Protein/(poly)peptide libraries

• US 6,696,248 B1
• Filed: 01/24/2000
• Issued: 02/24/2004
• Est. Priority Date: 08/18/1995
• Status: Expired due to Term

• Current Assignee: Morphosys Ag
• Original Assignee: Morphosys Ag
• Inventors: Plückthun, Andreas, Knappik, Achim, Pack, Peter, Moroney, Simon, Ge, Liming
• Primary Examiner(s): Zeman, Mary K.
• Assistant Examiner(s): Clow, Lori A.
• Application Number:
  US09/490,070
• Time in Patent Office: 1,492 Days
• Field of Search: 435/6, 435/320.1, 435/DIG.1, 536/23.1, 536/24.1, 536/24.5
• US Class Current: 435/6.16
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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

Abstract

The present invention relates to synthetic DNA sequences which encode one or more collections of homologous proteins/(poly)peptides, and methods for generating and applying libraries of these DNA sequences. In particular, the invention relates to the preparation of a library of human-derived antibody genes by the use of synthetic consensus sequences which cover the structural repertoire of antibodies encoded in the human genome. Furthermore, the invention relates to the use of a single consensus antibody gene as a universal framework for highly diverse antibody libraries.

Claims

1. A library of nucleic acids,

wherein said library encodes a plurality of human immunoglobulin variable domain amino acid sequences, each amino acid sequence comprising four consensus framework regions interspaced by three complementary determining regions CDR1, CDR2, and CDR3,

wherein each of said nucleic acids comprises DNA cleavage sites at the boundary between each consensus framework region and each complementary determining region, and wherein each of said cleavage sites is unique within said nucleic acid but common to all nucleic acid sequences of said library at corresponding positions.

2. The library according to claim 1, wherein said human immunoglobulin amino acid sequences are human VH immunoglobulin sequences.

3. The library according to claim 2, wherein said human VH immunoglobulin sequences comprise consensus framework regions selected from the group consisting of the framework region sequences of VH1A (SEQ ID NO:

57), VH1B (SEQ ID NO;

     59), VH2 (SEQ ID NO;

     61), VH3 (SEQ ID NO;

     63), VH4 (SEQ ID NO;

     65), VH5 (SEQ ID NO;

     67), and VH6 (SEQ ID NO;

     69).

4. The library according to claim 3, wherein said CDR1 is selected from the group consisting ofthe CDR1 sequences of VH1-12-1, VH1-13-16, VH2-31-10, VH3-13-8, VH4-11-7, CH5-12-1, and VH6-35-1.

5. The library according to claim 3, wherein said CDR2 is selected from the group consisting of the CDR2 sequences of VH1-12-1, VH1-13-6, VH2-31-3, VH3-13-8, VH4-11-8, VH4-31-17, VH5-12-1, and VH6-35-1.

6. The library according to claim 3, wherein said CDR1 and said CDR2 are independently selected from the group consisting of the CDR1 and CDR2 sequences of VH1-12-1, VH1-13-16, VH2-31-10, VH3-13-8, VH4-11-7, CH5-12-1, and VH6-35-1.

7. The library according to claim 6, wherein said CDR3 is selected from random amino acid sequences.

8. The library according to claim 2, wherein said CDR1 is selected from the group consisting of VH CDR1 germline sequences.

9. The library according to claim 8, wherein said CDR1 is selected from the group consisting of the CDR1 region sequences of VH1-12-1, VH1-13-16, VH2-31-10, VH3-13-8, VH4-11-7, CH5-12-1, and VH6-35-1.

10. The library according to claim 2, wherein said CDR2 is selected from the group consisting of VH CDR2 germline sequences.

11. The library according to claim 10, wherein said CDR2 is selected from the group consisting of the CDR2 region sequences of VH1-12-1, VH1-13-6, VH2-31-3, VH3-13-8, VH4-11-8, VH4-31-17, VH5-12-1, and VH6-35-1.

12. The library according to claim 2, wherein said CDR3 is selected from random amino acid sequences.

13. The library according to claim 2, wherein said human VH immunoglobulin sequences comprise the framework, CDR1 and CDR2 amino acid sequences encoded by a nucleic acid selected from the group consisting of VH1A (SEQ ID NO:

56), VH1B (SEQ ID NO;

     58), VH2 (SEQ ID NO;

     60), VH3 (SEQ ID NO;

     62), VH4 (SEQ ID NO;

     64), VH5 (SEQ ID NO;

     66), and VH6 (SEQ ID NO;

     68).

14. The library according to claim 13, wherein said CDR3 is selected from random amino acid sequences.

15. The library according to claim 1, wherein said human immunoglobulin sequences are human Vκ

immunoglobulin sequences.

16. The library according to claim 15, wherein said human Vκ

immunoglobulin sequences comprise framework region sequences selected from the group consisting of the framework region sequences of Vκ

1 (SEQ ID NO;

     43), Vκ

2 (SEQ ID NO;

     45), Vκ

3 (SEQ ID NO;

     47), and Vκ

4 (SEQ ID NO;

     49).

17. The library according to claim 16, wherein said CDR1 is selected from the group consisting of Vκ

CDR1 germline sequences.

18. The library according to claim 17, wherein said CDR1 is selected from the group consisting of the CDR1 region sequences of Vκ

1-14, Vκ

2-6, Vκ

3-1, and Vκ

4-1.

19. The library according to claim 16, wherein said CDR2 is selected from the group consisting of Vκ

CDR2 germline sequences.

20. The library according to claim 19, wherein said CDR2 is selected from the group consisting of the CDR2 region sequences of Vκ

1-14, Vκ

2-6, Vκ

3-1, and Vκ

4-1.

21. The library according to claim 16 wherein said CDR1 and said CDR2 sequences are independently selected from the group consisting of the CDR1 and CDR2 region sequences of Vκ

1-14, Vκ

2-6, Vκ

3-1, and Vκ

4-1.

22. The library according to claim 21, wherein said CDR3 is selected from random amino acid sequences.

23. The library according to claim 15, wherein said CDR1 is selected from the group consisting of Vκ

CDR1 germline sequences.

24. The library according to claim 23, wherein said CDR1 is selected from the group consisting of the CDR1 region sequences of Vκ

1-14, Vκ

2-6, Vκ

3-1, and Vκ

4-1.

25. The library according to claim 15, wherein said CDR2 is selected from the group consisting of Vκ

CDR2 germline sequences.

26. The library according to claim 25, wherein said CDR2 is selected from the group consisting of the CDR2 region sequences of Vκ

1-14, Vκ

2-6, Vκ

3-1, and Vκ

4-1.

27. The library according to claim 15, wherein said CDR3 is selected from random amino acid sequences.

28. The library according to claim 15, wherein said human Vκ

immunoglobulin sequences comprise the framework, CDR1 and CDR2 sequences encoded by a nucleic acid selected from the group consisting of Vκ

1 (SEQ ID NO;

     42), Vκ

2 (SEQ ID NO;

     44), Vκ

3 (SEQ ID NO;

     46), and Vκ

4 (SEQ ID NO;

     48).

29. The library according to claim 28, wherein said CDR3 is selected from random amino acid sequences.

30. The library according to claim 1, wherein said human immunoglobulin sequences are human Vλ

immunoglobulin sequences.

31. The library according to claim 30, wherein said human Vλ

immunoglobulin sequences comprise consensus framework regions selected from the group consisting of the framework region sequences of Vλ

1 (SEQ ID NO;

     51), Vλ

2 (SEQ ID NO;

     53), and Vλ

3 (SEQ ID NO;

     55).

32. The library according to claim 31, wherein said CDR1 is selected from the group consisting of Vλ

CDR1 germline sequences.

33. The library according to claim 32, wherein said CDR1 is selected from the group consisting of the CDR1 region sequences of VHUMLV86, DPL11, and DPL23.

34. The library according to claim 31, wherein said CDR2 is selected from the group consisting of Vλ

CDR2 germline sequences.

35. The library according to claim 34, wherein said CDR2 is selected from the group consisting of the CDR2 region sequences of DPL5, DPL12, and HUMLV318.

36. The library according to claim 31, wherein said CDR1 and said CDR2 are independently selected from the group consisting of the CDR1 and CDR2 region sequences of VHUMLV86, DPL11, and DPL23.

37. The library according to claim 36, wherein said CDR3 is selected from random amino acid sequences.

38. The library according to claim 30, wherein said CDR1 is selected from the group consisting of Vλ

CDR1 germline sequences.

39. The library according to claim 38, wherein said CDR1 is selected from the group consisting of the CDR1 region sequences of VHUMLV86, DPL11, and DPL23.

40. The library according to claim 30, wherein said CDR2 is selected from the group consisting of Vλ

CDR2 germline sequences.

41. The library according to claim 40, wherein said CDR2 is selected from the group consisting of the CDR2 region sequences of DPL5, DPL12, and HUMLV318.

42. The library according to claim 30, wherein said CDR3 is selected from random amino acid sequences.

43. The library according to claim 30, wherein said human Vλ

immunoglobulin sequences comprise the framework, CDR1 and CDR2 sequences encoded by a nucleic acid selected from the group consisting of Vλ

1 (SEQ ID NO;

     50), Vλ

2 (SEQ ID NO;

     52), and Vλ

3 (SEQ ID NO;

     54).

44. The library according to claim 43, wherein said CDR3 is selected from random amino acid sequences.

45. The library according to claim 1, wherein said human immunoglobulin sequences comprise human VH, Vλ

, and Vκ

immunoglobulin sequences.

46. The library according to claim 45,

wherein said human VH inimunoglobulin sequences comprise consensus framework regions selected from the group consisting of the framework region sequences of VH1A (SEQ ID NO:

57), VH1B (SEQ ID NO;

     59), VH2 (SEQ ID NO;

     61), VH3 (SEQ ID NO;

     63), VH4 (SEQ ID NO;

     65), VH5 (SEQ ID NO;

     67), and VH6 (SEQ ID NO;

     69),

wherein said human Vκ

immunoglobulin sequences comprise framework region sequences selected from the group consisting of the framework region sequences of Vκ

1 (SEQ ID NO;

     43), Vκ

2 (SEQ ID NO;

     45), Vκ

3 (SEQ ID NO;

     47), and Vκ

4 (SEQ ID NO;

     49), and

wherein said human Vλ

immunoglobulin sequences comprise framework region sequences selected from the group consisting of the framework region sequences of Vλ

1 (SEQ ID NO;

     51), Vλ

2 (SEQ ID NO;

     53), and Vλ

3 (SEQ ID NO;

     55).

47. An expression library, comprising a library of nucleic acids according to claim 46, wherein each of said nucleic acids is contained within an expression vector.

48. An expression library according to claim 47 wherein said vector is a phage display vector.

49. The library according to claim 45,

wherein said human VH immunoglobulin sequences comprise consensus framework, CDR1, and CDR2 regions selected from the group consisting of the framework region sequences of VH1A (SEQ ID NO:

57), VH1B (SEQ ID NO;

     59), VH2 (SEQ ID NO;

     61), VH3 (SEQ ID NO;

     63), VH4 (SEQ ID NO;

     65), VH5 (SEQ ID NO;

     67), and VH6 (SEQ ID NO;

     69),

wherein said human Vκ

immunoglobulin sequences comprise framework, CDR1, and CDR2 region sequences selected from the group consisting of the framework region sequences of Vκ

1 (SEQ ID NO;

     43), Vκ

2 (SEQ ID NO;

     45), Vκ

3 (SEQ ID NO;

     47), and Vκ

4 (SEQ ID NO;

     49), and

wherein said human Vλ

immunoglobulin sequences comprise framework, CDR1, and CDR2 region sequences selected from the group consisting of the framework region sequences of Vλ

1 (SEQ ID NO;

     51), Vλ

2 (SEQ ID NO;

     53), and Vλ

3 (SEQ ID NO;

     55).

50. An expression library, comprising a library of nucleic acids according to claim 49, wherein each of said nucleic acids is contained within an expression vector.

51. An expression library according to claim 50 wherein said vector is a phage display vector.

52. An expression library, comprising a library of nucleic acids according to claim 45, wherein each of said nucleic acids is contained within an expression vector.

53. An expression library according to claim 52 wherein said vector is a phage display vector.

54. An expression library, comprising a library of nucleic acids wherein each of said nucleic acids is contained within an expression vector,

wherein said library of nucleic acids encodes a plurality of human immunoglobulin variable domain amino acid sequences, each amino acid sequence comprising four consensus framework regions interspaced by three complementarity determining regions CDR1, CDR2, and CDR3,

wherein each of said nucleic acids comprises DNA cleavage sites at the boundary between each consensus framework region and complementarity determining region, and wherein each of said cleavage sites is unique within said nucleic acid but common to all nucleic acid sequences of said library at corresponding positions.

55. An expression library according to claim 54 wherein said vector is a phage display vector.

56. A collection of host cells transformed with a library according to claim 55.

57. A collection of host cells according to claim 56, wherein said host cells are E. coli.

58. A collection of host cells transformed with a library according to claim 54.

59. A collection of host cells according to claim 58, wherein said host cells are E. coli.

60. The expression library according to claim 54, wherein a plurality of VH families and/or a plurality of VL families are represented by said amino acid sequences.

61. The expression library according to claim 54, wherein a plurality of antibody subgroups is represented by said amino acid sequences.

62. A method of producing an antibody library, comprising culturing a collection of host cells transformed with an expression library members, wherein said expression library comprises a library of nucleic acids, wherein each of said nucleic acids is contained within an expression vector, and

wherein said library of nucleic acids encodes a plurality of human immunoglobulin variable domain amino acid sequences, each amino acid sequence comprising four consensus framework regions interspaced by three complementarity determining regions CDR1, CDR2, and CDR3,

wherein each of said nucleic acids comprises DNA cleavage sites at the boundary between each consensus framework region and complementarity determining region, and wherein each of said cleavage sites is unique within said nucleic acid but common to all nucleic acid sequences of said library at corresponding positions.

63. The method according to claim 62, wherein said expression vector is a phage display vector and said expression library is an antibody phage display library.

64. The method according to claim 62, wherein said nucleic acids encoding said immunoglobulin variable domain amino acid sequences comprise codons that are frequently used in said host cell.

65. The method according to claim 62, wherein a plurality of VH families and/or a plurality of VL families are represented by said amino acid sequences.

66. The method according to claim 62, wherein a plurality of antibody subgroups is represented by said amino acid sequences.

67. A library of nucleic acids,

wherein said library encodes a plurality of human immunoglobulin variable domain amino acid sequences,

wherein a plurality of VH families and/or a plurality of VL families are represented by said amino acid sequences;

wherein each amino acid sequences comprises four consensus framework regions interspaced by three complementarity determining regions CDR1, CDR2, and CDR3;

wherein each of said nucleic acids comprises DNA cleavage sites at the boundary between each consensus framework region and each complementarity determining region; and

wherein each of said cleavage sites is unique within said nucleic acid but common to all nucleic acid sequences of said library at corresponding positions.

68. The library according to claim 67, wherein at least one consensus framework regions amino acid sequences is selected from the group consisting of VH1A (SEQ ID NO:

57), VH1B (SEQ ID NO;

     59), VH2 (SEQ ID NO;

     61), VH3 (SEQ ID NO;

     63), VH4 (SEQ ID NO;

     65), VH5 (SEQ ID NO;

     67), and VH6 (SEQ ID NO;

     69).

69. The library according to claim 67, wherein at least one consensus framework region amino acid sequence is selected from the group consisting of Vκ

1 (SEQ ID NO;

     43), Vκ

2 (SEQ ID NO;

     45), Vκ

3 (SEQ ID NO;

     47), and Vκ

4 (SEQ ID NO;

     49).

70. The library according to claim 67, wherein at least one consensus framework region amino acid sequence is selected from the group consisting of Vλ

1 (SEQ ID NO;

     51), Vλ

2 (SEQ ID NO;

     53), and Vλ

3 (SEQ ID NO;

     55).

71. A library of nucleic acids,

wherein said library encodes a plurality of human immunoglobulin variable domain amino acid sequences;

wherein said each amino acid sequence comprises (i) a consensus consequence comprising four consensus framework regions, and (ii) three complementarity determining regions CDR1, CDR2, and CDR3 interspaced, respectively, between said consensus framework regions;

wherein each of said nucleic acids comprises DNA cleavage sites at the boundary between each consensus framework region and each complementarity determining region; and

wherein each of said cleavage sites is unique within said nucleic acid but common to all nucleic acid sequences of said library at corresponding positions; and

wherein a plurality of antibody subgroups is represented by said amino acid sequences.

72. The library according to claim 71, wherein at least one consensus framework regions amino acid sequences is selected from the group consisting of VH1A (SEQ ID NO:

57), VH1B (SEQ ID NO;

     59), VH2 (SEQ ID NO;

     61), VH3 (SEQ ID NO;

     63), VH4 (SEQ ID NO;

     65), VH5 (SEQ ID NO;

     67), and VH6 (SEQ ID NO;

     69).

73. The library according to claim 71, wherein at least one consensus framework region amino acid sequence is selected from the group consisting of Vκ

1 (SEQ ID NO;

     43), Vκ

2 (SEQ ID NO;

     45), Vκ

3 (SEQ ID NO;

     47), and Vκ

4 (SEQ ID NO;

     49).

74. The library according to claim 71, wherein at least one consensus framework region amino acid sequence is selected from the group consisting of Vλ

1 (SEQ ID NO;

     51), Vλ

2 (SEQ ID NO;

     53), and Vλ

3 (SEQ ID NO;

     55).