Claims for Patent: 9,150,849

Summary for Patent: 9,150,849

Title:	Directed evolution using proteins comprising unnatural amino acids
Abstract:	The invention provides methods and compositions for screening polypeptide libraries that include variants comprising unnatural amino acids. In addition, the invention provides vector packaging systems and methods for packaging a nucleic acid in a vector. Compositions of vectors produced by the methods and systems are also provided.
Inventor(s):	Liu; Chang (San Diego, CA), Tsao; Meng-Lin (Merced, CA), Smider; Vaughn (San Diego, CA), Schultz; Peter G. (La Jolla, CA)
Assignee:	The Scripps Research Institute (La Jolla, CA)
Application Number:	12/290,673
Patent Claims:	1. A recombinant polypeptide expression library with normalized expression levels of a plurality of expressed polypeptide variants comprising unnatural amino acids, wherein the library is expressed from an expression vector, the library comprising: a) a plurality of expressed target polypeptide variants, wherein at least one of the expressed target polypeptide variants comprises at least one unnatural amino acid residue, and b) a packaging or specificity polypeptide comprising at least one same unnatural amino acid as the at least one expressed target polypeptide variant comprising the at least one unnatural amino acid residue of a), which packaging or specificity polypeptide is essential for viability of the expression vector; wherein expressed polypeptides comprising only natural amino acids are present in the library at a molar ratio of 10:1, or less than 10:1, relative to polypeptides in the library comprising the at least one unnatural amino acid residue of a). 2. The recombinant polypeptide expression library of claim 1, wherein at least two of the expressed target polypeptide variants comprise at least one unnatural amino acid residue. 3. The recombinant polypeptide expression library of claim 1, wherein at least three of the expressed target polypeptide variants comprise at least one unnatural amino acid residue. 4. The recombinant polypeptide expression library of claim 1, wherein more than three of the expressed target polypeptide variants comprise at least one unnatural amino acid residue. 5. The recombinant polypeptide expression library of claim 1, wherein at least one of the expressed target polypeptide variants comprises at least two different unnatural amino acid residues. 6. The recombinant polypeptide expression library of claim 1, wherein at least one of the expressed target polypeptide variants comprises more than two different unnatural amino acid residues. 7. The recombinant polypeptide expression library of claim 1, wherein the expressed target polypeptide variants comprise antibody fragment variants, Alpha-1 antitrypsin variants, Angiostatin variants, Antihemolytic factor variants, Apolipoprotein variants, Apoprotein variants, Atrial natriuretic factor variants, Atrial natriuretic polypeptide variants, Atrial peptide variants, C-X-C chemokine variants, T39765 variants, NAP-2 variants, ENA-78 variants, Gro-a variants, Gro-b variants, Gro-c variants, IP-10 variants, GCP-2 variants, NAP-4 variants, SDF-1 variants, PF4 variants, MIG variants, Calcitonin variants, c-kit ligand variants, cytokine variants, CC chemokine variants, Monocyte chemoattractant protein-1 variants, Monocyte chemoattractant protein-2 variants, Monocyte chemoattractant protein-3 variants, Monocyte inflammatory protein-1 alpha variants, Monocyte inflammatory protein-1 beta variants, RANTES variants, 1309 variants, R83915 variants, R91733 variants, HCC1 variants, T58847 variants, D31065 variants, T64262 variants, CD40 variants, CD40 ligand variants, C-kit Ligand variants, Collagen variants, Colony stimulating factor (CSF) variants, Complement factor 5a variants, Complement inhibitor variants, Complement receptor 1 variants, cytokine variants, epithelial Neutrophil Activating Peptide-78 variants, GRO.alpha. variants, MGSA variants, GRO.beta. variants, GRO.gamma. variants, MIP1-.alpha. variants, MIP1-.beta. variants, MCP-1 variants, Epidermal Growth Factor (EGF) variants, epithelial Neutrophil Activating Peptide variants, Erythropoietin (EPO) variants, Exfoliating toxin variants, Factor IX variants, Factor VII variants, Factor VIII variants, Factor X variants, Fibroblast Growth Factor (FGF) variants, Fibrinogen variants, Fibronectin variants, G-CSF variants, GM-CSF variants, Glucocerebrosidase variants, Gonadotropin variants, growth factor variants, growth factor receptor variants, Hedgehog protein variants, Hemoglobin variants, Hepatocyte Growth Factor (HGF) variants, Hirudin variants, Human serum albumin variants, ICAM-1 variants, ICAM-1 receptor variants, LFA-1 variants, LFA-1 receptor variants, Insulin variants, Insulin-like Growth Factor (IGF) variants, IGF-I variants, IGF-II variants, interferon variants, IFN-.alpha. variants, IFN-.beta. variants, IFN-.gamma. variants, interleukin variants, IL-1 variants, IL-2 variants, IL-3 variants, IL-4 variants, IL-5 variants, IL-6 variants, IL-7 variants, IL-8 variants, IL-9 variants, IL-10 variants, IL-11 variants, IL-12 variants, Keratinocyte Growth Factor (KGF) variants, Lactoferrin variants, leukemia inhibitory factor variants, Luciferase variants, Neurturin variants, Neutrophil inhibitory factor (NIF) variants, oncostatin M variants, Osteogenic protein variants, oncogene product variants, Parathyroid hormone variants, PD-ECSF variants, PDGF variants, peptide hormone variants, Human Growth Hormone variants, Pleiotropin variants, Protein A variants, Protein G variants, variants of Pyrogenic exotoxins A, B, or C, Relaxin variants, Renin variants, SCF/c-kit variants, Soluble complement receptor I variants, Soluble I-CAM 1 variants, Soluble interleukin receptor variants, Soluble TNF receptor variants, Somatomedin variants, Somatostatin variants, Somatotropin variants, Streptokinase variants, Superantigen variants, Staphylococcal enterotoxin variants, SEA variants, SEB variants, SEC1 variants, SEC2 variants, SEC3 variants, SED variants, SEE variants, steroid hormone receptor variants, Superoxide dismutase variants, Toxic shock syndrome toxin variants, Thymosin alpha 1 variants, Tissue plasminogen activator variants, tumor growth factor (TGF) variants, TGF-.alpha. variants, TGF-.beta. variants, Tumor Necrosis Factor variants, Tumor Necrosis Factor alpha variants, Tumor necrosis factor beta variants, Tumor necrosis factor receptor (TNFR) variants, VLA-4 protein variants, VCAM-1 protein variants, Vascular Endothelial Growth Factor (VEGEF) variants, Urokinase variants, Mos variants, Ras variants, Raf variants, Met variants, p53 variants, Tat variants, Fos variants, Myc variants, Jun variants, Myb variants, Rel, estrogen receptor variants, progesterone receptor variants, testosterone receptor variants, aldosterone receptor variants, LDL receptor variants, variants of inflammatory molecules, variants of signal transduction molecules, variants of transcriptional activators, variants of a transcriptional suppressors, hyalurin variants, CD44 variants, and corticosterone variants. 8. The recombinant polypeptide expression library of claim 1, wherein the unnatural amino acid in the at least one of the polypeptide variants of a) comprises an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc.beta.-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an .alpha.-hydroxy containing acid; an amino thio acid containing amino acid; an .alpha.,.alpha. disubstituted amino acid; a .beta.-amino acid; sulfotyrosine, 4-borono-phenylalanine, or a cyclic amino acid other than proline. 9. The recombinant polypeptide expression library of claim 1, wherein the recombinant polypeptide expression library comprises a plurality of recombinant M13 phage, wherein each phage displays an expressed target polypeptide variant on its outer surface. 10. The phage display library of claim 9, wherein the expressed target polypeptide variants comprise antibody fragment variants. 11. The recombinant polypeptide expression library of claim 1, wherein the recombinant polypeptide expression library comprises a plurality of recombinant M13 phage, wherein each phage displays more than one expressed polypeptide variants on its outer surface, and wherein the more than one expressed variants are the same. 12. The recombinant polypeptide expression library of claim 1, wherein each of the expressed target polypeptide variants comprises at least one unnatural amino acid residue.

Details for Patent 9,150,849

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Microbix Biosystems Inc.	KINLYTIC	urokinase	For Injection	021846	01/16/1978		2027-11-02
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2027-11-02