Monovalent phage display

• US 5,821,047 A
• Filed: 06/05/1995
• Issued: 10/13/1998
• Est. Priority Date: 12/03/1990
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Lowman, Henry B., Wells, James A., Matthews, David J., Greene, Ronald, Henner, Dennis J., Bass, Steven, Garrard, Lisa J.
• Primary Examiner(s): Kemmerer, Elizabeth C.
• Assistant Examiner(s): Lathrop, Brian
• Application Number:
  US08/463,587
• Time in Patent Office: 1,226 Days
• Field of Search: 435/5, 435/68.1, 435/69.1, 435/69.7, 435/69.8, 435/172.1, 435/235.1, 435/239, 436/802, 530/350, 530/387.1, 530/387.9, 536/24.1, 935/31
• US Class Current: 435/5
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/735 containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 2750/00011 Details
• CPC Class Codes: C12N 2795/14022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2795/14122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: G01N 2333/61 Growth hormones [GH] (Somatotropin)
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• CPC Class Codes: Y10S 436/802 Protein-bacteriophage conjugates
• CPC Class Codes: Y10S 930/12 Growth hormone, growth factor other than t-cell or b-cell growth factor, and growth hormone releasing factor; related peptides

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.

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 polylpeptide, and a second gene encoding at least a portion of a phage 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 host cells with the plasmids;

(d) infecting the transformed host cells with an amount of a helper phage having a gene encoding the phage coat protein sufficient to produce recombinant phagemid particles wherein no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;

(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 transforming the host cells, the conditions adjusted so that no more than a minor amount of phagemid particles display one or more copies of the fusion protein on the surface of the particle;

(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 host cells with the phagemid particles that bind and repeating steps (d) through (g).

3. The method of claim 2 wherein the steps are repeated one or more times.

4. The method of claim 1 wherein the expression vector further comprises a secretory signal sequence.

5. The method of claim 1 wherein the transcription regulatory element is a promoter system selected from the group consisting of lac Z, pho A, tryptophan, tac, λ

_PL, bacteriophage T7, and combinations thereof.

6. The method of claim 1 wherein the first gene encodes a mammalian protein.

7. The method of claim 6 wherein the protein is 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 VII, lung surfactant, urokinasae, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombinm, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinasae, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, β

-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein (A) and D, rheumatoid factors, nerve growth factor -β

(INGF-β

), platelet-growth factor, transforming growth factor (TGF), 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, heregulin 2 (HER2) ligands, osteoinductive factors, interferonalpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interluekins (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, and C, immunoglobulins, and fragments of the above-listed proteins.

8. The method of claim 7 wherein the protein is a human protein.

9. The method of claim 8 wherein the protein comprises more than about 100 amino acid residues.

10. The method of claim 6 wherein the protein 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.

11. The method of claim 10 wherein the rigid secondary structures comprise structures selected from the group consisting of α

-(3.6₁₃)helix, 3₁₀ helix, π

-(4.4₁₆)helix, parallel and anti-parallel β

-pleated sheets, reverse turns, and non-ordered structures.

12. The method of claim 10 wherein the mutations are produced at more than one codon.

13. The method of claim 12 wherein the mutations are produced on more than one rigid secondary structure.

14. The method of claim 1 wherein the helper phage is selected from the group consisting of M13KO7, M13R408, M13-VCS, and Phi X174.

15. The method of claim 14 wherein the helper phage is M13KO7 and the coat protein is the M13 phage gene III coat protein.

16. The method of claim 15 wherein the host cells are E. coli.

17. The method of claim 16 wherein the plasmid is under tight control of the transcription regulatory element.

18. The method of claim 17 wherein the amount of phagemid particles displaying more than one copy of the fusion protein is less than 20% the amount of phagemid particles displaying a single copy of the fusion protein.

19. The method of claim 18 wherein the amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% of the amount of phagemid particles displaying a single copy of the fusion protein.

20. The method of claim 19 wherein the amount of phagemid particles displaying more than one copy of the fusion protein is less than about 1% of the phagemid particles displaying a single copy of the fusion protein.

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 said second gene encoding at least a portion of a phage coat protein.

22. The method of claim 21 wherein said mRNA suppressible terminator codon is selected from the following:

UAG (amber), UAA (ocher) and UGA (opel).

23. The method of claim 22 wherein said suppressible terminator codon results in the detectable production of a fusion polypeptide containing said polypeptide and said coat protein when said expression vector is grown in a suppressor host cell;

and, when grown in a non-suppressor host cell said polypeptide is synthesized substantially without fusion to said phage coat protein.

24. The method of claim 1 wherein about 10% or less of the recombinant phagemid particles display one or more copies of the fusion protein on the surface of the particle.

25. The method of claim 1 wherein the number of fusion proteins per recombinant phagemid particle is about 0.1 (number of bulk fusion proteins/number of phagemid particles).

26. The method of claim 1, wherein the multiplicity of infection with said helper phage is 10-3,000 plaque forming units per cell.

27. The method of claim 1, wherein the polypeptide is an antibody or fragment thereof.

28. The method of claim 1, wherein the polypeptide comprises an antibody Fab portion.

29. The method of claim 1, wherein the polypeptide comprises an antibody Fab portion which recognizes and binds to a cell receptor.

30. A group of phagemid particles, each particle 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 phage coat protein, wherein a DNA tripler 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, wherein no more than a minor amount of phagemid particles in said group display one or more copies of the fusion protein on the surface of the particle.

31. The method of claim 30 wherein said first gene encodes a mammalian protein.

32. The method of claim 31 wherein the protein is 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 VII, lung surfactant, urokinasae, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinasae, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, b-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A and D, rheumatoid factors, nerve growth factor -β

(NGF-β

), platelet-growth factor, transforming growth factor (TGF), TGF-alpha and TGF-beta, insulin-like growth -I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferonalpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interluekins (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, and C, immunoglobulins, and fragments of the above-listed proteins.

33. The method of claim 32 wherein said protein is a human protein.

34. The method of claim 33 wherein the protein comprises more than about 100 amino acid residues.

35. The phagemid of claim 31 wherein said protein comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target.

36. The method of claim 35 wherein said rigid secondary structures comprises structures selected from the group;

α

-(3.6₁₃)helix, 3₁₀ helix, x-(4.4₁₆)helix, parallel and anti-parallel β

-pleated sheets, reverse turns, and non-ordered structures.

37. The phagemid of claim 30 wherein the replicable expression vector is under tight control of the transcription regulatory element.

38. The method of claim 37 wherein the amount of phagemid particles displaying more than one copy of the fusion protein on the surface of the particles is less than 20% of the amount of phagemid particles displaying a single copy of the fusion protein.

39. The phagemid of claim 38 wherein the number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particle is less than about 10% of the amount of phagemid particles displaying a single copy of the fusion protein.

40. The phagemid of claim 39 wherein the number of phagemid particles displaying more than one copy of the fusion protein is less than 1% of the phagemid particles displaying a single copy of the fusion protein.

41. The method of claim 30, wherein the polypeptide is an antibody or fragment thereof.

42. The method of claim 30, wherein the polypeptide comprises an antibody Fab portion.

43. The method of claim 30, wherein the polypeptide comprises an antibody Fab portion which recognizes and binds to a cell receptor.

44. A method for selecting novel binding polypeptides comprising:

(a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a protein 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 phage coat protein;

(b) mutating the DNA encoding the protein of interest at one or more selected positions thereby forming a family of related vectors;

(c) transforming suitable host cells with the vectors;

(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein,;

(e) culturing the transformed infected host cells under conditions suitable for forming capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display one or more copies of the fusion protein on the surface of the particle;

(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.

45. The method of claim 44 wherein the expression vector further comprises a secretory signal sequence operably linked to the DNA encoding each subunit of the protein of interest.

46. The method of claim 45 wherein the protein of interest is a mammalian protein.

47. The method of claim 46 wherein the protein of interest is selected from the group consisting of insulin, relaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, monoclonal and polyclonal antibodies, lung surfactant, integrin receptors, insulin-like growth factor-I and -II, and fragments of the above-listed proteins.

48. The method of claim 47 wherein the protein of interest is a humanized antibody.

49. The method of claim 48 wherein the protein of interest is a humanized Fab fragment capable of binding to the HER-2 receptor (human epidermal growth factor receptor-2).

50. The method of claim 44, wherein the polypeptide is an antibody or fragment thereof.

51. The method of claim 44, wherein the polypeptide comprises an antibody Fab portion.

52. The method of claim 44, wherein the polypeptide comprises an antibody Fab portion which recognizes and binds to a cell receptor.

53. 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 operably connected to a linking amino acid sequence, and second gene encoding at least a portion of a phage 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 host cells with the plasmids;

(d) infecting the transformed host cells with a helper phage having a gene encoding the 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 transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display one or more copies of the fusion protein on the surface of the particle;

(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 amino acid sequence of at least a portion of the bound phagemid particles, and

(h) isolating the hydrolyzed phagemid particles.

54. The method of claim 53 further comprising infecting suitable host cells with the hydrolyzed phagemid particles and repeating steps (d) through (h).

55. The method of claim 53, wherein the polypeptide is an antibody or fragment thereof.

56. The method of claim 53, wherein the polypeptide comprises an antibody Fab portion.

57. The method of claim 53, wherein the polypeptide comprises an antibody Fab portion which recognizes and binds to a cell receptor.

58. 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 phage coat protein, wherein said coat protein is 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 host cells with the plasmids;

(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein, wherein the helper phage is M13KO7;

(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 transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display one or more copies of the fusion protein on the surface of the particle;

(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.

59. The method of claim 58, wherein said conditions are adjusted so that no more than 10% of phagemid particles display one or more copies of the fusion protein on the surface of the particle.

60. The method of claim 58, wherein the conditions are adjusted so that the number of fusion proteins per recombinant phagemid particle is about 0.1 (number of bulk fusion proteins/number of phagemid particles).

61. The method of claim 58, wherein the polypeptide is an antibody or fragment thereof.

62. The method of claim 58, wherein the polypeptide comprises an antibody Fab portion.

63. The method of claim 58, wherein the polypeptide comprises an antibody Fab portion which recognizes and binds to a cell receptor.

64. A group of phagemid particles, each particle 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 phage coat protein, wherein no more than a minor amount of phagemid particles in said group display one or more copies of the fusion protein on the surface of the particle.

65. The method of claim 64, wherein the polypeptide is an antibody or fragment thereof.

66. The method of claim 64, wherein the polypeptide comprises an antibody Fab portion.

67. The method of claim 64, wherein the polypeptide comprises an antibody Fab portion which recognizes and binds to a cell receptor.

68. A method for selecting novel binding polypeptides comprising steps of:

(a) constructing a family of variant replicable plasmids 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 phage coat protein, wherein the variant replicable plasmids comprise variant first genes encoding variant polypeptides;

(b) transforming suitable host cells with the plasmids;

(c) infecting the transformed host cells with an amount of helper phage encoding the phage coat protein sufficient to produce recombinant phagemid particles wherein no more than about 20 percent of the phagemid particles display one or more copies of the fusion protein on the surface of the phagemid particles;

(d) 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 transforming the host cells;

(e) contacting the recombinant phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and

(f) separating phagemid particles that bind to the target molecule from those that do not bind.

69. The method of claim 68, wherein the polypeptide is an antibody or binding fragment thereof.

70. The method of claim 69, wherein the phage coat protein is M13 phage gene III coat protein or M13 phage gene VII coat protein.

71. The method of claim 68, wherein no more than about 10% of the phagemid particles display one copy or more of the fusion protein on the surface of the phagemid particles.

72. The method of claim 68, wherein the polypeptide comprises an antibody Fab portion.

73. The method of claim 68, wherein the polypeptide comprises an antibody Fab portion which recognizes and binds to a cell receptor.