# US 8,465,734 B2 · Methods for increasing protein polyethylene glycol (PEG) conjugation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,465,734 B2 - Document ID: US8465734B2 - Status: Granted - Kind: B2 - Application number: 12/875,377 - Filed: 2010-09-03 - Published: 2010-12-30 - Granted: 2013-06-18 - Earliest claimed: 2004-03-17 - Assignees: AntiCancer, Inc. - Applicants: Shukuan Li; Zhijian Yang; Xinghua Sun; Yuying Tan; Shigeo Yagi - Inventors: Shukuan Li; Zhijian Yang; Xinghua Sun; Yuying Tan; Shigeo Yagi - Web page: https://patentgrep.com/patent/US8465734B2 ## CPC - A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol - A61K 38/51: Lyases (4) - A61P 35/00: Antineoplastic agents - C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids} - C12N 9/88: Lyases (4.) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to highly conjugated proteins and methods for making such proteins. In particular, the present invention relates to methods for linking additional sites to a protein for conjugation with activated polyethylene glycol (PEG) linkers, without denaturing the protein. The invention also relates to highly conjugated proteins with decreased immunogenicity and increased circulating half-life. ## Claims ### Claim 1 A modified methioninase protein wherein said modification comprises at least one first polyethylene glycol (PEG) polymer coupled to at least one amino group of said methioninase and at least one second PEG polymer coupled to a carboxyl group of said methioninase through a polyamine linker, wherein one amino group of said polyamine linker forms an amide with said carboxyl group of the methioninase and at least one additional amino group is coupled to said second PEG polymer. ### Claim 2 (depends on 1) The modified methioninase of claim 1 wherein more than one first PEG polymer are coupled to more than one amino group of said methioninase. ### Claim 3 (depends on 1) The modified methioninase of claim 1 wherein more than one second PEG polymer are coupled to more than one amino group of said polyamine linker. ### Claim 4 (depends on 2) The modified methioninase of claim 2 wherein more than one second PEG polymer are coupled to more than one amino group of said polyamine linker. ### Claim 5 (depends on 1) The modified methioninase of claim 1 which comprises six to eight first PEG polymers and six second PEG polymers. ### Claim 6 (depends on 1) The modified methioninase of claim 1 which is prepared by a process which comprises coupling at least one second PEG polymer to at least one amino group of a polyamine linker that forms an amide with at least one carboxyl group of said methioninase, and wherein said methioninase comprises at least one first PEG polymer coupled to at least one amino group of said methioninase. ### Claim 7 (depends on 6) The modified methioninase of claim 6 wherein said process comprises coupling more than one second PEG polymer to more than one amino group of said polyamine linker. ### Claim 8 (depends on 1) The modified methioninase of claim 1 which is a recombinant methioninase. ### Claim 9 (depends on 1) The modified methioninase of claim 1 wherein the poly functional amine linker is diaminobutane. ### Claim 10 (depends on 1) A pharmaceutical composition comprising the modified methioninase of claim 1 and a pharmaceutically acceptable excipient. ### Claim 11 (depends on 1) A method to modulate tumor activity comprising administering to a subject in need thereof a therapeutically affected amount of the modified methioninase of claim 1 or a pharmaceutical composition thereof. ### Claim 12 (depends on 11) The method of claim 11 wherein the subject is human.