# US 8,771,683 B2 · Therapeutic apyrase constructs, apyrase agents, and production methods > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,683 B2 - Document ID: US8771683B2 - Status: Granted - Kind: B2 - Application number: 13/522,311 - Filed: 2011-01-13 - Published: 2013-06-06 - Granted: 2014-07-08 - Earliest claimed: 2010-01-13 - Assignees: APT Therapeutics, Inc. - Applicants: Ridong Chen; Soon Seog Jeong - Inventors: Ridong Chen; Soon Seog Jeong - Web page: https://patentgrep.com/patent/US8771683B2 ## CPC - C12N 9/14: Hydrolases (3) - A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors - C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract This invention provides a new class of enhanced apyrases (EN-apyrases) with superior pharmacokinetic, pharmacodynamic, and pharmacochemical properties and which can be purified using simplified procedures. The invention further provides constructs for transforming a cell to produce these EN-apyrases. The EN-apyrase construct comprises sequences encoding a signal sequence, a linker, and a soluble apyrase. Also provided are preparations of apyrases and methods for producing apyrase in culture cells and purification thereof. ## Claims ### Claim 1 A composition comprising an EN-apyrase, wherein the EN-apyrase is a soluble CD39L3 or a homolog thereof, which has a homogeneous N-terminus wherein more than 80% of the EN-apyrase molecules and homologs have the same N-terminus beginning with EVLP (positions 20-23 of SEQ ID NO:24) and wherein (a) the EN-apyrase has an average isoelectric point in the range of about 3.0 to about 4.5; and/or (b) the EN-apyrase has an in vivo half life in rabbits or pigs at least twice that of HEK sol-CD39L3-01, measured by apyrase assay, wherein the homolog of soluble CD39L3 is a sequence having 1 to 5 conservative substitutions with respect to positions 49-485 of SEQ ID NO:1 and/or is at least 80% identical to positions 49-485 of SEQ ID NO:1, and which retains ADP'ase and ATP'ase activity. ### Claim 2 (depends on 1) The EN-apyrase of claim 1 which is produced by a transformed CHO cell line. ### Claim 3 (depends on 1) The EN apyrase of claim 1 wherein the EN-apyrase consists of the amino acid sequence of positions 20-457 of SEQ ID NO:24. ### Claim 4 (depends on 1) A nucleic acid construct for production of an EN-apyrase of claim 1 comprising a nucleotide sequence encoding a signal sequence, a linker, and said soluble CD39L3 apyrase or homolog, wherein the linker has the sequence EVLP as its C-terminus and wherein said linker or a portion thereof may represent a sequence present in the native soluble apyrase, and an operably linked promoter functional in CHO cells so as to generate said EN-apyrase. ### Claim 5 (depends on 4) CHO cells comprising the construct of claim 4. ### Claim 6 (depends on 4) The construct of claim 4 wherein the EN-apyrase has the amino acid sequence of positions 20-457 of SEQ ID NO:24. ### Claim 7 (depends on 5) A method to prepare an EN-apyrase which method comprises culturing the cells of claim 5, and collecting the cultured medium. ### Claim 8 (depends on 7) The method of claim 7 wherein during culturing the medium maintains a glutamine concentration at about 1.5-4 mM and a pH of between 7.0 and 7.8 and wherein the temperature is shifted from 37° C. to 31° C.-35° C. at days 4-6 of culturing. ### Claim 9 (depends on 8) The method of claim 8, wherein during culturing the medium maintains a glutamine concentration at about 2 mM and a pH of 7.4 and wherein the temperature of the culture is shifted from 37° C. to 34° C. at day 5 of culturing. ### Claim 10 (depends on 7) An EN-apyrase prepared by the method of claim 7. ### Claim 11 (depends on 8) An EN-apyrase prepared by the method of claim 8. ### Claim 12 (depends on 9) An EN-apyrase prepared by the method of claim 9. ### Claim 13 (depends on 1) A pharmaceutical composition comprising the EN-apyrase of claim 1. ### Claim 14 (depends on 10) A pharmaceutical composition comprising the EN-apyrase of claim 10. ### Claim 15 (depends on 13) A method to treat conditions benefited by apyrase activity, which method comprises administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 13. ### Claim 16 (depends on 14) A method to treat conditions benefited by apyrase activity, which method comprises administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 14. ### Claim 17 (depends on 1) A method to purify the EN-apyrase of claim 1 which method comprises subjecting culture medium containing said EN-apyrase to anion exchange chromatography, followed by treating EN-apyrase containing eluate from said anion exchange chromatography to purification by cation exchange chromatography. ### Claim 18 (depends on 1) The EN-apyrase of claim 1, wherein more than 90% of the EN-apyrase molecules have the same N-terminus.