Details for Patent: 5,750,373

Title:	Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
Abstract:	A method for selecting novel proteins such as growth hormone and antibody fragment variants having altered binding properties for their respective receptor molecules is provided. The method comprises fusing a gene encoding a protein of interest to the carboxy terminal domain of the gene III coat protein of the filamentous phage M13. The gene fusion is mutated to form a library of structurally related fusion proteins that are expressed in low quantity on the surface of a phagemid particle. Biological selection and screening are employed to identify novel ligands useful as drug candidates. Disclosed are preferred phagemid expression vectors and selected human growth hormone variants.
Inventor(s):	Garrard; Lisa J. (Burlingame, CA), Henner; Dennis J. (Pacifica, CA), Bass; Steven (Redwood City, CA), Greene; Ronald (Durham, NC), Lowman; Henry B. (Hercules, CA), Wells; James A. (Burlingame, CA), Matthews; David J. (San Francisco, CA)
Assignee:	Genentech, Inc. (South San Francisco, CA)
Filing Date:	Apr 30, 1993
Application Number:	08/050,058
Claims:	1. A method for selecting novel binding polypeptides comprising: (a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide, and a second gene encoding at least a portion of a wild-type M13 phage gene III coat protein; (b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids; (c) transforming suitable E. coli host cells with the plasmids; (d) infecting the transformed host cells with a M13K07 helper phage having a gene encoding the wild-type M13 phage gene III coat protein; (e) culturing the transformed infected E. coli host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of infecting the E. coli host, the phagemid particles displaying a mixture of the wild-type M13 phage gene III coat protein and the fusion protein; (f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and (g) separating the phagemid particles that bind from those that do not. 2. The method of claim 1 further comprising infecting suitable E. coli host cells with the phagemid particles that bind to the target and repeating steps (d) through (g). 3. The method of claim 2 wherein the steps are repeated more than once and where the polypeptide is heterologous to the phagemid. 4. The method of claim 1 wherein the expression vector further comprises a secretory signal sequence. 5. The method of claim 4 wherein the expression vector further comprises a third gene encoding a polypeptide, the third gene operably linked to a second secretory signal sequence. 6. The method of claim 5 further compromising mutating the vector at one or more selected positions within the third gene. 7. The method of claim 6 wherein the first gene encodes the variable domain of an IgG antibody heavy chain and at least a portion of the heavy chain constant region of an IgG antibody. 8. The method of claim 7 wherein the thord gene encodes the variable and constant regions of an IgG antibody. 9. The method of claim 8 wherein the vector is mutated in one or more of the codons encoding amino acids corresponding to the complemetarity-determining regions (CDRs). 10. The method of claim 1 wherein the first gene encodes a mammalian polypeptide. 11. The method of claim 6 wherein the polypeptide is a protein selected from the group consisting of: growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, the Fab portion of an IgG antibody, a humanized IgG antibody, the variable domain and the first domain of the consistent region of the heavy chain of a humanized IgG antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, .beta.-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-.beta., platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, HER2 ligands, osteoinductive factors, interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic peptides A, B, or C, and immuno globulins. 12. The method of claim 10 wherein the polypeptide is a human protein. 13. The method of claim 12 wherein the protein comprises more than about 100 amino acid residues. 14. The method of claim 1 wherein the polypeptide comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target, and the mutations are primarily produced at positions corresponding to codons encoding the amino acids. 15. The method of claim 14 wherein the rigid secondary structures comprise structures selected from the group consisting of; .alpha.-(3.6.sub.13)helix, 3.sub.10 helix, .pi.-(4.4.sub.16)helix, parallel and anti-parallel .beta.-pleated sheets, reverse turns, and non-ordered structures. 16. The method of claim 14 wherein the mutations are produced at more than one codon. 17. The method of claim 16 wherein the mutations are produced on more than one rigid secondary structure. 18. The method of claim 1 wherein the percentage of phagemid particles displaying more than a single copy of the fusion protein is less than about 1%. 19. The method of claim 1 wherein the percentage of phagemid particles displaying more than a single copy of the fusion protein is less than about 20%. 20. The method of claim 1 wherein the percentage of phagemid particles displaying more than a single copy of the fusion protein is less than about 10%. 21. The method of claim 1 further comprising in step (a), inserting a DNA triplet, encoding an mRNA suppressible terminator codon between said first gene encoding a polypeptide, and the second gene encoding a least a portion of a M13 phage gene III coat protein. 22. The method of claim 21 wherein the mRNA suppressible terminator codon is selected from the following: UAG (amber), UAA (ocher) and UGA (opel). 23. The method of claim 22 wherein the suppressible mutation results in the detectable production of a fusion polypeptide containing the polypeptide and the coat protein when the expression vector is grown in a suppressor host cell; and, when grown in a non-suppressor host cell the polypeptide is synthesized substantially without fusion to the phage coat protein. 24. A method for selecting noval binding polypeptides comprising (a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a polypeptide of interest containing one or more subunits, wherein the DNA encoding at least one of the subunits is fused to the DNA encoding at least a portion of a M13 phage gene III coat protein; (b) mutating the DNA encoding the polypeptide of interest at one or more selected positions thereby forming a family of related vectors; (c) transforming suitable E. coli host cells with the vectors; (d) infecting the transformed host cells with a M13K07 helper phage having a gene encoding the gene III phage coat protein; (e) culturing the transformed infected E. coli host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of infecting the host, at least a portion of the phagemid particles displaying a mixture of the wild-type phage coat protein and the fusion containing the protein of interest; (f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and (g) separating the phagemid particles that bind from those that do not. 25. The method of claim 24 wherein the expression vector further comprises a secretory signal sequence operably linked to the DNA encoding each subunit of the protein of interest. 26. The method of claim 25 wherein the protein of interest is a mammalian protein. 27. The method of claim 26 wherein the protein of interest is selected from the group; insulin, relaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, monoclonal and polyclonal antibodies, lung surfactant, integrin receptors and insulin-like growth factor-I and -II. 28. The method of claim 27 wherein the protein of interest is a humanized antibody. 29. The method of claim 28 wherein the protein of interest is a humanized Fab fragment capable of binding to the HER-2 receptor (human epidermal growth factor receptor-2). 30. A method for selecting novel protease substrate polypeptides comprising: (a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide operable connected to a linking amino acid sequence, and a second gene encoding at least a portion of M13 phage gene III coat protein; (b) mutating the vector at one or more selected positions within the amino acid linking sequence of the first gene thereby forming a family of related plasmids; (c) transforming suitable E. coli host cells with the plasmids; (d) infecting the transformed host cells with a M13K07 helper phage having a gene encoding the wild-type phage coat protein; (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of infecting the host, at least a portion of the phagemid particles displaying a mixture of the wild-type phage coat protein and the fusion protein; (f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and (g) contacting the bound phagemid particles with a protease capable of hydrolyzing the linking a amino acid sequence of at least a portion of the bound phagemid particles, and (h) isolating the hydrolyzed phagemid particles. 31. The method of claim 30 further comprising infecting suitable E. coli host cells with the hydrolyzed phagemid particles and repeating steps (d) through (h). 32. An M13 phagemid capable of infecting an E. coli host comprising a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide, and a second gene encoding at least a portion of a M13 phage gene III coat protein, wherein a DNA triplet codon encoding an mRNA suppressible terminator codon selected from UAG, UAA and UGA is inserted between the fused ends of the first and second genes, or is substituted for an amino acid encoding triplet codon adjacent to the gene fusion junction. 33. The phagemid of claim 32 wherein the first gene encodes a mammalian polypeptide. 34. The phagemid of claim 33 wherein the polypeptide is a protein selected from the group consisting of: growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), throid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, the Fab portion of an IgG antibody, a humanized IgG antibody, the variable domain and the first domain of the consistent region of the heavy chain of a humanized IgG antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, .beta.-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-.beta., platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth-1 and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic peptides A, B or C and immuno globulins. 35. The phagemid of claim 34 wherein the fusion protein comprises a human protein. 36. The phagemid of claim 35 wherein the protein comprises more than about 100 amino acid residues. 37. The phagemid of claim 32 wherein the fusion protein comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target. 38. The phagemid of claim 37 wherein the rigid secondary structures comprises structures selected from the group consisting of; .alpha.-(3.6.sub.13)helix, 3.sub.10 helix, .pi.-(4.4.sub.16)helix, parallel and anti-parallel .beta.-pleated sheets, reverse turns and non-ordered structures. 39. The phagemid of claim 32 wherein the host is the E. coli wild type or suppressor type. 40. The phagemid of claim 39 wherein the plasmid is under tight control of the transcription regulatory element. 41. The phagemid of claim 40 wherein the number of phagemid displaying more than one copy of the fusion protein on the surface of the particles is less than 1%. 42. The phagemid of claim 40 wherein the percentage of phagemid displaying more than a single copy of the fusion protein is less than about 10%. 43. The phagemid of claim 40 wherein the percentage of phagemid particles displaying more than a single copy of the fusion protein is less than about 20%. 44. A human growth hormone variant wherein hGH amino acids 172, 174, 176 and 178 respectively are as a group sequentially selected from the group consisting of: (1)R,S,F,R; (2)R,A,Y,R; (3)K,T,Y,K; (4)R,S,Y,R; (5)K,A,Y,R; (6)R,F,F,R; (7)K,Q,Y,R; (8)R,T,Y,H; (9)Q,R,Y,R; (10)K,K,Y,K; (11)R,S,F,S and (12)K,S,N,R. 45. A human growth variant wherein hGH amino acids 10, 14, 18, and 21 respectively are as a group sequentially selected from the group consisting of: (1)H,G,N,N; (2)A,W,D,N; (3)F,S,F,L; (4)Y,T,V,N and (5)I,N,I,N. 46. A human growth variant wherein hGH amino acids 174 is serine and 176 is tyrosine and hGH amino acids 167, 171, 175 and 179 respectively are as a group sequentially selected from the group consisting of: (1)N,S,T,T; (2)E,S,T,I; (3)K,S,T,L; (4)N,N,T,T; (5)R,D,I,I and (6)N,S,T,Q. 47. A human growth hormone (hGH) variant wherein hGH amino acid glutamate.sub.174 is replaced by serine.sub.174 and phenylalanine.sub.176 is replaced by tyrosine.sub.176 and one or more of the eight naturally occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 are replaced by another natural amino acid. 48. The hGH variant of claim 47 wherein the eight naturally occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 respectively are as a group replaced with a corresponding amino acid sequentially selected from the group consisting of: 49. The hGH variant of claim 48 wherein the human growth hormone variant further contains leucine.sub.15 replaced by arginine.sub.15 and lysine.sub.168 replaced by arginine.sub.168. 50. The hGH variant of claim 49 wherein the human growth hormone variant further contains phenylalamine.sub.176.