNAP failed (Whyte, et al., 1982, J. Pediatrics 101:379-386; Whyte, et al., 1984, J. Pediatrics 105:926-933; Weninger, et al., 1989, Acta Paediatrica Scandinavica Suppl. 360:154-160). Furthermore, literature at the time of the present invention indicated that untargeted NPP1 did not demonstrate efficacy in in vitro mineralization assays (WO 2012 / 125182 to Quinn et al., e.g., Figure 23 therein), thus demonstrating that bone targeting is essential for the in vivo biological activity of NPP1-containing biologics. However, in certain embodiments, the results of the present invention indicate that bone targeting is not required for therapeutic efficacy.

[0089] The arterial calcification of GACI may be accompanied by extravascular calcification in the skin and retina, typical of another rare disorder, PXE. PXE is closely related to GACI but, unlike GACI, causes ectopic elastic tissue calcification affecting the skin, eyes, and cardiovascular system. PXE is slow-onset and relatively common compared to GACI, with an incidence of 1 / 25,000–1 / 75,000. Clinical symptoms begin with the development of small yellowish papules on the skin, which coalesce into larger, leathery plaques, followed by retinal angioid streaks in the eyes, which can lead to hemorrhage, scarring, neovascularization, progressive vision loss, and blindness. The cardiovascular system may also be affected by progressive medium-sized arterial calcification, which can lead to hypertension, claudication, unrecurring intestinal bleeding, and (rarely) premature myocardial infarction. The genetic basis of PXE is a loss-of-function mutation in the abcc6 gene. This mutation impairs MRP6 protein function and reduces extracellular nucleotriphosphate (NTP) concentrations in vitro and in vivo, thereby reducing ENPP1 substrate concentrations and limiting extracellular production of PPi.

[0090] The NPP1-asj mouse model of GACI shares both the genetic etiology and pathological features of human GACI; however, these mice also develop periarticular calcification, which is not characteristic of GACI but is reminiscent of human diseases of disorganized periarticular calcification, such as osteoarthritis and ossification of the posterior longitudinal ligament (OPLL). Initially, mice carrying an ENPP1 missense mutation (V246D) were named "asj" mice, for "associated with stiffened joints," describing the progressive periarticular calcification that develops in the mouse forelimbs. While the murine ENPP1 mutation was used to model paraspinal calcification in ttw / ttw mice to gain insight into OPLL, the identification of the ENPP1 mutation in GACI led to a reassessment of the presence of vascular calcification in these animals. Uitto and colleagues demonstrated that NPP1-asj mice exhibit a high level of Ca +2 High in Mg +2We found that when ENPP1-containing mice were fed a specialized diet low in ENPP1, they reproduced many of the essential characteristics of human GACI. ENPP1 protein levels are inversely correlated with the severity of cartilage mineralization and osteoarthritis in humans, and ENPP1 gene variants account for a significant proportion of hand osteoarthritis in patient populations with a genetic predisposition to the disease. In certain embodiments, ENPP1 enzyme replacement therapy is a viable treatment strategy for several types of osteoarthritis caused by ENPP1 deficiency and / or reduced extracellular PPi concentrations. Such conditions include, but are not limited to, PXE, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, arteriosclerosis that occurs with aging, and calciphylaxis caused by end-stage renal disease.

[0091] composition In certain embodiments, the compositions of the present invention comprise a compound of formula (I): Protein-Z-Domain-XY (I) or a solvate or salt (e.g., a pharmaceutically acceptable salt) thereof, wherein in (I): The protein is at least one selected from the group consisting of NPP121 (SEQ ID NO: 15), NPP71 (SEQ ID NO: 17), NPP71 lacking NPP1 N-terminal GLK (SEQ ID NO: 19), and NPP51 (SEQ ID NO: 24); The domain is at least one selected from the group consisting of a human IgG Fc domain (Fc), a human serum albumin protein (ALB), and fragments thereof; X and Z are, independently, null or a polypeptide containing 1 to 20 amino acids; and Y is absent or TIFF2026015456000003.tif32156, wherein m is an integer of 1 to 15 and n is an integer of 1 to 10.

[0092] In certain embodiments, the compositions of the present invention comprise a compound of formula (II): Protein-Z-Domain-XY (II) or a pharmaceutical salt thereof, wherein in (II), The protein is at least one selected from the group consisting of NPP121 (SEQ ID NO: 15), NPP71 (SEQ ID NO: 17), NPP71 lacking NPP1 N-terminal GLK (SEQ ID NO: 19), and NPP51 (SEQ ID NO: 24); The domain is at least one selected from the group consisting of a human IgG Fc domain (Fc), a human serum albumin protein (ALB), and fragments thereof; X and Z are, independently, null or a polypeptide containing 1 to 20 amino acids; and Y is TIFF2026015456000004.tif33156, wherein m is an integer of 1 to 15 and n is an integer of 1 to 10.

[0093] In certain embodiments, the domain comprises a human IgG Fc domain or a fragment thereof. In other embodiments, the domain consists essentially of a human IgG Fc domain or a fragment thereof. In yet other embodiments, the domain consists of a human IgG Fc domain or a fragment thereof.

[0094] In certain embodiments, the domain comprises a human serum albumin protein or a fragment thereof. In other embodiments, the domain consists essentially of a human serum albumin protein or a fragment thereof. In yet other embodiments, the domain consists of a human serum albumin protein or a fragment thereof.

[0095] In certain embodiments, Y is a negatively charged bone-targeting sequence. In certain embodiments, Y is absent. In certain embodiments, Y is absent and the compound of Formula (I) or (II) lacks a negatively charged bone-targeting sequence. In yet other embodiments, the polyaspartic acid domain and SEQ ID NOs:4-14 are non-limiting examples of negatively charged bone-targeting sequences.

[0096] In certain embodiments, the protein has a mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 relative to SEQ ID NO: 1. In other embodiments, the protein or variant thereof is truncated to remove the nuclease domain. In yet other embodiments, the protein or variant thereof is truncated to remove the nuclease domain from about residue 524 to about residue 885 relative to SEQ ID NO:1, leaving only the catalytic domain from about residue 186 to about residue 586 relative to SEQ ID NO:1, which helps preserve the catalytic activity of the protein.

[0097] In certain embodiments, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:15)-Z-(Fc or a fragment thereof), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, relative to SEQ ID NO:1. In other embodiments, Z is a tripeptide. In yet other embodiments, Z is LIN. In yet another embodiment, in (I) or (II), the protein-Z-domain comprises SEQ ID NO:16, or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1.

[0098] In certain embodiments, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:17)-Z-(Fc or a fragment thereof), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, relative to SEQ ID NO:1. In other embodiments, Z is a tripeptide. In yet other embodiments, Z is LIN. In yet another embodiment, in (I) or (II), the protein-Z-domain comprises SEQ ID NO:18, or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1.

[0099] In certain embodiments, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:19)-Z-(Fc or a fragment thereof), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, relative to SEQ ID NO:1. In other embodiments, Z is a tripeptide. In yet other embodiments, Z is LIN. In yet another embodiment, in (I) or (II), the protein-Z-domain comprises SEQ ID NO:20, or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1.

[0100] In certain embodiments, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:24)-Z-(Fc or a fragment thereof), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, relative to SEQ ID NO:1. In other embodiments, Z is a tripeptide. In yet other embodiments, Z is LIN. In yet another embodiment, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:24)-Z-(SEQ ID NO:26), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1.

[0101] In certain embodiments, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:15)-Z-(ALB or a fragment thereof), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1. In other embodiments, Z is a tripeptide. In yet another embodiment, Z is one selected from the group consisting of SEQ ID NOs: 28 to 30. In yet another embodiment, in (I) or (II), the protein-Z-domain comprises SEQ ID NO: 21 or a variant thereof, which, compared to SEQ ID NO: 1, comprises at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536.

[0102] In certain embodiments, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:17)-Z-(ALB or a fragment thereof), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1. In other embodiments, Z is a tripeptide. In still other embodiments, Z is one selected from the group consisting of SEQ ID NOs: 28-30. In yet another embodiment, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:17)-Z-(SEQ ID NO:27), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1, and Z is one selected from the group consisting of SEQ ID NOs:28 to 30.

[0103] In certain embodiments, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:19)-Z-(ALB or a fragment thereof), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1. In other embodiments, Z is a tripeptide. In yet another embodiment, Z is one selected from the group consisting of SEQ ID NOs: 28 to 30. In yet another embodiment, in (I) or (II), the protein-Z-domain comprises SEQ ID NO: 22 or a variant thereof, which, compared to SEQ ID NO: 1, comprises at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536.

[0104] In certain embodiments, in (I) or (II), the protein-Z-domain comprises (SEQ ID NO:24)-Z-(ALB or a fragment thereof), or a variant thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536, compared to SEQ ID NO:1. In other embodiments, Z is a tripeptide. In yet another embodiment, Z is one selected from the group consisting of SEQ ID NOs: 28 to 30. In yet another embodiment, in (I) or (II), the protein-Z-domain comprises SEQ ID NO: 25 or a variant thereof, which, compared to SEQ ID NO: 1, comprises at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536.

[0105] In certain embodiments, X and Z are independently absent or a polypeptide comprising 1 to 18 amino acids. In other embodiments, X and Z are independently absent or a polypeptide comprising 1 to 16 amino acids. In still other embodiments, X and Z are independently absent or a polypeptide comprising 1 to 14 amino acids. In still other embodiments, X and Z are independently absent or a polypeptide comprising 1 to 12 amino acids. In still other embodiments, X and Z are independently absent or a polypeptide comprising 1 to 10 amino acids. In still other embodiments, X and Z are independently absent or a polypeptide comprising 1 to 8 amino acids. In still other embodiments, X and Z are independently absent or a polypeptide comprising 1 to 6 amino acids. In still other embodiments, X and Z are independently absent or a polypeptide comprising 1 to 5 amino acids. In still other embodiments, X and Z are independently absent or a polypeptide comprising 1 to 4 amino acids. In yet other embodiments, X and Z are independently absent or a polypeptide comprising 1-3 amino acids. In yet other embodiments, X and Z are independently absent or a polypeptide comprising 1-2 amino acids. In yet other embodiments, X and Z are independently absent or a single amino acid.

[0106] In certain embodiments, m is 1. In other embodiments, m is 2. In still other embodiments, m is 3. In still other embodiments, m is 4. In still other embodiments, m is 5. In still other embodiments, m is 6. In still other embodiments, m is 7. In still other embodiments, m is 8. In still other embodiments, m is 9. In still other embodiments, m is 10. In still other embodiments, m is 11. In still other embodiments, m is 12. In still other embodiments, m is 13. In still other embodiments, m is 14. In still other embodiments, m is 15. In still other embodiments, each occurrence of m is independently selected from 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 11 to 15, 12 to 15, 13 to 15, 14 to 15, 1 to 14, 2 to 14, 3 to 14, 4 to 14, 5 to 14, 6 to 14, 7 to 14, 8 to 15, ~14, 9~14, 10~14, 11~14, 12~14, 13~14, 1~13, 2~13, 3~13, 4~13, 5~13, 6~13, 7~13, 8~13, 9~13, 10~13, 11~13, 12~13, 1~12, 2~12, 3~12, 4~12, 5~12, 6~12, 7~12, 8~12, 9 ~12, 10~12, 11~12, 1~11, 2~11, 3~11, 4~11, 5~11, 6~11, 7~11, 8~11, 9~11, 10~11, 1~10, 2~10, 3~10, 4~10, 5~10, 6~10, 7~10, 8~10, 9~10, 1~9, 2~9, 3~9, 4~9, 5~9, 6~9, 7 is selected from the group consisting of integers of up to 9, 8 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 1 to 4, 2 to 4, 3 to 4, 1 to 3, 2 to 3, and 1 to 2.

[0107] In certain embodiments, n is 1. In other embodiments, n is 2. In still other embodiments, n is 3. In still other embodiments, n is 4. In still other embodiments, n is 5. In still other embodiments, n is 6. In still other embodiments, n is 7. In still other embodiments, n is 8. In still other embodiments, n is 9. In still other embodiments, n is 10. In still other embodiments, each occurrence of n is independently selected from the group consisting of integers from 1 to 10, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 1 to 9, from 2 to 9, from 3 to 9, from 4 to 9, from 5 to 9, from 6 to 9, from 7 to 9, from 8 to 9, from 1 to 8, from 2 to 8, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 1 to 7, from 2 to 7, from 3 to 7, from 4 to 7, from 5 to 7, from 6 to 7, from 1 to 6, from 2 to 6, from 3 to 6, from 4 to 6, from 5 to 6, from 1 to 5, from 2 to 5, from 3 to 5, from 4 to 5, from 1 to 4, from 2 to 4, from 3 to 4, from 1 to 3, from 2 to 3, and 1 to 2.

[0108] In certain embodiments, the protein or variant thereof is modified with a segment containing a furin cleavage site in the extracellular domain of NPP2 compared to SEQ ID NO: 1. In other embodiments, the protein or variant thereof is not modified with a segment containing a furin cleavage site in the extracellular domain of NPP2 compared to SEQ ID NO: 1.

[0109] In certain embodiments, the protein or variant thereof is modified with a segment containing a signal peptidase cleavage site in the extracellular region of NPP2 compared to SEQ ID NO: 1. In other embodiments, the protein or variant thereof is not modified with a segment containing a signal peptidase cleavage site in the extracellular region of NPP2 compared to SEQ ID NO: 1.

[0110] In certain embodiments, the compound of formula (I) or (II) is soluble. In other embodiments, the compound of formula (I) or (II) is a recombinant polypeptide. In still other embodiments, the compound of formula (I) or (II) comprises an NPP1 polypeptide or a variant thereof lacking the NPP1 transmembrane domain. In still other embodiments, the compound of formula (I) or (II) comprises an NPP1 polypeptide or a variant thereof in which the NPP1 transmembrane domain or variant thereof has been removed (and / or truncated) and replaced with the transmembrane domain of another polypeptide (e.g., as a non-limiting example, NPP2).

[0111] In certain embodiments, the compound of formula (I) or (II) comprises an NPP1 polypeptide or a variant thereof, which further comprises multiple transmembrane domains.

[0112] In certain embodiments, the C-terminus of NPP1 is fused to the Fc domain of human immunoglobulin 1 (IgG1).

[0113] In certain embodiments, the C-terminus of NPP1 is fused to human serum albumin.

[0114] In certain embodiments, a fragment and / or variant of NPP1 is fused to human serum albumin or a variant and / or fragment thereof. Human serum albumin may be conjugated to the NPP1 protein via a chemical linker, including but not limited to, a natural or engineered disulfide bond, or by genetic fusion with NPP1 or a fragment and / or variant thereof.

[0115] In certain embodiments, a compound of formula (I) or (II) comprises an NPP1 polypeptide or a variant thereof that comprises the transmembrane domain of NPP1 and another polypeptide (e.g., as a non-limiting example, NPP2).

[0116] In certain embodiments, the compound of formula (I) has a sequence selected from the group consisting of SEQ ID NOs: 21, 22, and 25.

[0117] In certain embodiments, the compound of formula (I) has a sequence selected from the group consisting of SEQ ID NO:21, 22, 25, (SEQ ID NO:17)-Z-(SEQ ID NO:27).

[0118] In certain embodiments, the compound of formula (I) has a sequence selected from the group consisting of SEQ ID NO:16, 18, 20, and (SEQ ID NO:24)-Z-(SEQ ID NO:26).

[0119] In certain embodiments, the compounds of the invention have multiple transmembrane domains. In other embodiments, the compounds of the invention are further pegylated. In yet other embodiments, the compounds of the invention have multiple transmembrane domains and are further pegylated.

[0120] In certain embodiments, the compound of formula (I) or (II) has a pH of about 3.4 (±0.4) s -1 enzyme -1 Greater than or equal to k cat has a value k cat is determined by measuring the rate of ATP hydrolysis of the compound.

[0121] In certain embodiments, compounds of Formula (I) or (II) have a K M has a value of K M is determined by measuring the rate of ATP hydrolysis of the compound.

[0122] In certain embodiments, the compounds of formula (I) or (II) are formulated as liquid formulations.

[0123] Additionally, the present invention provides a dry product form of a pharmaceutical composition comprising a therapeutic amount of a compound of formula (I) or (II), which can be reconstituted into a solution of the compound in liquid form.

[0124] method The present invention provides methods for treating or preventing disorders and diseases in a subject in which increased activity or levels of an NPP1 polypeptide, a fragment, derivative, variant, or variant fragment thereof is desired. In certain embodiments, the subject is administered a therapeutically effective amount of at least one compound of the present invention.

[0125] The present invention further provides a method for treating or preventing a disease or disorder associated with pathological calcification or pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of formula (I) or (II), wherein the disease includes GACI, IIAC, OPLL, hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

[0126] The present invention further provides a method for treating or preventing a disease or disorder associated with pathologic calcification or pathologic ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of Formula (I) or (II), wherein the disease comprises PXE, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, arteriosclerosis that occurs with aging, or calciphylaxis resulting from end-stage renal disease.

[0127] Additionally, the present invention provides methods for reducing or preventing cardiac and / or arterial calcification in infants with generalized arterial calcification of infancy (GACI). In certain embodiments, the methods comprise administering to the infant a therapeutically effective amount of a compound comprising (or consisting of) an ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide comprising (or fused to) an IgG Fc domain, wherein the compound lacks a polyaspartic acid domain, and wherein administration of the compound increases extracellular pyrophosphate (PPi) levels, thereby reducing or preventing cardiac and / or arterial calcification in the infant.

[0128] Additionally, the present invention provides methods for reducing or preventing cardiac and / or arterial calcification in infants with generalized arterial calcification of infancy (GACI). In certain embodiments, the methods comprise administering to the infant a therapeutically effective amount of a compound comprising (or consisting of) an ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide comprising (or fused to) a human serum albumin domain or a fragment thereof, wherein the compound lacks a polyaspartic acid domain, and wherein administration increases extracellular pyrophosphate (PPi) levels, thereby reducing or preventing cardiac and / or arterial calcification in the infant.

[0129] In certain embodiments, the disorder or disease comprises at least one selected from the group consisting of GACI, IIAC, OPLL, hypophosphatemic rickets, osteoarthritis, progeria, and calcification of atherosclerotic plaques. In other embodiments, the disorder or disease comprises at least one selected from the group consisting of PXE, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, arterial stiffness that occurs with aging, progeria, and calciphylaxis due to end-stage renal disease.

[0130] In certain embodiments, the compound is administered to the subject for short or long periods of time. In other embodiments, the compound is administered to the subject topically, locally, or systemically. In yet other embodiments, administration is subcutaneous. In yet other embodiments, the subject is a mammal. In yet other embodiments, the mammal is a human.

[0131] In certain embodiments, compounds of Formula (I) or (II), fragments or variants thereof have reduced Ap3A hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or fragment thereof. In other embodiments, compounds of Formula (I) or (II), fragments or variants thereof have substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or fragment thereof. In still other embodiments, compounds of Formula (I) or (II), fragments or variants thereof have reduced Ap3A hydrolysis activity and substantially the same ATP hydrolysis activity compared to the corresponding wild-type NPP1 polypeptide or fragment thereof.

[0132] In certain embodiments, the NPP1 polypeptide comprises a cleavage product of a precursor NPP1 polypeptide comprising the NPP2 transmembrane domain.

[0133] In certain embodiments, the NPP2 transmembrane domain comprises residues 12-30 of NCBI Accession No. NP_001124335 (SEQ ID NO:2), which is Equivalent to TIFF2026015456000005.tif4128.

[0134] In certain embodiments, the therapeutically effective amount administered comprises about 3-15 mg / kg of NPP1-Fc polypeptide once daily.

[0135] In certain embodiments, administration reduces the infant's extracellular pyrophosphate concentration to levels within the range found in infants not suffering from GACI, hi certain embodiments, the infant exhibits and / or has been diagnosed with "failure to thrive" prior to administration.

[0136] Those skilled in the art will understand, based on the disclosure provided herein, that the present invention is useful in subjects who have been or will be treated in whole (e.g., systemically) or in part (e.g., locally, tissue, organ) for pathological calcification or pathological ossification. In certain aspects, the present invention is useful in the treatment or prevention of pathological calcification or pathological ossification. Those skilled in the art will appreciate, based on the teachings provided herein, that diseases and disorders treatable by the compositions and methods described herein include any disease or disorder in which reduced calcification or ossification promotes a positive therapeutic outcome.

[0137] Armed with this disclosure, including the methods detailed herein, those skilled in the art will recognize that the present invention is not limited to the treatment of diseases or disorders once established. In particular, symptoms of the disease or disorder need not be manifest to the extent that they harm the subject. Indeed, the disease or disorder need not be detected in the subject before a therapeutic agent is administered. That is, noticeable symptoms of the disease or disorder need not occur before the present invention may provide benefit. Thus, as discussed elsewhere herein, a compound of formula (I) or (II) or a variant thereof can be administered to a subject before the disease or disorder develops, thereby preventing the disease or disorder from developing, and therefore the present invention includes methods for preventing diseases and disorders in a subject, as more fully described herein.

[0138] Given the disclosure herein, one of skill in the art will recognize that preventing a disease or disorder in a subject includes administering a compound of Formula (I) or (II) or a variant thereof to the subject as a prophylactic measure against the disease or disorder.

[0139] The present invention encompasses the administration of a compound of Formula (I) or (II) or a variant thereof to practice the methods of the present invention. Based on the disclosure provided herein, one of ordinary skill in the art will understand how to formulate and administer a compound of Formula (I) or (II) or a variant thereof to a subject. However, the present invention is not limited to any particular method of administration or treatment regimen. This is particularly true as one of ordinary skill in the art of pharmacology will recognize that, given the disclosure provided herein, including reduction to practice with art-recognized models of pathological calcification or pathological ossification, methods for administering the compounds of the present invention can be determined by one of ordinary skill in the art of pharmacology.

[0140] Pharmaceutical Compositions and Formulations The present invention provides pharmaceutical compositions comprising compounds of formula (I) or (II) that are present in the methods of the invention.

[0141] Such pharmaceutical compositions are in a form suitable for administration to subject.Or, pharmaceutical compositions can further comprise one or more pharmaceutically acceptable carriers, one or more additional components, or any combination thereof.As is well known in the art, various components of pharmaceutical compositions can be present in the form of physiologically acceptable salts, for example, in combination with physiologically acceptable cations or anions.

[0142] In embodiments, pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day, hi other embodiments, pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0143] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients present in the pharmaceutical compositions of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the compositions may contain from about 0.1% to about 100% (w / w) active ingredient.

[0144] Pharmaceutical compositions useful in the methods of the present invention may be developed for inhaled, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intravenous, or other routes of administration, as appropriate. Other contemplated formulations include injected nanoparticles, liposomal preparations, resealed red blood cells containing the active ingredient, and immune-based formulations. The route of administration will be readily apparent to one of skill in the art and will depend on 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, etc.

[0145] The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology, or any method hereafter developed in the art of pharmacology. Generally, such preparatory methods include the step of bringing into association the active ingredient with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into a desired single-dose or multi-dose unit.

[0146] As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient administered to a subject or a convenient fraction of such a dosage, such as 1 / 2 or 1 / 3 of such a dosage. The unit dosage form may be a unit dosage form 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.

[0147] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and veterinary pharmacologists of ordinary skill can design and implement such modifications with no more than routine experimentation, if any. Subjects to which the pharmaceutical compositions of the present invention are intended for administration include, but are not limited to, humans and other primates, and commercially relevant mammals, including, for example, cattle, pigs, horses, sheep, cats, and dogs.

[0148] In certain embodiments, the composition is formulated with one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an active agent and a pharmaceutically acceptable carrier. 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.

[0149] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. The activity of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are often used in the compositions. Prolonged absorption of injectable compositions can be achieved by including in the composition an absorption-delaying agent, for example, aluminum monostearate and gelatin.

[0150] The formulations may be used in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable administration method known to those skilled in the art. Pharmaceutical preparations may be sterilized and, if desired, may be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances, and / or aromatic substances. These may, if desired, be combined with other active agents, such as other analgesics.

[0151] " Additional components " used herein include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions, such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifiers, demulcents; buffer solutions; salts; thickeners; bulking agents; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric materials or hydrophobic materials.Other " additional components " that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

[0152] The compositions of the present invention may contain about 0.005% to 2.0% preservative by weight of the total composition. Preservatives are used to prevent deterioration when exposed to environmental contaminants. Examples of preservatives useful in accordance with the present invention include, but are not limited to, preservatives selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. A specific preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0153] The compositions may also include antioxidants and chelating agents that inhibit degradation of the compounds. Exemplary antioxidants for some compounds are BHT, BHA, α-tocopherol, and ascorbic acid, in an exemplary range of about 0.01% to 0.3% by weight, relative to the total weight of the composition, e.g., BHT in the range of 0.03% to 0.1% by weight. Chelating agents may be present in an amount of 0.01% to 0.5% by weight, relative to the total weight of the composition. Exemplary chelating agents include edetate (e.g., edetate disodium) and citric acid, in an exemplary range of about 0.01% to 0.20% by weight, e.g., in the range of 0.02% to 0.10% by weight, relative to the total weight of the composition. Chelating agents are useful for chelating metal ions present in the composition that may be detrimental to the shelf life of the formulation. BHT and edetate disodium are exemplary antioxidants and chelating agents, respectively, for some compounds, although other suitable equivalent antioxidants and chelating agents may be substituted as known to those skilled in the art.

[0154] Liquid suspensions can be prepared by conventional methods of suspending active ingredients in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further contain one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners. Oily suspensions may further contain thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum arabic, and cellulose derivatives (e.g., sodium carboxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose). Known dispersing or wetting agents include, but are not limited to, natural phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, long-chain aliphatic alcohols, partial esters derived from fatty acids and hexitols, or partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and gum acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl parahydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0155] Liquid solutions of active ingredients dissolved in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, with the main difference being that the active ingredient is dissolved rather than suspended in the solvent.As used herein, "oily" liquids are liquids that contain carbon-containing liquid molecules and exhibit less polarity than water.The liquid solutions of the pharmaceutical compositions of the present invention may contain each of the components described for liquid suspensions, and it is understood that the suspending agent does not necessarily help dissolve the active ingredient in the solvent.Aqueous solvents include, for example, water and isotonic saline.Oil solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0156] The powder and granular preparations of the pharmaceutical composition of the present invention can be prepared by known methods.Such preparations can be directly administered to a subject, or can be used, for example, to form tablets, to fill capsules, or to prepare aqueous or oily suspensions or solutions by adding aqueous or oily vehicles.These preparations can further comprise one or more of dispersing or wetting agents, suspending agents, and preservatives, respectively.These preparations can also comprise additional excipients, such as bulking agents and sweeteners, flavoring agents, or coloring agents.

[0157] The pharmaceutical compositions of the present invention may also be prepared, packaged, or sold in the form of an oil-in-water emulsion or a water-in-oil emulsion.The oil phase may be vegetable oil, such as olive oil or peanut oil, mineral oil, such as liquid paraffin, or a combination thereof.Such compositions may further contain one or more emulsifiers, such as natural gums, such as gum arabic or gum tragacanth, natural phosphatides, such as soybean phosphatides or lecithin phosphatides, esters or partial esters derived from the combination of fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.These emulsions may also contain additional ingredients, including, for example, sweeteners or flavoring agents.

[0158] Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods of attaching or bonding the chemical composition to a surface, methods of incorporating the chemical composition into the structure of the material during its synthesis (i.e., with, for example, physiologically degradable materials), and methods of imbibing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

[0159] Administration / Dosage Dosage regimens can affect what constitutes an effective amount. For example, several divided doses as well as staggered doses may be administered daily or continuously. Alternatively, the dose may be a continuous infusion or a bolus injection. Furthermore, the dosage of the therapeutic formulation may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0160] The compositions of the present invention can be administered to a patient, e.g., a mammal, e.g., a human, using known procedures at dosages and for periods of time effective to treat the patient's disease or disorder. The effective amount of a therapeutic compound required to achieve a therapeutic effect may vary depending on factors such as the activity of the particular compound used, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, or materials used in combination with the compound, the nature of the disease or disorder, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, as well as similar factors well known in the medical field. Dosage regimens may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily. Alternatively, the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dosage range for a therapeutic compound of the present invention is about 0.01 to 50 mg / kg body weight / day. One of ordinary skill in the art would be able to study the relevant factors and make a determination regarding the effective amount of a therapeutic compound without undue experimentation.

[0161] The compound may be administered to animals as frequently as several times a day, or 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 few months or once a year, or less frequently. It is understood that the amount of compound administered per day may be administered, in non-limiting examples, every day, every other day, every two days, every three days, every four days, or every five days. For example, when administered every other day, a dose of 5 mg per day may be administered starting on Monday, a first dose of 5 mg per day thereafter may be administered on Wednesday, and a second dose of 5 mg per day thereafter may be administered on Friday. The frequency of administration is readily apparent to those skilled in the art and depends on any number of factors, including, but not limited to, the type and severity of the disease being treated and the type and age of the animal.

[0162] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient.

[0163] A medical doctor, e.g., a physician or veterinarian, having ordinary skill in the art can easily determine the effective amount of pharmaceutical composition required and prescribe the effective amount of pharmaceutical composition. For example, the physician or veterinarian could start dosing the compound of the present invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0164] In certain embodiments, it is particularly advantageous to formulate the compounds in unit dosage forms for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to a physically discrete unit suitable for unitary administration to the patient to be treated. Each unit contains a predetermined amount of therapeutic compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical vehicle. The unit dosage forms of the present invention are determined by and directly influenced by (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the constraints inherent in the art of compounding / prescribing such therapeutic compounds to treat a disease or disorder in a patient.

[0165] In certain embodiments, the compositions of the present invention are administered to patients in dosages of 1 to 5 or more times daily. In other embodiments, the compositions of the present invention are administered to patients in dosage ranges including, but not limited to, once daily, once every two days, once every three days, to once weekly and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present invention will vary from subject to subject, depending on many factors, including, but not limited to, age, disease or disorder being treated, gender, general health, and other factors. Therefore, the present invention should not be construed as limited to any particular dosing regimen, and the exact dosage and composition administered to any patient will be determined by the attending physician, taking into account all other factors relevant to the patient.

[0166] The compound of the present invention for administration may be administered in the following dosages: about 1 μg to about 7,500 mg, about 20 μg to about 7,000 mg, about 40 μg to about 6,500 mg, about 80 μg to about 6,000 mg, about 100 μg to about 5,500 mg, about 200 μg to about 5,000 mg, about 400 μg to about 4,000 mg, about 800 μg to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg. mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments therebetween.

[0167] In some embodiments, the dosage of the compound of the present invention is from about 0.5 μg to about 5,000 mg. In some embodiments, the dosage of the compound of the present invention used in the compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of a second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0168] In certain aspects, the present invention relates to packaged pharmaceutical compositions comprising a container for holding a therapeutically effective amount of a compound of the present invention, alone or in combination with a second pharmaceutical agent, and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

[0169] The term "container" includes any receptacle for housing a pharmaceutical composition. For example, in certain embodiments, the container is a packaging container that holds the pharmaceutical composition. In other embodiments, the container is not a packaging container that holds the pharmaceutical composition. That is, the container is a receptacle, such as a box or vial, that holds a packaged or unpackaged pharmaceutical composition and instructions for using the pharmaceutical composition. Furthermore, packaging techniques are well known in the art. It should be understood that instructions for using the pharmaceutical composition may be included on the surface of the packaging container that holds the pharmaceutical composition. Thus, the instructions establish an enhanced functional relationship with the packaged product. However, it should be understood that the instructions may also include information regarding the ability of the compound to perform its intended function, for example, treating, preventing, or alleviating a disease or disorder in a patient.

[0170] Administration route Routes of administration for any of the compositions of the present invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal (intranasal), and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0171] Suitable 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, etc. It should be understood that the formulations and compositions useful in the present invention are not limited to the specific formulations and compositions described herein.

[0172] Oral administration For oral application, tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel caps are particularly suitable.Other formulations suitable for oral administration include, but are not limited to, powder or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, oral rinses, or emulsions.Compositions intended for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for tablet manufacture.Such excipients include, for example, inert diluents, such as lactose; granulating and disintegrating agents, such as cornstarch; binders, such as starch; and lubricants, such as magnesium stearate.

[0173] Tablets may be uncoated, or may be coated using known methods to delay disintegration in the subject's gastrointestinal tract, thereby allowing the sustained release and absorption of active ingredients.For example, materials such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.Furthermore, for example, tablets may be coated using the methods described in U.S. Patent Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets.Tablets may further comprise sweeteners, flavoring agents, coloring agents, preservatives, or any combination thereof to provide pharmaceutically acceptable and palatable preparations.

[0174] Hard capsules containing the active ingredient may be made using a physiologically degradable composition such as gelatin. Such hard capsules contain the active ingredient and may further contain additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

[0175] Soft gelatin capsules containing the active ingredient may be made using a physiologically degradable composition such as gelatin, and such soft capsules contain the active ingredient, which may be mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0176] For oral administration, the compounds of the present invention may be in the form of tablets or capsules prepared by conventional means using pharmaceutically acceptable excipients, such as binders, fillers, lubricants, disintegrants, or wetting agents. If desired, tablets may be coated using appropriate methods and coating materials, such as the OPADRY™ film coating system (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type, and OPADRY™ White, 32K18400) available from Colorcon, West Point, Pa.

[0177] Liquid preparations for oral administration may be in the form of a solution, syrup, or suspension. Liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl parahydroxybenzoate or sorbic acid). Liquid formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged, or sold in liquid form or in the form of a dry product intended to be reconstituted with water or another suitable vehicle before use.

[0178] Tablets containing an active ingredient may be made, for example, by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by mixing the active ingredient in a free-flowing form, such as a powder or granular preparation, with one or more binders, lubricants, excipients, surfactants, and dispersants, and compressing the mixture in a suitable machine. Molded tablets may be made by molding a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture in a suitable machine. Pharmaceutically acceptable excipients used in tablet manufacture include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, and lubricants. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, gum acacia, pre-gelatinized corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0179] Granulation methods are well known in the pharmaceutical field for modifying starting powders of active ingredients or other particulate materials.This powder is typically mixed with a binder material to form larger, long-lasting, freely flowing agglomerates or granules, called "granulations".For example, the "wet" granulation process using a solvent is generally characterized in that the powder is mixed with a binder material, moistened with water or an organic solvent under conditions that form a moist granular mass, and then the solvent must be evaporated from the moist granular mass.

[0180] Generally, melt granulation essentially involves using a material that is solid or semi-solid at room temperature (i.e., has a relatively low softening point or melting point range) to facilitate the granulation of powdered or other materials in the absence of added water or other liquid solvents. When the low-melting solid is heated to a temperature in the melting point range, it liquefies and acts as a binder or granulation medium. The liquefied solid remains and spreads over the surface of the powdered material it comes into contact with, and upon cooling, forms a solid granular mass that binds the initial materials together. The resulting melt granules can then be fed into a tablet press or placed in a capsule to prepare an oral dosage form. Melt granulation improves the dissolution rate and bioavailability of the active ingredient (i.e., drug) by forming a solid dispersion or solid solution.

[0181] U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules with improved flow properties. The granules are obtained when the wax is mixed in a molten state with certain flow-improving additives, followed by cooling and granulation of the mixture. In certain embodiments, the wax itself melts only when the wax and additive are melted and combined; in other cases, both the wax and additive melt.

[0182] The present invention also includes multi-layer tablets comprising a layer that provides delayed release of one or more compounds useful in the methods of the invention and an additional layer that provides immediate release of one or more compounds useful in the methods of the invention. A wax / pH-sensitive polymer mixture can be used to entrap the active ingredient to provide a stomach-insoluble composition that ensures delayed release of the active ingredient.

[0183] Parenteral administration As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physically creating a hole in the tissue of a subject and administering the pharmaceutical composition through the hole in the tissue. Thus, parenteral administration includes, but is not limited to, administering a pharmaceutical composition by injection of the pharmaceutical composition, applying the pharmaceutical composition through a surgical incision, applying the pharmaceutical composition through a non-surgical wound that penetrates the tissue, etc. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and kidney dialysis infusion.

[0184] Pharmaceutical compositions suitable for parenteral administration include the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry form (i.e., powder or granules) for reconstitution with an appropriate vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0185] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations may be prepared using a non-toxic, parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides. Other useful parenterally administrable formulations include those in which the active ingredient is in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0186] Further dosage forms Additional dosage forms of the present invention include those described in U.S. Patent Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of the present invention also include those described in U.S. Patent Application Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of the present invention also include those described in PCT Application Nos. WO03 / 35041, WO03 / 35040, WO03 / 35029, WO03 / 35177, WO03 / 35039, WO02 / 96404, WO02 / 32416, WO01 / 97783, WO01 / 56544, WO01 / 32217, WO98 / 55107, WO98 / 11879, WO97 / 47285, WO93 / 18755, and WO90 / 11757.

[0187] Sustained-Release Formulations and Drug Delivery Systems Slow-release or sustained-release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques. In some cases, the dosage form used can be provided as a slow or sustained release formulation of one or more active ingredients therein, for example, by using different ratios of hydropropylmethyl cellulose, other polymer matrices, gels, dialysis membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres, or a combination thereof, to provide a desired release profile. Suitable sustained-release formulations known to those skilled in the art, including the sustained-release formulations described herein, can be easily selected for use with the pharmaceutical compositions of the present invention. Therefore, the present invention includes single-unit dosage forms suitable for oral administration, such as tablets, capsules, gel caps, and caplets, that are adapted for sustained release.

[0188] Most sustained-release pharmaceutical products share a common goal of improving drug therapy over that achieved by their non-controlled counterparts.Ideally, the use of optimally designed sustained-release preparations in medical treatment is characterized by the use of a minimum amount of active pharmaceutical ingredient to cure or manage a condition in the shortest time.The advantages of sustained-release preparations include a prolonged period of drug activity, less frequent administration, and high patient compliance.In addition, sustained-release preparations can be used to affect the onset time of action or other characteristics, such as the blood concentration of the drug, and therefore can affect the occurrence of side effects.

[0189] Most sustained-release formulations are designed to initially release an amount of drug that rapidly produces the desired therapeutic effect, and then gradually and continuously release another amount of drug that maintains this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

[0190] The sustained release of the active ingredient can be stimulated by various inducers, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "sustained release ingredient" is defined herein in the context of the present invention as a compound that facilitates the sustained release of the active ingredient, including, but not limited to, a polymer, a polymer matrix, a gel, a dialysis membrane, a liposome, or a microsphere, or a combination thereof.

[0191] In certain embodiments, the formulations of the present invention may be, but are not limited to, short-acting formulations, rapid-offset formulations, and sustained release formulations, including sustained release formulations, delayed release formulations, and pulsatile release formulations.

[0192] The term "sustained release" is used in its conventional sense to refer to a drug formulation that gradually releases a drug over an extended period of time, resulting in a substantially constant blood concentration of the drug over an extended period of time, although this is not necessarily the case. The period may be as long as one month or longer, and should result in a longer release than the equivalent amount of drug administered in a bolus form. In the case of sustained release, the compound may be formulated with a suitable polymeric or hydrophobic material that imparts sustained release properties to the compound. Thus, the compound for use in the method of the present invention may be administered in the form of microparticles, for example, by injection, or in the form of a wafer or disk by implantation. In certain embodiments of the present invention, the compound of the present invention is administered to a patient using a sustained release formulation, alone or in combination with another pharmaceutical agent.

[0193] The term delayed release is used herein in its conventional sense to refer to a drug formulation that first releases drug after administration after some delay, which may include, but is not necessarily, from about 10 minutes to about 12 hours. The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that releases drug after administration to produce a pulsatile plasma profile of drug. The term immediate release is used herein in its conventional sense to refer to a drug formulation that releases drug immediately after administration.

[0194] As used herein, short-term refers to any period of time up to or 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 the drug is administered.

[0195] As used herein, rapid off-action refers to any period of time up to or 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 and partial increments thereof, after the drug is administered.

[0196] Those skilled in the art will recognize, or can recognize using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the accompanying claims. For example, variations in reaction conditions, including but not limited to reaction times, reaction sizes / volumes, and experimental reagents, e.g., solvents, catalysts, pressure, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxidizing agents, according to art-recognized alternatives and using no more than routine experimentation, should be understood to be within the scope of this application.

[0197] Whenever values ​​and ranges are provided herein, it should be understood that all values ​​and ranges encompassed by these values ​​and ranges are intended to be included within the scope of the present invention. Furthermore, all values ​​falling within these ranges, as well as the upper or lower limits of a range of values, are also included herein.

[0198] The following examples further illustrate aspects of the present invention, but do not limit the teachings or disclosure of the present invention presented herein. [Example]

[0199] The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention is not limited to these examples, but includes all variations that become apparent as a result of the teachings provided herein.

[0200] Methods and Materials: ENPP1-asj GACI Mouse Model: To generate ENPP1-WT and ENPP1-asj / asj sib pairs exposed in utero to an acceleration diet (TD00.442, Harlan Laboratories, Madison, WI), heterozygous ENPP1-asj / + breeding pairs were maintained on the acceleration diet throughout the experiment. Litters were genotyped on day 8 and weaned on day 21. After weaning, sib pairs were divided into experimental cohorts, and all experimental animals were maintained on the acceleration diet until the end of the study.

[0201] ENPP1-Fc design: Modified human NPP1 and modified mouse NPP1 (human: NCBI accession NP_006199; mouse: NCBI accession NP_03839), which were engineered to express soluble recombinant proteins, were fused with IgG1 by subcloning to obtain the pFUSE-hIgG1-Fc1 or pFUSE-mIgG1-Fc1 plasmids (InvivoGen, San Diego CA), respectively.

[0202] Protein production: Shake flask: Stable transfection of ENPP1-Fc was established in HEK293 cells under zeocin selection, and adherent HEK293 cells were adapted to suspension growth. Adapted cells were used to inoculate liquid cultures in FreeStyle medium (Gibco #12338-018) in shake flasks at 37°C and 5% CO2, with agitation at 120 rpm and high humidity. Cultures were expanded stepwise to the desired target volume and then maintained for an additional 12 days to allow extracellular protein accumulation. During the maintenance phase, cultures were supplemented with CD EfficientFeed C AGT (Gibco #A13275-05) to enhance protein production.

[0203] Bioreactor: Cells were grown in a 10-liter bioreactor equipped with dissolved oxygen and pH control devices. Dissolved oxygen was maintained at 40% air saturation by supplying the culture with an air and oxygen mixture at no more than 3 liters / min with an agitation rate of 80 RPM. pH was controlled at 7.4 by sparging with CO2 if the pH exceeded 7.4. Culture growth was followed by measurements of cell count, cell viability, glucose concentration, and lactate concentration. The final yield for both production methods was approximately 5 mg of purified ENPP1-Fc per liter of culture.

[0204] Protein purification: The liquid culture was centrifuged at 4300 xg for 15 minutes, and the supernatant was filtered through a 0.2 μm membrane and resuspended in Pellicon® 3 0.11 m 2Tangential flow concentration was performed using Ultracell® 30 kD cassettes (Millipore, Billerica MA). The concentrated supernatant was then purified by a combination of chromatographic methods in a multistep process. These techniques were performed sequentially and may include any of the following: affinity chromatography using Protein A or Protein G, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, hydrophobic exchange chromatography, high-pressure liquid chromatography (HPLC), precipitation, extraction, lyophilization, and / or crystallization. Using any one of these steps in succession, a person trained in protein chemistry could purify the described composition to homogeneity, resulting in the absence of contaminating protein bands on a silver-stained gel (see Figure 10 for a non-limiting example). The resulting protein samples were then tested using the Pierce LAL Chromogenic Endotoxin Quantitation Kit (cat. 88282) to ensure complete endotoxin-free status.

[0205] Enzymology: Steady-state hydrolysis of ATP by human NPP1 was confirmed by HPLC. Briefly, the enzymatic reaction was initiated by adding 10 nM NPP1 to various concentrations of ATP dissolved in a reaction buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 4.5 mM KCl, 14 μM ZnCl2, 1 mM MgCl2, and 1 mM CaCl2. At various time points, 50 μL of the reaction solution was removed and quenched with an equal volume of 3 M formic acid. The quenched reaction solution was loaded onto a C-18 (5 μm, 250 x 4.6 mm) column (Higgins Analytical) equilibrated with 15 mM ammonium acetate (pH 6.0) and eluted with a 0% to 20% methanol gradient. Substrate and product were monitored by UV absorbance at 259 nm and quantified according to the integration of their corresponding peaks and a calibration curve.

[0206] Vehicle: mENPP1-Fc was formulated in a vehicle that delivered a volume of 16 μl of vehicle per gram of body weight. The vehicle consisted of AmericanBio 10X PBS (Stock #AB11072) diluted to 1x with endotoxin-free water and supplemented with 14 μM CaCl2 and 14 μM ZnCl2.

[0207] dosage: Animals were dosed with vehicle or with mouse ENPP1-Fc (mENPP1-Fc) formulated in vehicle. Starting on day 14, mice were dosed with daily subcutaneous injections of a dose level of 500 au / Kg mENPP1-Fc.

[0208] Enzyme activity: In certain embodiments, enzymes useful in the present invention have enzymatic activity with a Michaelis Menton constant as described in Albright, et al., 2015, Nature Comm. 6:10006 (for ATP hydrolysis, K M Approximately 2μM;k cat 3.46(±0.44)s -1 ).

[0209] Quantitation of plasma PPi: Finally, ENPP1-WT and dosed ENPP1-asj / asj animals were bled retroorbitally using a heparinized micropipette. The blood was immediately dispensed into heparinized Eppendorf tubes and placed on wet ice. Samples were spun at 4000 rpm for 5 minutes in a microcentrifuge precooled to 4°C, and plasma was collected, diluted with a volume of 50 mM Tris-acetate, pH 8.0, and frozen at -80°C. Serum PPi quantification was performed as previously described (Cheung & Suhadolnik, 1977, Anal Biochem 83:61-63).

[0210] Micro-CT scan: In vivo 99m PYP Imaging: In animal cohorts, bone imaging agents99m Tc-pyrophosphate (Pharmalucence, Inc.) was evaluated using a preclinical microSPECT / CT hybrid imaging system equipped with a dual 1-mm pinhole collimator (X-SPECT, Gamma Medica-Ideas). Each animal received an i.p. injection of 2–5 mCi of radiolabeled tracer and was imaged 1–1.5 h after injection. CT scans (512 projections at 50 kVp, 800 μA, and a magnification factor of 1.25) were acquired for anatomical colocalization with SPECT images. SPECT imaging was acquired using a counterclockwise rotation of 180° per collimator head, 32 projections, 60 s per projection, a ROR of 7.0 cm, a FOV of 8.95 cm, and an energy window of 140 keV ± 20. CT images were reconstructed using FLEX XO CT software (Gamma Medica-Ideas) using a filtered back-projection algorithm. SPECT images were reconstructed using FLEX SPECT software (5 iterations, 4 subsets), subsequently fused with CT images, and analyzed using AMIRA software and offline in-house scripts. Data were corrected for attenuation and injected dose to obtain percent injected dose (%ID).

[0211] 99m Quantification of PYP uptake: 99mFor PYP mouse scans, animals were imaged 2 hours after injection. The resulting SPECT scans were imported into NIH's ImageJ image processing software, and ROIs were drawn around the head (target organ) and whole body of each animal. The percent injected activity (PIA), often referred to as the "percent injected dose" (%ID), was calculated by comparing the ratio of head counts to whole body counts to measure the affinity of the radiotracer to be taken up by the ROI (head), and expressed as %ID. The total counts per scan were considered a whole-body measure of the injected dose.

[0212] array: NPP1 amino acid sequence (NCBI accession NP_006199) (SEQ ID NO: 1) TIFF2026015456000006.tif90159

[0213] NPP2 amino acid sequence (NCBI accession NP_001124335) (SEQ ID NO:2) TIFF2026015456000007.tif90159

[0214] NPP4 amino acid sequence (NCBI accession AAH18054.1) (SEQ ID NO:3) TIFF2026015456000008.tif43159(DSS) n (SEQ ID NO: 4), where n is an integer from 1 to 10. (ESS) n (SEQ ID NO: 5), where n is an integer from 1 to 10. (RQQ) n (SEQ ID NO:6), where n is an integer from 1 to 10. (KR) n (SEQ ID NO:7), where n is an integer from 1 to 10. R m (SEQ ID NO: 8). m is an integer from 1 to 15. TIFF2026015456000009.tif31128E m (SEQ ID NO: 14). m is an integer from 1 to 15.

[0215] NPP121 amino acid sequence (SEQ ID NO:15) TIFF2026015456000010.tif218150TIFF2026015456000011.tif91150

[0216] single underline :Transition position( ** ), residues exchanged with residues 1–27 of NPP2 to cleave at ; double underline :NPP1 protein (beginning and end)

[0217] NPP121-Fc amino acid sequence (SEQ ID NO:16) TIFF2026015456000012.tif97150TIFF2026015456000013.tif217150TIFF2026015456000014.tif64150 single underline :Transition position( ** ), residues exchanged with residues 1–27 of NPP2 to cleave at ; double underline : NPP1 protein (beginning and end); bold: hIgG1(Fc)

[0218] NPP71 amino acid sequence (SEQ ID NO:17) TIFF2026015456000015.tif118143 single underline :NPP7; double underline :NPP1 protein (beginning and end)

[0219] NPP71-Fc amino acid sequence (SEQ ID NO:18) TIFF2026015456000016.tif144143 single underline :NPP7; double underline : NPP1 protein (beginning and end); bold: hIgG1(Fc)

[0220] (NPP1 N-terminal GLK-less NPP71) amino acid sequence (SEQ ID NO:19) TIFF2026015456000017.tif118143 single underline :NPP7; double underline :NPP1 protein (beginning and end) (the first three amino acids, GLK, at the N-terminus of NPP1 are omitted)

[0221] (NPP1 N-terminal GLK-less NPP71)-Fc amino acid sequence (SEQ ID NO:20) TIFF2026015456000018.tif144143 single underline :NPP7; double underline : NPP1 protein (beginning and end) (the first 3 amino acids at the N-terminus of NPP1 were omitted); bold: hIgG1(Fc)

[0222] NPP121-ALB amino acid sequence (SEQ ID NO:21) TIFF2026015456000019.tif157159Bold italics: NPP1 cytoplasmic and transmembrane; single underline :Transition position( ** ), residues exchanged with residues 1–27 of NPP2 to cleave at ; double underline :NPP1 transmembrane; normal:NPP1 extracellular domain; Bold Underline : linker; bold: albumin

[0223] (NPP1 N-terminal GLK-less NPP71)-ALB amino acid sequence (SEQ ID NO:22) TIFF2026015456000020.tif145159 double underline :NPP7; plain letters:NPP1; bold letters:spacer sequence; single underline :albumin

[0224] IISLFTFAVGVNICLGFTA(SEQ ID NO:23)

[0225] NPP51 amino acid sequence (SEQ ID NO:24) TIFF2026015456000021.tif84159 underline :NPP5;Normal:NPP1

[0226] NPP51-ALB amino acid sequence (SEQ ID NO:25) TIFF2026015456000022.tif146159 double underline :NPP5;Normal:NPP1;Bold:Spacer; single underline :albumin

[0227] Human IgG Fc domain, Fc (SEQ ID NO:26) TIFF2026015456000023.tif132159

[0228] Example 1: ENPP1-asj PXE Mouse Model Certain polypeptides of the present invention (e.g., ENPP1-Fc) were tested in mouse models of PXE and osteoarthritis (OA). Similar to humans with PXE, PXE mice exhibit loss-of-function mutations in the multi-pass membrane transporter ABCC6. ANK mice were used as a mammalian model of OA.

[0229] To generate ENPP1-WT and ENPP1-asj / asj sib pairs exposed in utero to the "Acceleration Diet" (TD00.442, Harlan Laboratories, Madison WI), heterozygous ENPP1-asj / + breeding pairs were maintained on the "Acceleration Diet" throughout the experiment. Litters were genotyped on day 8 and weaned on day 21. After weaning, sib pairs were divided into experimental cohorts, and all experimental animals were maintained on the Acceleration Diet until the end of the study. Study animals were administered selected polypeptides of the invention, and bones were analyzed as described herein.

[0230] As illustrated in Figure 7, initially, both PXE and ANK mice have low PPi, a biomarker reported in the literature to explain the pathogenesis of PXE (Jansen, et al., 2014, Arterioscler. Thromb. Vasc. Biol. 34:1985-1989).

[0231] Two PXE mice were administered ENPP1-Fc for one week. The average plasma PPi in these animals increased to about 4 μM. This indicates that administration of the polypeptide of the present invention to mammals increases the extracellular level of PPi and treats PXE.

[0232] The ability of this polypeptide to increase PPi was unexpected, since the biological mechanism of low PPi was thought to be related to low ATP concentration (Jansen, et al., 2013, PNAS USA 110(50):20206-20211). Indeed, the prior art has proposed that correcting plasma PPi in this disease is sufficient to treat PXE (Jansen, et al., 2013, PNAS USA 110(50):20206-20211). Based on the prior art at the time of the present invention, one skilled in the art would believe that the ENPP1 enzyme cannot produce PPi in the context of PXE because there is insufficient substrate in the extracellular space. As demonstrated herein, this is clearly not the case.

[0233] Example 2: When fed the acceleration diet, the daily weight gain of ENPP1-asj / asj mice diverged from that of their WT sibling pairs on day 26. At this time, ENPP1-asj / asj mice underwent a "failure to thrive" event and began to lose weight (Figure 1A). After day 26, ENPP1-asj / asj animals exhibited progressive stiffness and decreased physical activity. All ENPP1-asj / asj animals died between days 35 and 71, with a median lifespan of 58 days (Figure 1G). The presence of calcification in ENPP1-asj / asj and ENPP1-WT mice was assessed postmortem by micro-CT scanning and tissue sections taken from the heart, aorta, and kidney. Micro-CT imaging revealed that approximately one-third of ENPP1-asj / asj mice had visible calcification in the heart, and two-thirds had visible calcification in the aorta (Table 2). Upon histological examination, these percentages increased to 100%. Histological examination also revealed significant, nearly circumferential calcification in the aortic walls of many animals (Figures 1D–1E). Histological examination also revealed that 100% of coronary arteries had arterial wall calcification, and 70% of animals had focal or confluent areas of myocardial necrosis consistent with myocardial infarction (Figures 1F–1G). In contrast, ENPP1-WT mice did not exhibit any of these abnormalities. These findings demonstrate that this animal model recapitulates human GACI, which is characterized by prominent calcification of large and medium-sized arteries and cardiac demise.

[0234] To produce soluble recombinant ENPP1 for in vivo use, ENPP1 was fused to the Fc domain of IgG1 (hereafter referred to as ENPP1-Fc, Figure 2B), and the fusion protein was expressed in a stable mammalian (HEK293) cell line. The combined effects of switching protein expression from insect cells to mammalian cells and fusing ENPP1 to the Fc domain of IgG1 increased the affinity of ENPP1 for the ATP substrate by more than two orders of magnitude, while simultaneously increasing the K catThe activity of ENPP1-Fc was also reduced by 1 / 4 to 1 / 3, altering the Michaelis-Menten reaction kinetics (Figures 2C-2E). Although the activity of ENPP1-Fc was observed to decrease over 30 days when stored at 4°C, the enzyme could be frozen at -80°C and retain nearly full activity upon thawing (Figure 2C). Therefore, the enzyme was stored as a frozen stock solution until needed after purification.

[0235] After purification, ENPP1-Fc was diluted in PBS supplemented with Zn and Mg (PBS plus The concentrate was dialyzed against PBS, concentrated to 5-7 mg / ml, and frozen at -80°C in 200-500 μl aliquots. Immediately before use, the aliquots were thawed and resuspended in PBS. plus The specific activity of the solution was adjusted to 31.25 au / ml (or approximately 0.7 mg / ml depending on the preparation) by dilution with HCl.

[0236] Dosing was performed according to activity units (au) per kg of animal body weight to account for variations in specific activity among different protein preparations. Because enzyme specific activity varies among protein preparations and clinical responses are highly dependent on enzyme specific activity, protein preparations with specific activities below 40 au / mg were rejected. To determine the initial dosing level for proof-of-concept studies, a dose-escalation study was performed in a limited number of animals (1–2 per dose level). While both the human and mouse versions of ENPP1 were used in the dose-escalation study, the proof-of-concept study was performed using the mouse isoform of ENPP1-Fc (mENPP1-Fc). 14-day-old ENPP1-asj / asj mice were dosed daily with subcutaneous injections of mENPP1-Fc and weekly with intraperitoneal (IP) injections of GK1.5. The latter was added to minimize immune rejection of the recombinant protein. Subcutaneous dosing of mEnpp1-Fc at 500 au / Kg once daily demonstrated a strong early response in body weight without the "failure to thrive" crisis observed in untreated ENPP1-asj / asj animals.

[0237] Based on the results of the dose-escalation study, a cohort of eight NPP1-asj / asj animals was dosed with 500 au / kg mNPP1-Fc once daily and weekly IP injections of GK1.5 (Figure 3). Control groups (NPP1-WT + vehicle and NPP1-asj / asj + vehicle) were dosed daily with vehicle and weekly with GK1.5 in the same manner as the dosed cohorts. The study duration was shortened to 55 days. All eight treated NPP1-asj / asj animals survived the entire 55-day study. In this therapeutic study, a significant clinical response was observed in the treated NPP1 asj / asj animals, whereas the median lifespan of untreated NPP1 asj / asj animals was shortened from 58 days to 37 days. This was likely due to the weekly IP injections of the GK1.5 immunosuppressant. All untreated ENPP1-asj / asj animals also experienced a growth failure crisis on day 26, characterized by weight loss and restricted mobility that progressed indefinitely to paralysis and death over the next 30 days. Over the 55-day study, all but one untreated ENPP1-asj / asj animal died; in contrast, all treated ENPP1-asj / asj mice gained weight and showed no signs of reduced mobility or stiffness compared with ENPP1-WT mice.

[0238] At the end of the study, 100% of vehicle-treated ENPP1-asj / asj mice exhibited calcification in the heart, aorta, and coronary arteries, and 77% of animals showed histological evidence of myocardial infarction (Table 1). In most cases, this took the form of small areas of cardiomyocyte necrosis that disappeared adjacent to the cardiac calcification (Figures 3C-3D, 4C-4E). However, two animals (22%) had large, full-thickness myocardial infarctions in the right ventricular free wall (Figures 4C-4D). Myocardial fibrosis in the myocardial tissue adjacent to coronary artery calcification was a common finding (Figure 4E). This indicates that ischemia due to coronary artery calcification likely accounts for myocardial disease. In contrast, none of the ENPP1-asj / asj animals treated with ENPP1-Fc showed cardiac, arterial, or aortic calcification by histology or postmortem micro-CT (Table 1 and Figures 3D and 4D).

[0239] In addition to survival, daily animal weights, and peripheral tissue structure, the response to treatment was assessed by vascular calcification, plasma [PPi] concentrations, and Tc99PPi ( 99m PYP) uptake was also assessed by postmortem high-resolution micro-CT scans (Figure 5 and Table 1). Biochemical and physiological responses were observed as measured by all of these parameters. In contrast to the significant calcification observed in the aorta, coronary arteries, and heart of the untreated ENPP1-asj / asj cohort, neither WT nor treated ENPP1-asj / asj animals were found to have vascular calcification by micro-CT (Figure 5A). Furthermore, serum PPi concentrations in treated ENPP1-asj / asj animals were elevated above those of WT animals (approximately 30 μM in treated ENPP1-asj / asj vs. approximately 10 μM in WT), significantly above the levels (<0.5 μM) of untreated ENPP1-asj / asj (Figure 5B). Furthermore, serum PPi concentrations in treated ENPP1-asj / asj animals (approximately 30 μM) were elevated significantly above untreated ENPP1-asj / asj levels (<0.5 μM) and above serum PPi concentrations in WT animals (approximately 10 μM) ( Fig. 5B ).

[0240] 99m PYP is a commonly used imaging agent in cardiac imaging and bone remodeling and was used as a marker of response to treatment because it was predicted that plasma [PPi] would be reduced and "open" PPi binding sites would be increased at sites of ectopic calcification in animals lacking functional ENPP1. 99m To test this hypothesis, we performed in vivo experiments in ENPP1-WT and naïve ENPP1-asj / asj animals to detect differences in PYP uptake between sib pairs. 99m PYP imaging was performed weekly (Figures 5C-5D). 99mAnalysis of PYP uptake was restricted to the head, which is composed of both cartilaginous bone (cranium) and soft tissue (vibrissae). The head is a known site of ectopic calcification in this mouse model of GACI. Furthermore, analysis was restricted to the head to simplify data collection because the head is transiently displaced during the 180° camera rotation that occurs during data collection. 99m This is because there is no overlap with internal organs that have shown PYP uptake (e.g., bladder, heart, and diaphragm).

[0241] By serially imaging ENPP1-WT and untreated ENPP1-asj / asj animals every week, we were able to determine the changes in the skull of ENPP1-asj / asj animals. 99m The uptake rate (%) of PYP relative to the injected dose was higher than that of ENPP1-WT animals, 99m PYP uptake did not change significantly during the study (Figures 5C-5D). 99m PYP uptake was compared at two time points: days 30-35 and at the end of the study (days 50-65). Comparison of these experimental groups demonstrated that ENPP1-Fc treatment significantly improved PYP uptake in GACI mice. 99m As evidenced by the restoration of PYP uptake to WT levels (Figures 5E-5F), this suggests that ENPP1-Fc treatment can abrogate the unregulated tissue and cranial mineralization in ENPP1-asj / asj mice by saturating open PPi binding sites with "cold" PPi, likely resulting from the therapeutic-induced increase in plasma PPi concentrations.

[0242] Table 1. Cardiovascular pathology, proof-of-concept studies TIFF2026015456000024.tif44151

[0243] (Table 2) Cardiovascular pathology, natural history research TIFF2026015456000025.tif44128

[0244] Example 3: Expression of albumin fusion proteins Human serum albumin (HSA) is a 585-amino acid protein that is responsible for a significant proportion of serum osmolality and also functions as a carrier of endogenous and exogenous ligands. Currently, HSA for clinical use is produced by extraction from human blood. The production of recombinant HSA (rHSA) in microorganisms has been disclosed in EP 0330451 and EP 0361991.

[0245] Albumin's role as a carrier molecule and its inert nature make it a desirable property for use as a stabilizer and transporter of polypeptides. The use of albumin as a component of fusion proteins to stabilize other proteins has been disclosed in WO93 / 15199, WO93 / 15200, and EP0413622. The use of an N-terminal fragment of HSA to fuse to a polypeptide has also been disclosed (EP0399666). Fusion with a polypeptide is achieved by genetic engineering, such that DNA encoding HSA or a fragment thereof is linked to DNA encoding the polypeptide. A suitable host is then transformed or transfected with the fusion nucleotide sequence arranged on an appropriate plasmid to express the fusion polypeptide. Nomura, et al., 1995, Biosci. Biotechnol. Biochem. 59(3):532-4, attempted to express human apolipoprotein E as a fusion protein with HSA or an HSA fragment in S. cerevisiae using an HSA presequence to induce secretion. Fusion to full-length HSA resulted in low levels of protein secreted into the medium (maximum yield of 6.3 mg / liter), whereas fusion to HSA(1-198) or HSA(1-390) did not result in secretion into the medium.

[0246] Human serum albumin may be a variant of normal HSA (hereinafter referred to as "HSA"). As used herein, "variant" includes conservative or non-conservative insertions, deletions, and substitutions, where such changes do not substantially alter one or more of the albumin's useful oncotic ligand-binding and non-immunogenic properties. In particular, "variant" includes naturally occurring polymorphic variants of human albumin and human albumin fragments, such as the fragments disclosed in EP0322094 (i.e., HA(1-n), where n is 369 to 419). Albumin or growth hormone (GH) may be derived from any vertebrate, particularly any mammal, such as human, cow, sheep, pig, hen, or salmon. The albumin and GH portions of the fusion may be derived from different animals.

[0247] "Conservative substitutions" are intended exchanges within groups such as Gly / Ala; Val / Ile / Leu; Asp / Glu; Asn / Gln; Ser / Thr; Lys / Arg; and Phe / Tyr. A variant typically has at least 75% (e.g., at least 80%, 90%, 95%, or 99%) sequence identity with a length of normal HSA, the length being the same as the variant, and is more identical than any other length of normal HSA, allowing for deletions and insertions as is conventional in the art. Generally, HSA variants are at least 100 amino acids long, and in some embodiments at least 150 amino acids long. HSA variants may consist of or include at least one entire HSA domain, e.g., domains 1 (1-194), 2 (195-387), 3 (388-585), 1 + 2 (1-387), 2 + 3 (195-585), or 1 + 3 (1-194, + 388-585). Each domain itself consists of two homologous subdomains: 1-105, 120-194, 195-291, 316-387, 388-491, and 512-585, with flexible inter-subdomain linker regions comprising residues Lys106 to Glu199, Glu292 to Val315, and Glu492 to Ala511. In some embodiments, the HSA portion of the NPP1 fusion comprises at least one subdomain or domain of HA, or a conservative modification thereof.

[0248] Many expression systems are known, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells.

[0249] The desired protein can be produced in conventional manner, for example, from a coding sequence inserted into the host chromosome or carried on a free plasmid.

[0250] Yeast can be transformed with the coding sequence of the desired protein by any useful method, such as electroporation. Methods for transforming yeast by electroporation are disclosed in Becker & Guarente, 1990, Methods Enzymol. 194:182. Successfully transformed cells, i.e., cells containing the DNA construct of the present invention, 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 can be examined for the presence of DNA using methods such as those described in Southern, 1975, J. Mol. Biol. 98:503 and / or Berent, et al., 1985, Biotech 3:208. Alternatively, the presence of protein in the supernatant can be detected using antibodies.

[0251] Useful yeast plasmid vectors include pRS403-406 and pRS413-416, which are generally available from Stratagene Cloning Systems, La Jolla, CA, USA. Plasmids pRS403, pRS404, pRS405, and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2, and URA3. Plasmid pRS413-416 is a Yeast Centromeric Plasmid (YCp).

[0252] Various methods have been developed to operably link DNA to a vector via complementary cohesive ends. For example, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and the DNA segment are then joined by hydrogen bonds between the complementary homopolymer tails to form a recombinant DNA molecule.

[0253] Synthetic linkers containing one or more restriction sites provide an alternative method for joining DNA segments to vectors. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding 3'-single-stranded ends with their 3'-5'-exonuclease activity and fill in recessed 3'-ends with their polymerization activity.

[0254] Thus, combining these activities produces 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 can catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.The product of the reaction is therefore a DNA segment with a polymer linker sequence at its end.These DNA segments are then cleaved with an appropriate restriction enzyme and ligated into an expression vector that has been cleaved with an enzyme that produces ends compatible with the ends of the DNA segments.

[0255] Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources, including International Biotechnologies Inc, New Haven, CT, USA.

[0256] A desirable method for modifying DNA according to the present invention, for example, to prepare an HA variant, is to use the polymerase chain reaction as disclosed in Saiki, et al., 1988, Science 239:487-491. In this method, the DNA to be enzymatically amplified is flanked by two specific oligonucleotides that are incorporated into the amplified DNA. The specific primers may contain restriction endonuclease recognition sites that can be used for cloning into an expression vector using methods known in the art.

[0257] ENPP1-ALB design: Modified human NPP1 and modified mouse NPP1 (human: NCBI accession NP_006199; mouse: NCBI accession NP_03839), which were engineered to express soluble recombinant proteins, were fused with human serum albumin (HSA) by subcloning to obtain pFUSE plasmids (InvivoGen, San Diego CA), respectively.

[0258] Protein production: Shake flask: Stable transfection of ENPP1-ALB is established in HEK293 cells under Zeocin selection. Adherent HEK293 cells can be adapted to suspension growth. Adapted cells are used to inoculate liquid cultures in FreeStyle medium (Gibco #12338-018) in shake flasks at 37°C and 5% CO2 with agitation at 120 rpm in high humidity. Cultures are expanded stepwise to the desired target volume and then maintained for an additional 12 days to accumulate extracellular protein. Cultures are supplemented with CD EfficientFeed C AGT (Gibco #A13275-05) to enhance protein production during the maintenance phase.

[0259] Bioreactor: Cells are grown in a 10-liter bioreactor equipped with dissolved oxygen and pH control devices. Dissolved oxygen is maintained at 40% air saturation by supplying the culture with an air / oxygen mixture not exceeding 3 liters / minute at an agitation rate of 80 RPM. pH is controlled at 7.4 by sparging with CO2 if it exceeds 7.4. Culture growth is followed by measurements of cell count, cell viability, glucose concentration, and lactate concentration.

[0260] Protein purification: The liquid culture was centrifuged at 4300 x g for 15 minutes, and the supernatant was filtered through a 0.2 μm membrane and concentrated by tangential flow using a Pellicon® 3 0.11 m2 Ultracell® 30 kD cassette (Millipore, Billerica, MA). The concentrated supernatant was loaded onto a Protein-AG column. The concentrated supernatant was eluted with a buffer containing 50 mM sodium citrate, 150 mM NaCl, 3 mM ZnCl2, 3 mM CaCl2, pH 3.5. Fractions containing enzyme activity were pooled and dialyzed against IXPBS buffer pH 7.4, 11 μM ZnCl2, 20 μM CaCl2, then concentrated to 6 mg / ml, distributed into small aliquots, and stored at -80°C.

[0261] The resulting protein samples are tested using the Pierce LAL Chromogenic Endotoxin Quantitation Kit (cat. 88282) to ensure all are endotoxin free.

[0262] Enzymology The purified NPP1-albumin fusion protein is characterized according to the experimental protocols discussed in Examples 1 and 2 elsewhere herein.

[0263] The disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed in connection with specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

[0264] Sequence information SEQUENCE LISTING <110> Yale University <120> Compositions for Treating Pathological Calcification Conditions, and Methods Using Same <150> US 62 / 163,500 <151> 2015-05-19 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 925 <212> PRT <213> Homo sapiens <400> 1 Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser Arg Gly Gly Glu Gly 1 5 10 15 Gly Arg Ala Pro Arg Glu Gly Pro Ala Gly Asn Gly Arg Asp Arg Gly 20 25 30 Arg Ser His Ala Ala Glu Ala Pro Gly Asp Pro Gln Ala Ala Ala Ser 35 40 45 Leu Leu Ala Pro Met Asp Val Gly Glu Glu Pro Leu Glu Lys Ala Ala 50 55 60 Arg Ala Arg Thr Ala Lys Asp Pro Asn Thr Tyr Lys Val Leu Ser Leu 65 70 75 80 Val Leu Ser Val Cys Val Leu Thr Thr Ile Leu Gly Cys Ile Phe Gly 85 90 95 Leu Lys Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys 100 105 110 Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu 115 120 125 Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu 130 135 140 His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr 145 150 155 160 Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys 165 170 175 Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu 180 185 190 Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu 195 200 205 Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr 210 215 220 Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys 225 230 235 240 Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr 245 250 255 Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His 260 265 270 Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe 275 280 285 Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu 290 295 300 Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe 305 310 315 320 Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile 325 330 335 Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala 340 345 350 Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr 355 360 365 Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro 370 375 380 Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val 385 390 395 400 Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu 405 410 415 Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys 420 425 430 Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys 435 440 445 Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp 450 455 460 Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys 465 470 475 480 Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro 485 490 495 Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe 500 505 510 Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys 515 520 525 Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met 530 535 540 Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu 545 550 555 560 Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu 565 570 575 Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn 580 585 590 His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val 595 600 605 His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu 610 615 620 Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr 625 630 635 640 Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr 645 650 655 Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys 660 665 670 Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu 675 680 685 Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser 690 695 700 Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu 705 710 715 720 Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser 725 730 735 Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile 740 745 750 Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser 755 760 765 Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr 770 775 780 Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp 785 790 795 800 Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys 805 810 815 Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe 820 825 830 Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys 835 840 845 Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn 850 855 860 Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu 865 870 875 880 Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr 885 890 895 Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu 900 905 910 Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp 915 920 925 <210> 2 <211> 888 <212> PRT <213> Homo sapiens <400> 2 Met Ala Arg Arg Ser Ser Phe Gln Ser Cys Gln Ile Ile Ser Leu Phe 1 5 10 15 Thr Phe Ala Val Gly Val Asn Ile Cys Leu Gly Phe Thr Ala His Arg 20 25 30 Ile Lys Arg Ala Glu Gly Trp Glu Glu Gly Pro Pro Thr Val Leu Ser 35 40 45 Asp Ser Pro Trp Thr Asn Ile Ser Gly Ser Cys Lys Gly Arg Cys Phe 50 55 60 Glu Leu Gln Glu Ala Gly Pro Pro Asp Cys Arg Cys Asp Asn Leu Cys 65 70 75 80 Lys Ser Tyr Thr Ser Cys Cys His Asp Phe Asp Glu Leu Cys Leu Lys 85 90 95 Thr Ala Arg Gly Trp Glu Cys Thr Lys Asp Arg Cys Gly Glu Val Arg 100 105 110 Asn Glu Glu Asn Ala Cys His Cys Ser Glu Asp Cys Leu Ala Arg Gly 115 120 125 Asp Cys Cys Thr Asn Tyr Gln Val Val Cys Lys Gly Glu Ser His Trp 130 135 140 Val Asp Asp Asp Cys Glu Glu Ile Lys Ala Ala Glu Cys Pro Ala Gly 145 150 155 160 Phe Val Arg Pro Pro Leu Ile Ile Phe Ser Val Asp Gly Phe Arg Ala 165 170 175 Ser Tyr Met Lys Lys Gly Ser Lys Val Met Pro Asn Ile Glu Lys Leu 180 185 190 Arg Ser Cys Gly Thr His Ser Pro Tyr Met Arg Pro Val Tyr Pro Thr 195 200 205 Lys Thr Phe Pro Asn Leu Tyr Thr Leu Ala Thr Gly Leu Tyr Pro Glu 210 215 220 Ser His Gly Ile Val Gly Asn Ser Met Tyr Asp Pro Val Phe Asp Ala 225 230 235 240 Thr Phe His Leu Arg Gly Arg Glu Lys Phe Asn His Arg Trp Trp Gly 245 250 255 Gly Gln Pro Leu Trp Ile Thr Ala Thr Lys Gln Gly Val Lys Ala Gly 260 265 270 Thr Phe Phe Trp Ser Val Val Ile Pro His Glu Arg Arg Ile Leu Thr 275 280 285 Ile Leu Gln Trp Leu Thr Leu Pro Asp His Glu Arg Pro Ser Val Tyr 290 295 300 Ala Phe Tyr Ser Glu Gln Pro Asp Phe Ser Gly His Lys Tyr Gly Pro 305 310 315 320 Phe Gly Pro Glu Met Thr Asn Pro Leu Arg Glu Ile Asp Lys Ile Val 325 330 335 Gly Gln Leu Met Asp Gly Leu Lys Gln Leu Lys Leu His Arg Cys Val 340 345 350 Asn Val Ile Phe Val Gly Asp His Gly Met Glu Asp Val Thr Cys Asp 355 360 365 Arg Thr Glu Phe Leu Ser Asn Tyr Leu Thr Asn Val Asp Asp Ile Thr 370 375 380 Leu Val Pro Gly Thr Leu Gly Arg Ile Arg Ser Lys Phe Ser Asn Asn 385 390 395 400 Ala Lys Tyr Asp Pro Lys Ala Ile Ile Ala Asn Leu Thr Cys Lys Lys 405 410 415 Pro Asp Gln His Phe Lys Pro Tyr Leu Lys Gln His Leu Pro Lys Arg 420 425 430 Leu His Tyr Ala Asn Asn Arg Arg Ile Glu Asp Ile His Leu Leu Val 435 440 445 Glu Arg Arg Trp His Val Ala Arg Lys Pro Leu Asp Val Tyr Lys Lys 450 455 460 Pro Ser Gly Lys Cys Phe Phe Gln Gly Asp His Gly Phe Asp Asn Lys 465 470 475 480 Val Asn Ser Met Gln Thr Val Phe Val Gly Tyr Gly Ser Thr Phe Lys 485 490 495 Tyr Lys Thr Lys Val Pro Pro Phe Glu Asn Ile Glu Leu Tyr Asn Val 500 505 510 Met Cys Asp Leu Leu Gly Leu Lys Pro Ala Pro Asn Asn Gly Thr His 515 520 525 Gly Ser Leu Asn His Leu Leu Arg Thr Asn Thr Phe Arg Pro Thr Met 530 535 540 Pro Glu Glu Val Thr Arg Pro Asn Tyr Pro Gly Ile Met Tyr Leu Gln 545 550 555 560 Ser Asp Phe Asp Leu Gly Cys Thr Cys Asp Asp Lys Val Glu Pro Lys 565 570 575 Asn Lys Leu Asp Glu Leu Asn Lys Arg Leu His Thr Lys Gly Ser Thr 580 585 590 Glu Ala Glu Thr Arg Lys Phe Arg Gly Ser Arg Asn Glu Asn Lys Glu 595 600 605 Asn Ile Asn Gly Asn Phe Glu Pro Arg Lys Glu Arg His Leu Leu Tyr 610 615 620 Gly Arg Pro Ala Val Leu Tyr Arg Thr Arg Tyr Asp Ile Leu Tyr His 625 630 635 640 Thr Asp Phe Glu Ser Gly Tyr Ser Glu Ile Phe Leu Met Pro Leu Trp 645 650 655 Thr Ser Tyr Thr Val Ser Lys Gln Ala Glu Val Ser Ser Val Pro Asp 660 665 670 His Leu Thr Ser Cys Val Arg Pro Asp Val Arg Val Ser Pro Ser Phe 675 680 685 Ser Gln Asn Cys Leu Ala Tyr Lys Asn Asp Lys Gln Met Ser Tyr Gly 690 695 700 Phe Leu Phe Pro Pro Tyr Leu Ser Ser Ser Pro Glu Ala Lys Tyr Asp 705 710 715 720 Ala Phe Leu Val Thr Asn Met Val Pro Met Tyr Pro Ala Phe Lys Arg 725 730 735 Val Trp Asn Tyr Phe Gln Arg Val Leu Val Lys Lys Tyr Ala Ser Glu 740 745 750 Arg Asn Gly Val Asn Val Ile Ser Gly Pro Ile Phe Asp Tyr Asp Tyr 755 760 765 Asp Gly Leu His Asp Thr Glu Asp Lys Ile Lys Gln Tyr Val Glu Gly 770 775 780 Ser Ser Ile Pro Val Pro Thr His Tyr Tyr Ser Ile Ile Thr Ser Cys 785 790 795 800 Leu Asp Phe Thr Gln Pro Ala Asp Lys Cys Asp Gly Pro Leu Ser Val 805 810 815 Ser Ser Phe Ile Leu Pro His Arg Pro Asp Asn Glu Glu Ser Cys Asn 820 825 830 Ser Ser Glu Asp Glu Ser Lys Trp Val Glu Glu Leu Met Lys Met His 835 840 845 Thr Ala Arg Val Arg Asp Ile Glu His Leu Thr Ser Leu Asp Phe Phe 850 855 860 Arg Lys Thr Ser Arg Ser Tyr Pro Glu Ile Leu Thr Leu Lys Thr Tyr 865 870 875 880 Leu His Thr Tyr Glu Ser Glu Ile 885 <210> 3 <211> 453 <212> PRT <213> Homo sapiens <400> 3 Met Lys Leu Leu Val Ile Leu Leu Phe Ser Gly Leu Ile Thr Gly Phe 1 5 10 15 Arg Ser Asp Ser Ser Ser Ser Leu Pro Pro Lys Leu Leu Leu Val Ser 20 25 30 Phe Asp Gly Phe Arg Ala Asp Tyr Leu Lys Asn Tyr Glu Phe Pro His 35 40 45 Leu Gln Asn Phe Ile Lys Glu Gly Val Leu Val Glu His Val Lys Asn 50 55 60 Val Phe Ile Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly 65 70 75 80 Leu Tyr Glu Glu Ser His Gly Ile Val Ala Asn Ser Met Tyr Asp Ala 85 90 95 Val Thr Lys Lys His Phe Ser Asp Ser Asn Asp Lys Asp Pro Phe Trp 100 105 110 Trp Asn Glu Ala Val Pro Ile Trp Val Thr Asn Gln Leu Gln Glu Asn 115 120 125 Arg Ser Ser Ala Ala Ala Met Trp Pro Gly Thr Asp Val Pro Ile His 130 135 140 Asp Thr Ile Ser Ser Tyr Phe Met Asn Tyr Asn Ser Ser Val Ser Phe 145 150 155 160 Glu Glu Arg Leu Asn Asn Ile Thr Met Trp Leu Asn Asn Ser Asn Pro 165 170 175 Pro Val Thr Phe Ala Thr Leu Tyr Trp Glu Glu Pro Asp Ala Ser Gly 180 185 190 His Lys Tyr Gly Pro Glu Asp Lys Glu Asn Met Ser Arg Val Leu Lys 195 200 205 Lys Ile Asp Asp Leu Ile Gly Asp Leu Val Gln Arg Leu Lys Met Leu 210 215 220 Gly Leu Trp Glu Asn Leu Asn Val Ile Ile Thr Ser Asp His Gly Met 225 230 235 240 Thr Gln Cys Ser Gln Asp Arg Leu Ile Asn Leu Asp Ser Cys Ile Asp 245 250 255 His Ser Tyr Tyr Thr Leu Ile Asp Leu Ser Pro Val Ala Ala Ile Leu 260 265 270 Pro Lys Ile Asn Arg Thr Glu Val Tyr Asn Lys Leu Lys Asn Cys Ser 275 280 285 Pro His Met Asn Val Tyr Leu Lys Glu Asp Ile Pro Asn Arg Phe Tyr 290 295 300 Tyr Gln His Asn Asp Arg Ile Gln Pro Ile Ile Leu Val Ala Asp Glu 305 310 315 320 Gly Trp Thr Ile Val Leu Asn Glu Ser Ser Gln Lys Leu Gly Asp His 325 330 335 Gly Tyr Asp Asn Ser Leu Pro Ser Met His Pro Phe Leu Ala Ala His 340 345 350 Gly Pro Ala Phe His Lys Gly Tyr Lys His Ser Thr Ile Asn Ile Val 355 360 365 Asp Ile Tyr Pro Met Met Cys His Ile Leu Gly Leu Lys Pro His Pro 370 375 380 Asn Asn Gly Thr Phe Gly His Thr Lys Cys Leu Leu Val Asp Gln Trp 385 390 395 400 Cys Ile Asn Leu Pro Glu Ala Ile Ala Ile Val Ile Gly Ser Leu Leu 405 410 415 Val Leu Thr Met Leu Thr Cys Leu Ile Ile Ile Met Gln Asn Arg Leu 420 425 430 Ser Val Pro Arg Pro Phe Ser Arg Leu Gln Leu Gln Glu Asp Asp Asp 435 440 445 Asp Pro Leu Ile Gly 450 <210> 4 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <220> <221> REPEAT <222> (1)..(3) <223> the tripeptide is present n times, wherein n is integer ranging from 1 to 10 <400> 4 Asp Ser Ser 1 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <220> <221> REPEAT <222> (1)..(3) <223> the tripeptide is present n times, wherein n is an integer ranging from 1 to 10 <400> 5 Glu Ser Ser 1 <210> 6 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <220> <221> REPEAT <222> (1)..(3) <223> the tripeptide is present n times, wherein n is integer ranging from 1 to 10 <400> 6 Arg Gln Gln 1 <210> 7 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <220> <221> REPEAT <222> (1)..(2) <223> the dipeptide is present n times, wherein n is integer ranging from 1 to 10 <400> 7 Lys Arg 1 <210> 8 <211> 1 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <220> <221> REPEAT <222> (1)..(1) <223> the amino acid is present n times, wherein n is integer ranging from 1 to 10 <400> 8 Arg 1 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 9 Asp Ser Ser Ser Glu Glu Lys Phe Leu Arg Arg Ile Gly Arg Phe Gly 1 5 10 15 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 10 Glu Glu Glu Glu Glu Glu Glu Pro Arg Gly Asp Thr 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 11 Ala Pro Trp His Leu Ser Ser Gln Tyr Ser Arg Thr 1 5 10 <210> 12 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 12 Ser Thr Leu Pro Ile Pro His Glu Phe Ser Arg Glu 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 13 Val Thr Lys His Leu Asn Gln Ile Ser Gln Ser Tyr 1 5 10 <210> 14 <211> 1 <212> PRT <213> Artificial Sequence <220> <223> chemically sysnthesized <220> <221> REPEAT <222> (1)..(1) <223> the amino acid is present m times, wherein m is an integer ranging from 1 to 15 <400> 14 Glu 1 <210> 15 <211> 925 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 15 Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser Arg Gly Gly Glu Gly 1 5 10 15 Gly Arg Ala Pro Arg Glu Gly Pro Ala Gly Asn Gly Arg Asp Arg Gly 20 25 30 Arg Ser His Ala Ala Glu Ala Pro Gly Asp Pro Gln Ala Ala Ala Ser 35 40 45 Leu Leu Ala Pro Met Asp Val Gly Glu Glu Pro Leu Glu Lys Ala Ala 50 55 60 Arg Ala Arg Thr Ala Lys Asp Pro Asn Thr Tyr Lys Ile Ile Ser Leu 65 70 75 80 Phe Thr Phe Ala Val Gly Val Asn Ile Cys Leu Gly Phe Thr Ala Gly 85 90 95 Leu Lys Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys 100 105 110 Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu 115 120 125 Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu 130 135 140 His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr 145 150 155 160 Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys 165 170 175 Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu 180 185 190 Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu 195 200 205 Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr 210 215 220 Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys 225 230 235 240 Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr 245 250 255 Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His 260 265 270 Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe 275 280 285 Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu 290 295 300 Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe 305 310 315 320 Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile 325 330 335 Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala 340 345 350 Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr 355 360 365 Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro 370 375 380 Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val 385 390 395 400 Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu 405 410 415 Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys 420 425 430 Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys 435 440 445 Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp 450 455 460 Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys 465 470 475 480 Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro 485 490 495 Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe 500 505 510 Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys 515 520 525 Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met 530 535 540 Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu 545 550 555 560 Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu 565 570 575 Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn 580 585 590 His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val 595 600 605 His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu 610 615 620 Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr 625 630 635 640 Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr 645 650 655 Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys 660 665 670 Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu 675 680 685 Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser 690 695 700 Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu 705 710 715 720 Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser 725 730 735 Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile 740 745 750 Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser 755 760 765 Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr 770 775 780 Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp 785 790 795 800 Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys 805 810 815 Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe 820 825 830 Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys 835 840 845 Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn 850 855 860 Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu 865 870 875 880 Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr 885 890 895 Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu 900 905 910 Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp 915 920 925 <210> 16 <211> 1155 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 16 Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser Arg Gly Gly Glu Gly 1 5 10 15 Gly Arg Ala Pro Arg Glu Gly Pro Ala Gly Asn Gly Arg Asp Arg Gly 20 25 30 Arg Ser His Ala Ala Glu Ala Pro Gly Asp Pro Gln Ala Ala Ala Ser 35 40 45 Leu Leu Ala Pro Met Asp Val Gly Glu Glu Pro Leu Glu Lys Ala Ala 50 55 60 Arg Ala Arg Thr Ala Lys Asp Pro Asn Thr Tyr Lys Ile Ile Ser Leu 65 70 75 80 Phe Thr Phe Ala Val Gly Val Asn Ile Cys Leu Gly Phe Thr Ala Gly 85 90 95 Leu Lys Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys 100 105 110 Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu 115 120 125 Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu 130 135 140 His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr 145 150 155 160 Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys 165 170 175 Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu 180 185 190 Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu 195 200 205 Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr 210 215 220 Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys 225 230 235 240 Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr 245 250 255 Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His 260 265 270 Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe 275 280 285 Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu 290 295 300 Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe 305 310 315 320 Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile 325 330 335 Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala 340 345 350 Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr 355 360 365 Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro 370 375 380 Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val 385 390 395 400 Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu 405 410 415 Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys 420 425 430 Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys 435 440 445 Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp 450 455 460 Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys 465 470 475 480 Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro 485 490 495 Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe 500 505 510 Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys 515 520 525 Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met 530 535 540 Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu 545 550 555 560 Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu 565 570 575 Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn 580 585 590 His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val 595 600 605 His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu 610 615 620 Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr 625 630 635 640 Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr 645 650 655 Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys 660 665 670 Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu 675 680 685 Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser 690 695 700 Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu 705 710 715 720 Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser 725 730 735 Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile 740 745 750 Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser 755 760 765 Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr 770 775 780 Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp 785 790 795 800 Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys 805 810 815 Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe 820 825 830 Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys 835 840 845 Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn 850 855 860 Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu 865 870 875 880 Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr 885 890 895 Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu 900 905 910 Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp Leu Ile Asn 915 920 925 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 930 935 940 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 945 950 955 960 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 965 970 975 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 980 985 990 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 995 1000 1005 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 1010 1015 1020 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 1025 1030 1035 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 1040 1045 1050 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 1055 1060 1065 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 1070 1075 1080 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 1085 1090 1095 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 1100 1105 1110 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 1115 1120 1125 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 1130 1135 1140 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1145 1150 1155 <210> 17 <211> 852 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 17 Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu 1 5 10 15 Ala Pro Gly Ala Gly Ala Gly Leu Lys Pro Ser Cys Ala Lys Glu Val 20 25 30 Lys Ser Cys Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg 35 40 45 Cys Asp Ala Ala Cys Val Glu Leu Gly Asn Cys Cys Leu Asp Tyr Gln 50 55 60 Glu Thr Cys Ile Glu Pro Glu His Ile Trp Thr Cys Asn Lys Phe Arg 65 70 75 80 Cys Gly Glu Lys Arg Leu Thr Arg Ser Leu Cys Ala Cys Ser Asp Asp 85 90 95 Cys Lys Asp Lys Gly Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln 100 105 110 Gly Glu Lys Ser Trp Val Glu Glu Pro Cys Glu Ser Ile Asn Glu Pro 115 120 125 Gln Cys Pro Ala Gly Phe Glu Thr Pro Pro Thr Leu Leu Phe Ser Leu 130 135 140 Asp Gly Phe Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro 145 150 155 160 Val Ile Ser Lys Leu Lys Lys Cys Gly Thr Tyr Thr Lys Asn Met Arg 165 170 175 Pro Val Tyr Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr 180 185 190 Gly Leu Tyr Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp 195 200 205 Pro Lys Met Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn 210 215 220 Pro Glu Trp Tyr Lys Gly Glu Pro Ile Trp Val Thr Ala Lys Tyr Gln 225 230 235 240 Gly Leu Lys Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile 245 250 255 Asn Gly Ile Phe Pro Asp Ile Tyr Lys Met Tyr Asn Gly Ser Val Pro 260 265 270 Phe Glu Glu Arg Ile Leu Ala Val Leu Gln Trp Leu Gln Leu Pro Lys 275 280 285 Asp Glu Arg Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser 290 295 300 Ser Gly His Ser Tyr Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu 305 310 315 320 Gln Arg Val Asp Gly Met Val Gly Met Leu Met Asp Gly Leu Lys Glu 325 330 335 Leu Asn Leu His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly 340 345 350 Met Glu Gln Gly Ser Cys Lys Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu 355 360 365 Gly Asp Val Lys Asn Ile Lys Val Ile Tyr Gly Pro Ala Ala Arg Leu 370 375 380 Arg Pro Ser Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly 385 390 395 400 Ile Ala Arg Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Lys Pro 405 410 415 Tyr Leu Lys His Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp 420 425 430 Arg Ile Glu Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala 435 440 445 Leu Asn Pro Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser 450 455 460 Asp Asn Val Phe Ser Asn Met Gln Ala Leu Phe Val Gly Tyr Gly Pro 465 470 475 480 Gly Phe Lys His Gly Ile Glu Ala Asp Thr Phe Glu Asn Ile Glu Val 485 490 495 Tyr Asn Leu Met Cys Asp Leu Leu Asn Leu Thr Pro Ala Pro Asn Asn 500 505 510 Gly Thr His Gly Ser Leu Asn His Leu Leu Lys Asn Pro Val Tyr Thr 515 520 525 Pro Lys His Pro Lys Glu Val His Pro Leu Val Gln Cys Pro Phe Thr 530 535 540 Arg Asn Pro Arg Asp Asn Leu Gly Cys Ser Cys Asn Pro Ser Ile Leu 545 550 555 560 Pro Ile Glu Asp Phe Gln Thr Gln Phe Asn Leu Thr Val Ala Glu Glu 565 570 575 Lys Ile Ile Lys His Glu Thr Leu Pro Tyr Gly Arg Pro Arg Val Leu 580 585 590 Gln Lys Glu Asn Thr Ile Cys Leu Leu Ser Gln His Gln Phe Met Ser 595 600 605 Gly Tyr Ser Gln Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val 610 615 620 Asp Arg Asn Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr 625 630 635 640 Gln Asp Phe Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Phe Tyr 645 650 655 Lys Asn Asn Thr Lys Val Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu 660 665 670 Asn Lys Asn Ser Ser Gly Ile Tyr Ser Glu Ala Leu Leu Thr Thr Asn 675 680 685 Ile Val Pro Met Tyr Gln Ser Phe Gln Val Ile Trp Arg Tyr Phe His 690 695 700 Asp Thr Leu Leu Arg Lys Tyr Ala Glu Glu Arg Asn Gly Val Asn Val 705 710 715 720 Val Ser Gly Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Cys Asp Ser 725 730 735 Leu Glu Asn Leu Arg Gln Lys Arg Arg Val Ile Arg Asn Gln Glu Ile 740 745 750 Leu Ile Pro Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Asp Thr 755 760 765 Ser Gln Thr Pro Leu His Cys Glu Asn Leu Asp Thr Leu Ala Phe Ile 770 775 780 Leu Pro His Arg Thr Asp Asn Ser Glu Ser Cys Val His Gly Lys His 785 790 795 800 Asp Ser Ser Trp Val Glu Glu Leu Leu Met Leu His Arg Ala Arg Ile 805 810 815 Thr Asp Val Glu His Ile Thr Gly Leu Ser Phe Tyr Gln Gln Arg Lys 820 825 830 Glu Pro Val Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Thr Phe 835 840 845 Ser Gln Glu Asp 850 <210> 18 <211> 1082 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 18 Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu 1 5 10 15 Ala Pro Gly Ala Gly Ala Gly Leu Lys Pro Ser Cys Ala Lys Glu Val 20 25 30 Lys Ser Cys Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg 35 40 45 Cys Asp Ala Ala Cys Val Glu Leu Gly Asn Cys Cys Leu Asp Tyr Gln 50 55 60 Glu Thr Cys Ile Glu Pro Glu His Ile Trp Thr Cys Asn Lys Phe Arg 65 70 75 80 Cys Gly Glu Lys Arg Leu Thr Arg Ser Leu Cys Ala Cys Ser Asp Asp 85 90 95 Cys Lys Asp Lys Gly Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln 100 105 110 Gly Glu Lys Ser Trp Val Glu Glu Pro Cys Glu Ser Ile Asn Glu Pro 115 120 125 Gln Cys Pro Ala Gly Phe Glu Thr Pro Pro Thr Leu Leu Phe Ser Leu 130 135 140 Asp Gly Phe Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro 145 150 155 160 Val Ile Ser Lys Leu Lys Lys Cys Gly Thr Tyr Thr Lys Asn Met Arg 165 170 175 Pro Val Tyr Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr 180 185 190 Gly Leu Tyr Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp 195 200 205 Pro Lys Met Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn 210 215 220 Pro Glu Trp Tyr Lys Gly Glu Pro Ile Trp Val Thr Ala Lys Tyr Gln 225 230 235 240 Gly Leu Lys Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile 245 250 255 Asn Gly Ile Phe Pro Asp Ile Tyr Lys Met Tyr Asn Gly Ser Val Pro 260 265 270 Phe Glu Glu Arg Ile Leu Ala Val Leu Gln Trp Leu Gln Leu Pro Lys 275 280 285 Asp Glu Arg Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser 290 295 300 Ser Gly His Ser Tyr Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu 305 310 315 320 Gln Arg Val Asp Gly Met Val Gly Met Leu Met Asp Gly Leu Lys Glu 325 330 335 Leu Asn Leu His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly 340 345 350 Met Glu Gln Gly Ser Cys Lys Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu 355 360 365 Gly Asp Val Lys Asn Ile Lys Val Ile Tyr Gly Pro Ala Ala Arg Leu 370 375 380 Arg Pro Ser Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly 385 390 395 400 Ile Ala Arg Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Lys Pro 405 410 415 Tyr Leu Lys His Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp 420 425 430 Arg Ile Glu Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala 435 440 445 Leu Asn Pro Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser 450 455 460 Asp Asn Val Phe Ser Asn Met Gln Ala Leu Phe Val Gly Tyr Gly Pro 465 470 475 480 Gly Phe Lys His Gly Ile Glu Ala Asp Thr Phe Glu Asn Ile Glu Val 485 490 495 Tyr Asn Leu Met Cys Asp Leu Leu Asn Leu Thr Pro Ala Pro Asn Asn 500 505 510 Gly Thr His Gly Ser Leu Asn His Leu Leu Lys Asn Pro Val Tyr Thr 515 520 525 Pro Lys His Pro Lys Glu Val His Pro Leu Val Gln Cys Pro Phe Thr 530 535 540 Arg Asn Pro Arg Asp Asn Leu Gly Cys Ser Cys Asn Pro Ser Ile Leu 545 550 555 560 Pro Ile Glu Asp Phe Gln Thr Gln Phe Asn Leu Thr Val Ala Glu Glu 565 570 575 Lys Ile Ile Lys His Glu Thr Leu Pro Tyr Gly Arg Pro Arg Val Leu 580 585 590 Gln Lys Glu Asn Thr Ile Cys Leu Leu Ser Gln His Gln Phe Met Ser 595 600 605 Gly Tyr Ser Gln Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val 610 615 620 Asp Arg Asn Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr 625 630 635 640 Gln Asp Phe Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Phe Tyr 645 650 655 Lys Asn Asn Thr Lys Val Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu 660 665 670 Asn Lys Asn Ser Ser Gly Ile Tyr Ser Glu Ala Leu Leu Thr Thr Asn 675 680 685 Ile Val Pro Met Tyr Gln Ser Phe Gln Val Ile Trp Arg Tyr Phe His 690 695 700 Asp Thr Leu Leu Arg Lys Tyr Ala Glu Glu Arg Asn Gly Val Asn Val 705 710 715 720 Val Ser Gly Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Cys Asp Ser 725 730 735 Leu Glu Asn Leu Arg Gln Lys Arg Arg Val Ile Arg Asn Gln Glu Ile 740 745 750 Leu Ile Pro Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Asp Thr 755 760 765 Ser Gln Thr Pro Leu His Cys Glu Asn Leu Asp Thr Leu Ala Phe Ile 770 775 780 Leu Pro His Arg Thr Asp Asn Ser Glu Ser Cys Val His Gly Lys His 785 790 795 800 Asp Ser Ser Trp Val Glu Glu Leu Leu Met Leu His Arg Ala Arg Ile 805 810 815 Thr Asp Val Glu His Ile Thr Gly Leu Ser Phe Tyr Gln Gln Arg Lys 820 825 830 Glu Pro Val Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Thr Phe 835 840 845 Ser Gln Glu Asp Leu Ile Asn Asp Lys Thr His Thr Cys Pro Pro Cys 850 855 860 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 865 870 875 880 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 885 890 895 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 900 905 910 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 915 920 925 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 930 935 940 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 945 950 955 960 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 965 970 975 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 980 985 990 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 995 1000 1005 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 1010 1015 1020 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 1025 1030 1035 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 1040 1045 1050 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 1055 1060 1065 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1070 1075 1080 <210> 19 <211> 849 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 19 Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu 1 5 10 15 Ala Pro Gly Ala Gly Ala Pro Ser Cys Ala Lys Glu Val Lys Ser Cys 20 25 30 Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala 35 40 45 Ala Cys Val Glu Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys 50 55 60 Ile Glu Pro Glu His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu 65 70 75 80 Lys Arg Leu Thr Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp 85 90 95 Lys Gly Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys 100 105 110 Ser Trp Val Glu Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro 115 120 125 Ala Gly Phe Glu Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe 130 135 140 Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser 145 150 155 160 Lys Leu Lys Lys Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr 165 170 175 Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr 180 185 190 Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met 195 200 205 Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp 210 215 220 Tyr Lys Gly Glu Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys 225 230 235 240 Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile 245 250 255 Phe Pro Asp Ile Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu 260 265 270 Arg Ile Leu Ala Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg 275 280 285 Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His 290 295 300 Ser Tyr Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val 305 310 315 320 Asp Gly Met Val Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu 325 330 335 His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln 340 345 350 Gly Ser Cys Lys Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val 355 360 365 Lys Asn Ile Lys Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser 370 375 380 Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg 385 390 395 400 Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys 405 410 415 His Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu 420 425 430 Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro 435 440 445 Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val 450 455 460 Phe Ser Asn Met Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys 465 470 475 480 His Gly Ile Glu Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu 485 490 495 Met Cys Asp Leu Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His 500 505 510 Gly Ser Leu Asn His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His 515 520 525 Pro Lys Glu Val His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro 530 535 540 Arg Asp Asn Leu Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu 545 550 555 560 Asp Phe Gln Thr Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile 565 570 575 Lys His Glu Thr Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu 580 585 590 Asn Thr Ile Cys Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser 595 600 605 Gln Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn 610 615 620 Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe 625 630 635 640 Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn 645 650 655 Thr Lys Val Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn 660 665 670 Ser Ser Gly Ile Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro 675 680 685 Met Tyr Gln Ser Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu 690 695 700 Leu Arg Lys Tyr Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly 705 710 715 720 Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn 725 730 735 Leu Arg Gln Lys Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro 740 745 750 Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr 755 760 765 Pro Leu His Cys Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His 770 775 780 Arg Thr Asp Asn Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser 785 790 795 800 Trp Val Glu Glu Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val 805 810 815 Glu His Ile Thr Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val 820 825 830 Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu 835 840 845 Asp <210> 20 <211> 1079 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 20 Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu 1 5 10 15 Ala Pro Gly Ala Gly Ala Pro Ser Cys Ala Lys Glu Val Lys Ser Cys 20 25 30 Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala 35 40 45 Ala Cys Val Glu Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys 50 55 60 Ile Glu Pro Glu His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu 65 70 75 80 Lys Arg Leu Thr Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp 85 90 95 Lys Gly Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys 100 105 110 Ser Trp Val Glu Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro 115 120 125 Ala Gly Phe Glu Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe 130 135 140 Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser 145 150 155 160 Lys Leu Lys Lys Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr 165 170 175 Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr 180 185 190 Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met 195 200 205 Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp 210 215 220 Tyr Lys Gly Glu Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys 225 230 235 240 Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile 245 250 255 Phe Pro Asp Ile Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu 260 265 270 Arg Ile Leu Ala Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg 275 280 285 Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His 290 295 300 Ser Tyr Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val 305 310 315 320 Asp Gly Met Val Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu 325 330 335 His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln 340 345 350 Gly Ser Cys Lys Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val 355 360 365 Lys Asn Ile Lys Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser 370 375 380 Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg 385 390 395 400 Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys 405 410 415 His Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu 420 425 430 Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro 435 440 445 Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val 450 455 460 Phe Ser Asn Met Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys 465 470 475 480 His Gly Ile Glu Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu 485 490 495 Met Cys Asp Leu Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His 500 505 510 Gly Ser Leu Asn His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His 515 520 525 Pro Lys Glu Val His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro 530 535 540 Arg Asp Asn Leu Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu 545 550 555 560 Asp Phe Gln Thr Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile 565 570 575 Lys His Glu Thr Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu 580 585 590 Asn Thr Ile Cys Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser 595 600 605 Gln Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn 610 615 620 Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe 625 630 635 640 Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn 645 650 655 Thr Lys Val Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn 660 665 670 Ser Ser Gly Ile Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro 675 680 685 Met Tyr Gln Ser Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu 690 695 700 Leu Arg Lys Tyr Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly 705 710 715 720 Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn 725 730 735 Leu Arg Gln Lys Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro 740 745 750 Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr 755 760 765 Pro Leu His Cys Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His 770 775 780 Arg Thr Asp Asn Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser 785 790 795 800 Trp Val Glu Glu Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val 805 810 815 Glu His Ile Thr Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val 820 825 830 Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu 835 840 845 Asp Leu Ile Asn Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 850 855 860 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 865 870 875 880 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 885 890 895 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 900 905 910 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 915 920 925 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 930 935 940 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 945 950 955 960 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 965 970 975 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 980 985 990 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 995 1000 1005 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 1010 1015 1020 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 1025 1030 1035 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 1040 1045 1050 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 1055 1060 1065 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1070 1075 <210> 21 <211> 1550 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 21 Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser Arg Gly Gly Glu Gly 1 5 10 15 Gly Arg Ala Pro Arg Glu Gly Pro Ala Gly Asn Gly Arg Asp Arg Gly 20 25 30 Arg Ser His Ala Ala Glu Ala Pro Gly Asp Pro Gln Ala Ala Ala Ser 35 40 45 Leu Leu Ala Pro Met Asp Val Gly Glu Glu Pro Leu Glu Lys Ala Ala 50 55 60 Arg Ala Arg Thr Ala Lys Asp Pro Asn Thr Tyr Lys Ile Ile Ser Leu 65 70 75 80 Phe Thr Phe Ala Val Gly Val Asn Ile Cys Leu Gly Phe Thr Ala Gly 85 90 95 Leu Lys Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys 100 105 110 Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu 115 120 125 Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu 130 135 140 His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr 145 150 155 160 Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys 165 170 175 Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu 180 185 190 Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu 195 200 205 Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr 210 215 220 Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys 225 230 235 240 Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr 245 250 255 Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His 260 265 270 Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe 275 280 285 Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu 290 295 300 Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe 305 310 315 320 Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile 325 330 335 Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala 340 345 350 Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr 355 360 365 Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro 370 375 380 Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val 385 390 395 400 Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu 405 410 415 Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys 420 425 430 Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys 435 440 445 Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp 450 455 460 Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys 465 470 475 480 Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro 485 490 495 Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe 500 505 510 Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys 515 520 525 Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met 530 535 540 Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu 545 550 555 560 Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu 565 570 575 Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn 580 585 590 His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val 595 600 605 His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu 610 615 620 Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr 625 630 635 640 Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr 645 650 655 Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys 660 665 670 Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu 675 680 685 Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser 690 695 700 Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu 705 710 715 720 Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser 725 730 735 Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile 740 745 750 Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser 755 760 765 Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr 770 775 780 Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp 785 790 795 800 Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys 805 810 815 Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe 820 825 830 Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys 835 840 845 Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn 850 855 860 Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu 865 870 875 880 Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr 885 890 895 Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu 900 905 910 Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp Arg Ser Gly 915 920 925 Ser Gly Gly Ser Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val 930 935 940 Ser Gly Ser Ala Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys 945 950 955 960 Ser Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu Gln His Phe Lys 965 970 975 Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln Lys Cys Ser Tyr 980 985 990 Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr 995 1000 1005 Cys Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His 1010 1015 1020 Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Asn Leu Arg Glu 1025 1030 1035 Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro Glu 1040 1045 1050 Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Ser Leu 1055 1060 1065 Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe 1070 1075 1080 Lys Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val 1085 1090 1095 Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr 1100 1105 1110 Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala Glu Ala 1115 1120 1125 Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu 1130 1135 1140 Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys Ser Ser 1145 1150 1155 Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala 1160 1165 1170 Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr 1175 1180 1185 Lys Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His 1190 1195 1200 Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys 1205 1210 1215 Tyr Met Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr 1220 1225 1230 Cys Cys Asp Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu 1235 1240 1245 Val Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala 1250 1255 1260 Asp Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala 1265 1270 1275 Lys Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg 1280 1285 1290 His Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys 1295 1300 1305 Tyr Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro 1310 1315 1320 Ala Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu Val Glu 1325 1330 1335 Glu Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys 1340 1345 1350 Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg Tyr Thr 1355 1360 1365 Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala 1370 1375 1380 Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro Glu 1385 1390 1395 Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu 1400 1405 1410 Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His 1415 1420 1425 Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys 1430 1435 1440 Phe Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe 1445 1450 1455 Lys Ala Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro 1460 1465 1470 Glu Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu 1475 1480 1485 Val Lys His Lys Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val 1490 1495 1500 Met Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala 1505 1510 1515 Asp Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn Leu Val Thr 1520 1525 1530 Arg Cys Lys Asp Ala Leu Ala Arg Ser Trp Ser His Pro Gln Phe 1535 1540 1545 Glu Lys 1550 <210> 22 <211> 1474 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 22 Met Arg Gly Pro Ala Val Leu Leu Thr Val Ala Leu Ala Thr Leu Leu 1 5 10 15 Ala Pro Gly Ala Gly Ala Pro Ser Cys Ala Lys Glu Val Lys Ser Cys 20 25 30 Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala 35 40 45 Ala Cys Val Glu Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys 50 55 60 Ile Glu Pro Glu His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu 65 70 75 80 Lys Arg Leu Thr Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp 85 90 95 Lys Gly Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys 100 105 110 Ser Trp Val Glu Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro 115 120 125 Ala Gly Phe Glu Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe 130 135 140 Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser 145 150 155 160 Lys Leu Lys Lys Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr 165 170 175 Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr 180 185 190 Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met 195 200 205 Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp 210 215 220 Tyr Lys Gly Glu Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys 225 230 235 240 Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile 245 250 255 Phe Pro Asp Ile Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu 260 265 270 Arg Ile Leu Ala Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg 275 280 285 Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His 290 295 300 Ser Tyr Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val 305 310 315 320 Asp Gly Met Val Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu 325 330 335 His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln 340 345 350 Gly Ser Cys Lys Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val 355 360 365 Lys Asn Ile Lys Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser 370 375 380 Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg 385 390 395 400 Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys 405 410 415 His Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu 420 425 430 Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro 435 440 445 Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val 450 455 460 Phe Ser Asn Met Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys 465 470 475 480 His Gly Ile Glu Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu 485 490 495 Met Cys Asp Leu Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His 500 505 510 Gly Ser Leu Asn His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His 515 520 525 Pro Lys Glu Val His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro 530 535 540 Arg Asp Asn Leu Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu 545 550 555 560 Asp Phe Gln Thr Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile 565 570 575 Lys His Glu Thr Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu 580 585 590 Asn Thr Ile Cys Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser 595 600 605 Gln Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn 610 615 620 Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe 625 630 635 640 Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn 645 650 655 Thr Lys Val Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn 660 665 670 Ser Ser Gly Ile Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro 675 680 685 Met Tyr Gln Ser Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu 690 695 700 Leu Arg Lys Tyr Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly 705 710 715 720 Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn 725 730 735 Leu Arg Gln Lys Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro 740 745 750 Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr 755 760 765 Pro Leu His Cys Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His 770 775 780 Arg Thr Asp Asn Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser 785 790 795 800 Trp Val Glu Glu Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val 805 810 815 Glu His Ile Thr Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val 820 825 830 Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu 835 840 845 Asp Arg Ser Gly Ser Gly Gly Ser Met Lys Trp Val Thr Phe Leu Leu 850 855 860 Leu Leu Phe Val Ser Gly Ser Ala Phe Ser Arg Gly Val Phe Arg Arg 865 870 875 880 Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu 885 890 895 Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln 900 905 910 Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp 915 920 925 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys 930 935 940 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Asn Leu 945 950 955 960 Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro 965 970 975 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Ser Leu 980 985 990 Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe Lys 995 1000 1005 Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val Ala 1010 1015 1020 Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala 1025 1030 1035 Glu Gln Tyr Asn Glu Ile Thr Gln Cys Cys Ala Glu Ala Asp 1040 1045 1050 Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu Lys 1055 1060 1065 Ala Leu Will Be Val Arg Gln Arg Met Lys Cys Ser Ser Met 1070 1075 1080 Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 1085 1090 1095 Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr Lys 1100 1105 1110 Only Thr Asp With Thr Lys Val Asn Lys Glu Cys Cys His Gly 1115 1120 1125 Asp Leu Leu Glu Cys Ala Asp Asp Arg Glu Leu Ala Lys Tyr 1130 1135 1140 Met Cys Glu Asn Gln Ala Thr Is Served By Lys Leu Gln Thr Cys 1145 1150 1155 Cys Asp Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val 1160 1165 1170 Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp 1175 1180 1185 Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys 1190 1195 1200 Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His 1205 1210 1215 Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys Tyr 1220 1225 1230 Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro Ala 1235 1240 1245 Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu Val Glu Glu 1250 1255 1260 Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys Leu 1265 1270 1275 Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg Tyr Thr Gln 1280 1285 1290 Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala Arg 1295 1300 1305 Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp 1310 1315 1320 Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn 1325 1330 1335 Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His Val 1340 1345 1350 Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe 1355 1360 1365 Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys 1370 1375 1380 Ala Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu 1385 1390 1395 Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val 1400 1405 1410 Lys His Lys Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met 1415 1420 1425 Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp 1430 1435 1440 Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn Leu Val Thr Arg 1445 1450 1455 Cys Lys Asp Ala Leu Ala Arg Ser Trp Ser His Pro Gln Phe Glu 1460 1465 1470 Lys <210> 23 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 23 Ile Ile Ser Leu Phe Thr Phe Ala Val Gly Val Asn Ile Cys Leu Gly 1 5 10 15 Phe Thr Ala <210> 24 <211> 850 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 24 Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser 1 5 10 15 Leu Ser Thr Thr Phe Ser Leu Gln Pro Ser Cys Ala Lys Glu Val Lys 20 25 30 Ser Cys Lys Gly Arg Cys Phe Glu Arg Thr Phe Ser Asn Cys Arg Cys 35 40 45 Asp Ala Ala Cys Val Ser Leu Gly Asn Cys Cys Leu Asp Phe Gln Glu 50 55 60 Thr Cys Val Glu Pro Thr His Ile Trp Thr Cys Asn Lys Phe Arg Cys 65 70 75 80 Gly Glu Lys Arg Leu Ser Arg Phe Val Cys Ser Cys Ala Asp Asp Cys 85 90 95 Lys Thr His Asn Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln Asp 100 105 110 Lys Lys Ser Trp Val Glu Glu Thr Cys Glu Ser Ile Asp Thr Pro Glu 115 120 125 Cys Pro Ala Glu Phe Glu Ser Pro Pro Thr Leu Leu Phe Ser Leu Asp 130 135 140 Gly Phe Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val 145 150 155 160 Ile Ser Lys Leu Lys Asn Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro 165 170 175 Met Tyr Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly 180 185 190 Leu Tyr Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro 195 200 205 Lys Met Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro 210 215 220 Leu Trp Tyr Lys Gly Gln Pro Ile Trp Val Thr Ala Asn His Gln Glu 225 230 235 240 Val Lys Ser Gly Thr Tyr Phe Trp Pro Gly Ser Asp Val Glu Ile Asp 245 250 255 Gly Ile Leu Pro Asp Ile Tyr Lys Val Tyr Asn Gly Ser Val Pro Phe 260 265 270 Glu Glu Arg Ile Leu Ala Val Leu Glu Trp Leu Gln Leu Pro Ser His 275 280 285 Glu Arg Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser 290 295 300 Gly His Ser His Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu Gln 305 310 315 320 Lys Val Asp Arg Leu Val Gly Met Leu Met Asp Gly Leu Lys Asp Leu 325 330 335 Gly Leu Asp Lys Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met 340 345 350 Glu Gln Gly Ser Cys Lys Lys Tyr Val Tyr Leu Asn Lys Tyr Leu Gly 355 360 365 Asp Val Asn Asn Val Lys Val Val Tyr Gly Pro Ala Ala Arg Leu Arg 370 375 380 Pro Thr Asp Val Pro Glu Thr Tyr Tyr Ser Phe Asn Tyr Glu Ala Leu 385 390 395 400 Ala Lys Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Arg Pro Tyr 405 410 415 Leu Lys Pro Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp Arg 420 425 430 Ile Glu Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu 435 440 445 Asn Pro Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser Asp 450 455 460 Asn Leu Phe Ser Asn Met Gln Ala Leu Phe Ile Gly Tyr Gly Pro Ala 465 470 475 480 Phe Lys His Gly Ala Glu Val Asp Ser Phe Glu Asn Ile Glu Val Tyr 485 490 495 Asn Leu Met Cys Asp Leu Leu Gly Leu Ile Pro Ala Pro Asn Asn Gly 500 505 510 Ser His Gly Ser Leu Asn His Leu Leu Lys Lys Pro Ile Tyr Asn Pro 515 520 525 Ser His Pro Lys Glu Glu Gly Phe Leu Ser Gln Cys Pro Ile Lys Ser 530 535 540 Thr Ser Asn Asp Leu Gly Cys Thr Cys Asp Pro Trp Ile Val Pro Ile 545 550 555 560 Lys Asp Phe Glu Lys Gln Leu Asn Leu Thr Thr Glu Asp Val Asp Asp 565 570 575 Ile Tyr His Met Thr Val Pro Tyr Gly Arg Pro Arg Ile Leu Leu Lys 580 585 590 Gln His Arg Val Cys Leu Leu Gln Gln Gln Gln Phe Leu Thr Gly Tyr 595 600 605 Ser Leu Asp Leu Leu Met Pro Leu Trp Ala Ser Tyr Thr Phe Leu Ser 610 615 620 Asn Asp Gln Phe Ser Arg Asp Asp Phe Ser Asn Cys Leu Tyr Gln Asp 625 630 635 640 Leu Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Tyr Tyr Lys Ser 645 650 655 Asn Ser Lys Leu Ser Tyr Gly Phe Leu Thr Pro Pro Arg Leu Asn Arg 660 665 670 Val Ser Asn His Ile Tyr Ser Glu Ala Leu Leu Thr Ser Asn Ile Val 675 680 685 Pro Met Tyr Gln Ser Phe Gln Val Ile Trp His Tyr Leu His Asp Thr 690 695 700 Leu Leu Gln Arg Tyr Ala His Glu Arg Asn Gly Ile Asn Val Val Ser 705 710 715 720 Gly Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Tyr Asp Ser Leu Glu 725 730 735 Ile Leu Lys Gln Asn Ser Arg Val Ile Arg Ser Gln Glu Ile Leu Ile 740 745 750 Pro Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Gln Leu Ser Glu 755 760 765 Thr Pro Leu Glu Cys Ser Ala Leu Glu Ser Ser Ala Tyr Ile Leu Pro 770 775 780 His Arg Pro Asp Asn Ile Glu Ser Cys Thr His Gly Lys Arg Glu Ser 785 790 795 800 Ser Trp Val Glu Glu Leu Leu Thr Leu His Arg Ala Arg Val Thr Asp 805 810 815 Val Glu Leu Ile Thr Gly Leu Ser Phe Tyr Gln Asp Arg Gln Glu Ser 820 825 830 Val Ser Glu Leu Leu Arg Leu Lys Thr His Leu Pro Ile Phe Ser Gln 835 840 845 Glu Asp 850 <210> 25 <211> 1474 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 25 Met Thr Ser Lys Phe Leu Leu Val Ser Phe Ile Leu Ala Ala Leu Ser 1 5 10 15 Leu Ser Thr Thr Phe Ser Leu Gln Pro Ser Cys Ala Lys Glu Val Lys 20 25 30 Ser Cys Lys Gly Arg Cys Phe Glu Arg Thr Phe Ser Asn Cys Arg Cys 35 40 45 Asp Ala Ala Cys Val Ser Leu Gly Asn Cys Cys Leu Asp Phe Gln Glu 50 55 60 Thr Cys Val Glu Pro Thr His Ile Trp Thr Cys Asn Lys Phe Arg Cys 65 70 75 80 Gly Glu Lys Arg Leu Ser Arg Phe Val Cys Ser Cys Ala Asp Asp Cys 85 90 95 Lys Thr His Asn Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln Asp 100 105 110 Lys Lys Ser Trp Val Glu Glu Thr Cys Glu Ser Ile Asp Thr Pro Glu 115 120 125 Cys Pro Ala Glu Phe Glu Ser Pro Pro Thr Leu Leu Phe Ser Leu Asp 130 135 140 Gly Phe Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val 145 150 155 160 Ile Ser Lys Leu Lys Asn Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro 165 170 175 Met Tyr Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly 180 185 190 Leu Tyr Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro 195 200 205 Lys Met Asn Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro 210 215 220 Leu Trp Tyr Lys Gly Gln Pro Ile Trp Val Thr Ala Asn His Gln Glu 225 230 235 240 Val Lys Ser Gly Thr Tyr Phe Trp Pro Gly Ser Asp Val Glu Ile Asp 245 250 255 Gly Ile Leu Pro Asp Ile Tyr Lys Val Tyr Asn Gly Ser Val Pro Phe 260 265 270 Glu Glu Arg Ile Leu Ala Val Leu Glu Trp Leu Gln Leu Pro Ser His 275 280 285 Glu Arg Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser 290 295 300 Gly His Ser His Gly Pro Val Ser Ser Glu Val Ile Lys Ala Leu Gln 305 310 315 320 Lys Val Asp Arg Leu Val Gly Met Leu Met Asp Gly Leu Lys Asp Leu 325 330 335 Gly Leu Asp Lys Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met 340 345 350 Glu Gln Gly Ser Cys Lys Lys Tyr Val Tyr Leu Asn Lys Tyr Leu Gly 355 360 365 Asp Val Asn Asn Val Lys Val Val Tyr Gly Pro Ala Ala Arg Leu Arg 370 375 380 Pro Thr Asp Val Pro Glu Thr Tyr Tyr Ser Phe Asn Tyr Glu Ala Leu 385 390 395 400 Ala Lys Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Arg Pro Tyr 405 410 415 Leu Lys Pro Phe Leu Pro Lys Arg Leu His Phe Ala Lys Ser Asp Arg 420 425 430 Ile Glu Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu 435 440 445 Asn Pro Ser Glu Arg Lys Tyr Cys Gly Ser Gly Phe His Gly Ser Asp 450 455 460 Asn Leu Phe Ser Asn Met Gln Ala Leu Phe Ile Gly Tyr Gly Pro Ala 465 470 475 480 Phe Lys His Gly Ala Glu Val Asp Ser Phe Glu Asn Ile Glu Val Tyr 485 490 495 Asn Leu Met Cys Asp Leu Leu Gly Leu Ile Pro Ala Pro Asn Asn Gly 500 505 510 Ser His Gly Ser Leu Asn His Leu Leu Lys Lys Pro Ile Tyr Asn Pro 515 520 525 Ser His Pro Lys Glu Glu Gly Phe Leu Ser Gln Cys Pro Ile Lys Ser 530 535 540 Thr Ser Asn Asp Leu Gly Cys Thr Cys Asp Pro Trp Ile Val Pro Ile 545 550 555 560 Lys Asp Phe Glu Lys Gln Leu Asn Leu Thr Thr Glu Asp Val Asp Asp 565 570 575 Ile Tyr His Met Thr Val Pro Tyr Gly Arg Pro Arg Ile Leu Leu Lys 580 585 590 Gln His Arg Val Cys Leu Leu Gln Gln Gln Gln Phe Leu Thr Gly Tyr 595 600 605 Ser Leu Asp Leu Leu Met Pro Leu Trp Ala Ser Tyr Thr Phe Leu Ser 610 615 620 Asn Asp Gln Phe Ser Arg Asp Asp Phe Ser Asn Cys Leu Tyr Gln Asp 625 630 635 640 Leu Arg Ile Pro Leu Ser Pro Val His Lys Cys Ser Tyr Tyr Lys Ser 645 650 655 Asn Ser Lys Leu Ser Tyr Gly Phe Leu Thr Pro Pro Arg Leu Asn Arg 660 665 670 Val Ser Asn His Ile Tyr Ser Glu Ala Leu Leu Thr Ser Asn Ile Val 675 680 685 Pro Met Tyr Gln Ser Phe Gln Val Ile Trp His Tyr Leu His Asp Thr 690 695 700 Leu Leu Gln Arg Tyr Ala His Glu Arg Asn Gly Ile Asn Val Val Ser 705 710 715 720 Gly Pro Val Phe Asp Phe Asp Tyr Asp Gly Arg Tyr Asp Ser Leu Glu 725 730 735 Ile Leu Lys Gln Asn Ser Arg Val Ile Arg Ser Gln Glu Ile Leu Ile 740 745 750 Pro Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Gln Leu Ser Glu 755 760 765 Thr Pro Leu Glu Cys Ser Ala Leu Glu Ser Ser Ala Tyr Ile Leu Pro 770 775 780 His Arg Pro Asp Asn Ile Glu Ser Cys Thr His Gly Lys Arg Glu Ser 785 790 795 800 Ser Trp Val Glu Glu Leu Leu Thr Leu His Arg Ala Arg Val Thr Asp 805 810 815 Val Glu Leu Ile Thr Gly Leu Ser Phe Tyr Gln Asp Arg Gln Glu Ser 820 825 830 Val Ser Glu Leu Leu Arg Leu Lys Thr His Leu Pro Ile Phe Ser Gln 835 840 845 Glu Asp Gly Gly Ser Gly Gly Ser Met Lys Trp Val Thr Phe Leu Leu 850 855 860 Leu Leu Phe Val Ser Gly Ser Ala Phe Ser Arg Gly Val Phe Arg Arg 865 870 875 880 Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu 885 890 895 Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln 900 905 910 Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp 915 920 925 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys 930 935 940 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Asn Leu 945 950 955 960 Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro 965 970 975 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Ser Leu 980 985 990 Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe Lys 995 1000 1005 Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val Ala 1010 1015 1020 Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala 1025 1030 1035 Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala Glu Ala Asp 1040 1045 1050 Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu Lys 1055 1060 1065 Ala Leu Will Be Val Arg Gln Arg Met Lys Cys Ser Ser Met 1070 1075 1080 Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 1085 1090 1095 Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr Lys 1100 1105 1110 Only Thr Asp With Thr Lys Val Asn Lys Glu Cys Cys His Gly 1115 1120 1125 Asp Leu Leu Glu Cys Ala Asp Asp Arg Glu Leu Ala Lys Tyr 1130 1135 1140 Met Cys Glu Asn Gln Ala Thr Is Served By Lys Leu Gln Thr Cys 1145 1150 1155 Cys Asp Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val 1160 1165 1170 Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp 1175 1180 1185 Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys 1190 1195 1200 Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His 1205 1210 1215 Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys Tyr 1220 1225 1230 Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro Ala 1235 1240 1245 Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu Val Glu Glu 1250 1255 1260 Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys Leu 1265 1270 1275 Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg Tyr Thr Gln 1280 1285 1290 Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala Rec 1295 1300 1305 Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp 1310 1315 1320 Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn 1325 1330 1335 Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His Val 1340 1345 1350 Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe 1355 1360 1365 Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys 1370 1375 1380 Ala Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu 1385 1390 1395 Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val 1400 1405 1410 Lys His Lys Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met 1415 1420 1425 Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp 1430 1435 1440 Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn Leu Val Thr Arg 1445 1450 1455 Cys Lys Asp Ala Leu Ala Arg Ser Trp Ser His Pro Gln Phe Glu 1460 1465 1470 Lys <210> 26 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 26 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 27 <211> 618 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 27 Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala 1 5 10 15 Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30 His Arg Tyr Asn Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu 35 40 45 Ile Ala Phe Ser Gln Tyr Leu Gln Lys Cys Ser Tyr Asp Glu His Ala 50 55 60 Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Ala Ile Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala 100 105 110 Asp Cys Cys Thr Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu Ala 130 135 140 Glu Ala Met Cys Thr Ser Phe Lys Glu Asn Pro Thr Thr Phe Met Gly 145 150 155 160 His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys 180 185 190 Cys Ala Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly 195 200 205 Val Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys 210 215 220 Ser Ser Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr 245 250 255 Lys Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280 285 Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys Cys Asp 290 295 300 Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val Glu His Asp 305 310 315 320 Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp Phe Val Glu Asp 325 330 335 Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser 355 360 365 Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys 370 375 380 Cys Ala Glu Ala Asn Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu 385 390 395 400 Phe Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys 405 410 415 Asp Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu 420 425 430 Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu 450 455 460 Pro Glu Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile 465 470 475 480 Leu Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His 485 490 495 Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala 515 520 525 Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu Lys Glu 530 535 540 Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met Asp Asp Phe Ala 565 570 575 Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe 580 585 590 Ser Thr Glu Gly Pro Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala 595 600 605 Arg Ser Trp Ser His Pro Gln Phe Glu Lys 610 615 <210> 28 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 28 Leu Ile Asn 1 <210> 29 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 29 Gly Gly Ser Gly Gly Ser 1 5 <210> 30 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 30 Arg Ser Gly Ser Gly Gly Ser 1 5

Claims

1. Formula (I): Protein-Z-Domain-XY (I) or a salt thereof, In (I), The protein is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:24; The domain is selected from the group consisting of a human IgG Fc domain (Fc), a human serum albumin protein (ALB), and fragments thereof; X and Z are, independently, null or a polypeptide containing 1 to 20 amino acids; and Y is absent or wherein m is an integer from 1 to 15 and n is an integer from 1 to 10; The compound or a salt thereof.

2. The compound of claim 1, wherein the domain is Fc or a fragment thereof.

3. The compound of claim 1, wherein the domain is ALB or a fragment thereof.

4. 2. The compound of claim 1, wherein Y is absent and the compound lacks a negatively charged bone-targeting sequence.

5. 2. The compound of claim 1, wherein the protein has a mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 compared to SEQ ID NO:

1.

6. The compound of claim 1, wherein the nuclease domain of said protein or variant thereof is absent.

7. The compound of claim 1, wherein the nuclease domain from about residue 524 to about residue 885 of SEQ ID NO:1 is absent in the protein or variant thereof.

8. The compound of claim 1, wherein a segment of the extracellular region of NPP2 containing a furin or signal peptide cleavage site is or is not introduced into the protein or a variant thereof by substitution.

9. The domain is Fc or a fragment thereof, and the protein-Z-domain is (SEQ ID NO: 15)-Z-(Fc or a fragment thereof), (SEQ ID NO: 17)-Z-(Fc or a fragment thereof), (SEQ ID NO: 19)-Z-(Fc or a fragment thereof), (SEQ ID NO: 24)-Z-(Fc or a fragment thereof), or variants thereof, a variant comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 relative to NO:1; 2. The compound of claim 1, comprising:

10. the protein-Z-domain SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, (SEQ ID NO:24)-Z-(SEQ ID NO:26), or variants thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 compared to SEQ ID NO:

1.

10. The compound of claim 9, comprising:

11. The domain is ALB or a fragment thereof, and the protein-Z-domain is (SEQ ID NO:15)-Z-(ALB or a fragment thereof), (SEQ ID NO:17)-Z-(ALB or a fragment thereof), (SEQ ID NO:19)-Z-(ALB or a fragment thereof), (SEQ ID NO:24)-Z-(ALB or a fragment thereof), or variants thereof, a variant comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 relative to NO:1; 2. The compound of claim 1, comprising:

12. the protein-Z-domain SEQ ID NO:21, (SEQ ID NO:17)-Z-(SEQ ID NO:27), SEQ ID NO:22, SEQ ID NO:25, or variants thereof, comprising at least one mutation at at least one position selected from the group consisting of Ser532, Tyr529, Tyr451, Ile450, Ser381, Tyr382, Ser377, Phe346, Gly531, Ser289, Ser287, Ala454, Gly452, Gln519, Glu526, Lys448, Glu508, Arg456, Asp276, Tyr434, Gln519, Ser525, Gly342, Ser343, and Gly536 compared to SEQ ID NO:

1.

12. The compound of claim 11, comprising:

13. The compound has a viscosity of 3.4 (±0.4) s -1 enzyme -1 More than k cat having a value k cat 2. The compound of claim 1, wherein is determined by measuring the rate of ATP hydrolysis of the compound.

14. The compound has a K M having a value of K M 2. The compound of claim 1, wherein is determined by measuring the rate of ATP hydrolysis of the compound.

15. 15. A method for treating or preventing a disease or disorder associated with pathologic calcification or pathologic ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of any one of claims 1 to 14, wherein the disease comprises at least one selected from the group consisting of generalized arterial calcification of infancy (GACI), idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, and calcification of atherosclerotic plaques.

16. 16. The method of claim 15, wherein Y is absent and the compound lacks a negatively charged bone-targeting sequence.

17. 15. A method for treating or preventing a disease or disorder associated with pathological calcification or pathological ossification in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of at least one compound according to any one of claims 1 to 14, wherein the disease comprises at least one selected from the group consisting of PXE, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, arteriosclerosis that occurs with aging, calciphylaxis due to end-stage renal disease, and progeria.

18. 18. The method of claim 17, wherein Y is absent and the compound lacks a negatively charged bone-targeting sequence.

19. A method for reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in an infant suffering from at least one disease or disorder selected from the group consisting of GACI and PXE, comprising the step of administering to the infant a therapeutically effective amount of a predetermined polypeptide comprising an ectonucleotide pyrophosphate / phosphodiesterase-1 (NPP1) polypeptide and Fc, wherein the predetermined polypeptide lacks a polyaspartic acid domain, and administration of the predetermined polypeptide increases extracellular pyrophosphate (PPi) concentration in the infant, thereby reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in the infant.

20. 20. The method of claim 19, wherein the administration is subcutaneous.

21. 20. The method of claim 19, wherein said administration restores the infant's extracellular pyrophosphate concentration to a level within the range found in infants not suffering from GACI and / or PXE.

22. 20. The method of claim 19, wherein said infant exhibits and / or has been diagnosed with "failure to thrive" prior to said administration.

23. The method of claim 19, wherein the NPP1 polypeptide is a cleavage product of a precursor NPP1 polypeptide comprising an ectonucleotide pyrophosphate / phosphodiesterase-2 (NPP2) transmembrane domain.

24. 20. The method of claim 19, wherein the NPP2 transmembrane domain is residues 12 to 30 of NCBI Accession No. NP_001124335 (SEQ ID NO:2), which corresponds to SEQ ID NO:

23.

25. A method for reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in an infant suffering from at least one disease or disorder selected from the group consisting of GACI and PXE, comprising the step of administering to the infant a therapeutically effective amount of a predetermined polypeptide comprising an NPP1 polypeptide and an ALB, wherein the predetermined polypeptide lacks a polyaspartic acid domain, and said administration increases the extracellular PPi concentration in the infant, thereby reducing or preventing cardiac calcification, arterial calcification, and / or elastic fiber calcification in the infant.

26. 26. The method of claim 25, wherein the administering comprises subcutaneous administration.

27. 26. The method of claim 25, wherein said administration restores the infant's extracellular pyrophosphate concentration to a level within the range found in infants not suffering from GACI and / or PXE.

28. 26. The method of claim 25, wherein said infant exhibits and / or has been diagnosed with "failure to thrive" prior to said administration.

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