Protein scaffolds for antibody mimics and other binding proteins

• US 20070082365A1
• Filed: 10/03/2006
• Published: 04/12/2007
• Est. Priority Date: 12/10/1998
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Adnexus Therapeutics, Inc. (Bristol-Myers Squibb Company)
• Inventors: Wagner, Richard, Lipovsek, Dasa, Kuimelis, Robert
• Granted Patent: US 9,605,039 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• 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 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2317/22 from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2318/20 Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• 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

Disclosed herein are proteins that include an immunoglobulin fold and that can be used as scaffolds. Also disclosed herein are nucleic acids encoding such proteins and the use of such proteins in diagnostic methods and in methods for evolving novel compound-binding species and their ligands.

Claims

1-43. -43. (canceled)

44. A non-antibody protein comprising a domain having an immunoglobulin-like fold, said non-antibody protein deriving from a reference protein, having an immunoglobulin-like fold, by having a mutated amino acid sequence, wherein said domain has an immunoglobulin-like fold and a sequence that is at least 70% identical to a sequence selected from the group consisting of:

SEQ ID NOs;

80, 96, 67, 127, 128, 135, and 140, wherein said non-antibody protein binds with a Kd at least as tight as 1 μ

M to a compound that is not bound as tightly by said reference protein.

46. The derivative protein of claim 44, said derivative protein binding with a Kd at least as tight as 100 nM.

47. The derivative protein of claim 44, said derivative protein binding with Kd at least as tight as 1 nM.

48. The derivative protein of claim 44, said derivative protein binding with a Kd at least as tight as 100 pM.

49. The derivative protein of claim 44, said derivative protein binding with a Kd at least as tight as 20 pM.

50. The derivative protein of claim 44, wherein said domain has an immunoglobulin-like fold and a sequence that is at least 80% identical to a sequence selected from the group consisting of:

SEQ ID NOs;

80, 96, 67, 127, 128, 135, and 140.

51. The derivative protein of claim 44 containing one, two, or three mutated loops and wherein at least one of said loops contributes to the binding of said derivative protein to said compound.

52. The derivative protein of claim 51, wherein at least two of said loops contribute to said binding of said derivative protein to said compound.

53. The derivative protein of claim 51, wherein three of said loops contribute to said binding of said derivative protein to said compound.

54. The derivative protein of claim 44, wherein said derivative protein comprises a mutated fibronectin type III domain and wherein said domain is mutated in the BC, DE and FG loops.

55. The derivative protein of claim 54, wherein said FG loop is the same length as the wild-type FG loop.

56. The derivative protein of claim 44, wherein said reference protein lacks Cys.

57. The derivative protein of claim 56, wherein said derivative protein comprises a Cys.

58. The derivative protein of claim 44, wherein said domain has a molecular mass less than 10 kD.

59. The derivative protein of claim 44, wherein said domain has a molecular mass greater than 7.5 kD.

60. The derivative protein of claim 44, wherein said derivative protein is a monomer under physiological conditions.

61. The derivative protein of claim 44, wherein said derivative protein is a dimer under physiological conditions.

62. The derivative protein of claim 44, wherein said reference protein is a naturally-occurring mammalian protein.

63. The derivative protein of claim 44, wherein said domain is mutated and comprises up to 34% mutated amino acids as compared to the immunoglobulin-like fold of said reference protein.

64. The derivative protein of claim 44, wherein said derivative protein is derived from a fibronectin or fibronectin dimer.

65. The derivative protein of claim 44, wherein said derivative protein is fused to a second protein domain.

66. The derivative protein of claim 65, wherein said second protein domain is the constant region of an IgG (Fc).

67. The derivative protein of claim 65, wherein said second protein domain comprises a second immunoglobulin-like fold, said second protein domain deriving from said reference protein, having an immunoglobulin-like fold, by having a mutated amino acid sequence, wherein said second protein domain binds to said compound with a Kd at least as tight as 1 μ

M.

68. The derivative protein of claim 65, wherein said second protein domain comprises a second immunoglobulin-like fold, said second protein domain deriving from said reference protein, having an immunoglobulin-like fold, by having a mutated amino acid sequence, wherein said second protein domain binds to a second compound with a Kd at least as tight as 1 μ

M that is not bound as tightly as said reference protein.

69. The derivative protein of claim 65, wherein said second protein domain comprises a second immunoglobulin-like fold, said second protein domain deriving from a second reference protein, having an immunoglobulin-like fold, by having a mutated amino acid sequence, wherein said second protein domain binds to said compound with a Kd at least as tight as 1 μ

M.

70. The derivative protein of claim 65, wherein said second protein domain comprises a second immunoglobulin-like fold, said second protein domain deriving from a second reference protein, having an immunoglobulin-like fold, by having a mutated amino acid sequence, wherein said second protein domain binds to a second compound with a Kd at least as tight as 1 □

M that is not bound as tightly as said second reference protein.

71. A non-antibody protein comprising a domain having an immunoglobulin-like fold, said non-antibody protein deriving from a reference protein, having an immunoglobulin-like fold, by having mutated amino acid sequence, wherein said domain has an immunoglobulin-like fold and a sequence that is at least 80% identical to a sequence selected from the group consisting of:

SEQ ID NOs;

80, 96, 67, 127, 128, 135, and 140, wherein said non-antibody protein binds with a Kd at least as tight as 100 nM to a compound that is not bound as tightly by said reference protein.

72. The derivative protein of claim 71, said derivative protein binding with a Kd at least as 50 nM.

73. The derivative protein of claim 71, said derivative protein binding with a Kd at least as 50 pM.

74. The derivative protein of claim 71, wherein said domain has a molecular mass between 7.5-10 kD.

75. A loop structure on a protein, said loop comprising an amino acid sequence that is derived from a reference protein and at least 70% identical to a sequence selected from the group consisting of:

SEQ ID NOs;

80, 96, 67, 127, 128, 135, and 140, wherein said loop binds with a Kd at least as tight as 1 μ

M to a compound that is not bound as tightly by said reference protein.