Claims for Patent: 8,609,383

Summary for Patent: 8,609,383

Title:	Production of carrier-peptide conjugates using chemically reactive unnatural amino acids
Abstract:	Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.
Inventor(s):	Young; Travis (San Diego, CA), Schultz; Peter G. (La Jolla, CA)
Assignee:	The Scripps Research Institute (La Jolla, CA)
Application Number:	13/133,629
Patent Claims:	1. A method of making a covalently coupled carrier polypeptide-target polypeptide conjugate, the method comprising: incorporating a first unnatural amino acid residue into a carrier polypeptide during translation of the carrier polypeptide in a methylotrophic yeast cell, wherein incorporating the first unnatural amino acid into the carrier polypeptide during translation comprises: (a) providing a methylotrophic yeast cell comprising (i) the first unnatural amino acid, (ii) an orthogonal tRNA-synthetase (O--RS) derived from Escherichia coli, wherein providing an O--RS comprises providing an O--RS polynucleotide under the transcriptional control of a FLD1 promoter; (iii) an orthogonal tRNA (O-tRNA) derived from Escherichia coli, wherein the O--RS and the O-tRNA are encoded in a cassette on a single plasmid and stably integrated into the cell genome at an ARG4 gene, and wherein the O--RS preferentially aminoacylates the O-tRNA with the first unnatural amino acid in the methylotrophic yeast cell; and (iv) a nucleic acid encoding the carrier polypeptide, wherein the nucleic acid comprises a selector codon that is recognized by the O-tRNA, and wherein the nucleic acid is encoded in a cassette on a second plasmid and stably integrated into the cell genome; and, (b) translating the nucleic acid, thereby incorporating the first unnatural amino acid into the carrier polypeptide during translation in the methylotrophic yeast cell; incorporating a second unnatural amino acid residue into a target polypeptide during synthesis or translation of the target polypeptide, wherein the target polypeptide is different from the carrier polypeptide, and wherein the second unnatural amino acid is different from the first amino acid; and, reacting the first and second unnatural amino acid residues in vitro to produce the covalently coupled carrier polypeptide-target polypeptide conjugate. 2. The method of claim 1, wherein the carrier or target polypeptide is produced in a Candida cell, a Hansenula cell, a Pichia cell, or a Torulopsis cell. 3. The method of claim 1, wherein the carrier polypeptide is or is homologous to a human serum albumin (HSA), an antibody, a HER2 antibody, an OKT3 antibody, an antibody fragment, an Fab, an Fc, an scFv, an albumin, a serum albumin, a bovine serum albumin, an ovalbumin, a c-reactive protein, a conalbumin, a lactalbumin, a keyhole limpet hemocyanin (KLH), an ion carrier protein, an acyl carrier protein, a signal transducing adaptor protein, an androgen-binding protein, a calcium-binding protein, a calmodulin-binding protein, a ceruloplasmin, a cholesterol ester transfer protein, an f-box protein, a fatty acid-binding proteins, a follistatin, a follistatin-related protein, a GTP-binding protein, an insulin-like growth factor binding protein, an iron-binding protein, a latent TGF-beta binding protein, a light-harvesting protein complex, a lymphocyte antigen, a membrane transport protein, a neurophysin, a periplasmic binding protein, a phosphate-binding protein, a phosphatidylethanolamine binding protein, a phospholipid transfer protein, a retinol-binding protein, an RNA-binding protein, an s-phase kinase-associated protein, a sex hormone-binding globulin, a thyroxine-binding protein, a transcobalamin, a transcortin, a transferrin-binding protein, or a vitamin D-binding protein. 4. The method of claim 1, wherein the target polypeptide is or is homologous to a TSP-1, an ABT-510, a glugacon-like peptide-1 (GLP-1), a parathyroid hormone (PTH), a ribosome inactivating protein (RIP), an angiostatin, an Exedin-4, an apoprotein, an atrial natriuretic factor, an atrial natriuretic polypeptide, an atrial peptide, a C--X--C chemokine, a T39765, a NAP-2, an ENA-78, a gro-a, a gro-b, a gro-c, an IP-10, a GCP-2, a NAP-4, an a PF4, a MIG, a calcitonin, a c-kit ligand, a cytokine, a CC chemokine, a monocyte chemoattractant protein-1, a monocyte chemoattractant protein-2, a monocyte chemoattractant protein-3, a monocyte inflammatory protein-1 alpha, a monocyte inflammatory protein-1 beta, a RANTES, an I309, an R83915, an R91733, a T58847, a D31065, a T64262, a CD40 ligand, a complement inhibitor, a cytokine, an epithelial neutrophil activating peptide-78, a GRO'.UPSILON., a MGSA, a GRO.beta., a GRO.gamma., a MIP1-.alpha., a MIP1-.beta., an MCP-1, an epithelial neutrophil activating peptide, an erythropoietin (EPO), an exfoliating toxin, a fibroblast growth factor (FGF), an FGF21, a G-CSF, a gonadotropin, a growth factor, a Hirudin, an LFA-1, a human insulin, a human insulin-like growth factor (hIGF), an hIGF-I, an hIGF-II, a human interferon, an IFN-.alpha., an IFN-.beta., an IFN-.gamma., an interleukin, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, a keratinocyte growth factor (KGF), a leukemia inhibitory factor, a neurturin, a PDGF, a peptide hormone, a pleiotropin, a pyrogenic exotoxin A, a pyrogenic exotoxin B, a pyrogenic exotoxin C, a relaxin, a somatostatin, a superoxide dismutase, a thymosin alpha 1, a human tumor necrosis factor (hTNF), a human tumor necrosis factor alpha, a human tumor necrosis factor beta, a Ras, a Tat, an inflammatory molecule, a signal transduction molecule, a bovine pancreatic trypsin inhibitor (BPTI), or a BP320 antigen. 5. The method of claim 1, wherein the first and second unnatural amino acids are reacted via one or more of: an electrophile-nucleophile reaction, a ketone reaction with a nucleophile, an oxime ligation, an aldehyde reaction with a nucleophile, a reaction between a carbonyl group and a nucleophile, a reaction between a sulfonyl group and a nucleophile, an esterification reaction, a reaction between a hindered ester group and a nucleophile, a reaction between a thioester group and a nucleophile, a reaction between a stable imine group and a nucleophile, a reaction between an epoxide group and a nucleophile, a reaction between an aziridine group and a nucleophile, a reaction between an electrophile and an aliphatic or aromatic amine, a reaction between an electrophile and a hydrazide, a reaction between an electrophile and a carbohydrazide, a reaction between an electrophile and a semicarbazide, a reaction between an electrophile and a thiosemicarbazide, a reaction between an electrophile and a carbonylhydrazide, a reaction between an electrophile and a thiocarbonylhydrazide, a reaction between an electrophile and a sulfonylhydrazide, a reaction between an electrophile and a carbazide, a reaction between an electrophile and a thiocarbazide, a reaction between an electrophile and a hydroxylamine, a reaction between a nucleophile or nucleophiles such as a hydroxyl or diol and a boronic acid or ester, a transition metal catalyzed reaction, a palladium catalyzed reaction, a copper catalyzed heteroatom alkylation reaction, a cycloaddition reaction, a 1,3, cycloaddition reaction, a 2,3 cycloaddition reaction, an alkyne-azide reaction, a Diels-Alder reaction, or a Suzuki coupling reaction. 6. The method of claim 5, wherein the efficiency of said conjugation reaction is determined by a mass difference in yield between the mutant carrier polypeptide conjugate and a wild-type carrier polypeptide, and wherein said reaction is greater than 50% efficient, greater than 70% efficient, or greater than 90% efficient.

Details for Patent 8,609,383

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Eli Lilly And Company	HUMULIN R U-100	insulin human	Injection	018780	10/28/1982		2028-12-10
Eli Lilly And Company	HUMULIN R U-500	insulin human	Injection	018780	12/29/2015		2028-12-10
Eli Lilly And Company	HUMULIN R U-100	insulin human	Injection	018780	08/06/1998		2028-12-10
Eli Lilly And Company	HUMULIN R U-500	insulin human	Injection	018780	03/31/1994		2028-12-10