Protein scaffolds for antibody mimics and other binding proteins

• US 20050255548A1
• Filed: 11/15/2004
• Published: 11/17/2005
• Est. Priority Date: 12/10/1998
• Status: Abandoned Application

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Compound Therapeutics, Inc. (Bristol-Myers Squibb Company), Phylos, Inc. (Bristol-Myers Squibb Company)
• Inventors: Kuimelis, Robert G., Lipovsek, Dasa, Wagner, Richard W.
• Application Number:
  US10/989,723
• Publication Number:
  US 20050255548A1
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• 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 2319/30 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: C12N 15/62 DNA sequences coding for fusion proteins
• 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 a fibronectin type III domain having at least one randomized loop. 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

1-67. -67. (canceled)

68. A protein comprising:

(a) a first fibronectin type III (Fn3) domain wherein said first Fn3 domain has an amino acid sequence that has been altered relative to the amino acid sequence of a first naturally occurring Fn3 domain such that the first Fn3 domain binds to a first compound that is not bound by the first naturally occurring Fn3 domain; and

(b) a second Fn3 domain, wherein said second Fn3 domain has an amino acid sequence that has been altered relative to the amino acid sequence of a second naturally occurring Fn3 domain such that the second Fn3 domain binds to a compound that is not bound by the second naturally occurring Fn3 domain.

69. The protein of claim 68, wherein said protein further comprises one or more additional Fn3 domains.

70. The protein of claim 69, wherein each of said additional Fn3 domains has an amino acid sequence that has been altered relative to the amino acid sequence of a naturally occurring Fn3 domain such that each said additional Fn3 domain binds to a compound that is not bound by the corresponding naturally occurring Fn3 domain.

71. The protein of claim 68, wherein said first Fn3 domain has an amino acid sequence that is altered relative to the amino acid sequence of a first human Fn3 domain, such that the first Fn3 domain binds to a first compound that is not bound by the first human Fn3 domain.

72. The protein of claim 71, wherein said first Fn3 domain has an amino acid sequence that is at least 50% identical to the amino acid sequence of said first human Fn3 domain.

73. The protein of claim 71, wherein said first human Fn3 domain is an Fn3 domain of fibronectin.

74. The protein of claim 73, wherein said first human Fn3 domain is the tenth Fn3 module of human fibronectin (¹⁰Fn3).

75. The protein of claim 68, wherein the first and second Fn3 domains are altered in one or more loops relative to the first or second naturally occurring Fn3 domains, respectively.

76. The protein of claim 75, wherein the first and second Fn3 domains are altered in two or more loops relative to the first or second naturally occurring Fn3 domains, respectively.

77. The protein of claim 74, wherein the amino acid sequence of the first Fn3 domain is altered relative to the amino acid sequence of human 10Fn3 in one or more loops defined by amino acids 21-31, 51-6 and 76-88 in human Fn3.

78. The protein of claim 77, wherein the amino acid sequence of the first Fn3 domain is altered relative to the amino acid sequence of human ¹⁰Fn3 in one or more beta strands.

79. The protein of claim 73, wherein said second Fn3 domain has an amino acid sequence that is altered relative to the amino acid sequence of a second human Fn3 domain, such that the second human Fn3 domain binds to a second compound that is not bound by the second human Fn3 domain.

80. The protein of claim 79, wherein said second Fn3 domain has an amino acid sequence that is at least 50% identical to the amino acid sequence of said second human Fn3 domain.

81. The protein of claim 80, wherein said second human Fn3 domain is an Fn3 domain of fibronectin.

82. The protein of claim 81, wherein said second human Fn3 domain is the tenth Fn3 module of human fibronectin (¹⁰Fn3).

83. The protein of claim 68, wherein said first and second naturally occurring Fn3 domains are the same.

84. The protein of claim 68, wherein said first and second compounds are the same.

85. The protein of claim 68, wherein said first and second compounds are different.

86. The protein of claim 68, wherein said first compound is a first protein.

87. The protein of claim 86, wherein said second compound is a second protein.

88. The protein of claim 69, wherein said protein comprises:

(a) a first Fn3 domain wherein said first Fn3 domain has an amino acid sequence that has been altered relative to the amino acid sequence of the eighth Fn3 module of human fibronectin (⁸Fn3) such that the first Fn3 domain binds to a first compound that is not bound by human ⁸Fn3;

(b) a second Fn3 domain wherein said first Fn3 domain has an amino acid sequence that has been altered relative to the amino acid sequence of the ninth Fn3 module of human fibronectin (9Fn3) such that the second Fn3 domain binds to a second compound that is not bound by human ⁹Fn3; and

(c) a third Fn3 domain wherein said third Fn3 domain has an amino acid sequence that has been altered relative to the amino acid sequence of the tenth Fn3 module of human fibronectin (¹⁰Fn3) such that the third Fn3 domain binds to a first compound that is not bound by human ¹⁰Fn3.