PROTEIN SCAFFOLDS FOR ANTIBODY MIMICS AND OTHER BINDING PROTEINS

• US 20170275342A1
• Filed: 02/23/2017
• Published: 09/28/2017
• Est. Priority Date: 12/10/1998
• Status: Abandoned Application

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: LIPOVSEK, Dasa, WAGNER, Richard W., KUIMELIS, Robert G.
• Application Number:
  US15/440,730
• Publication Number:
  US 20170275342A1
• Time in Patent Office: Days
• 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. An isolated protein comprising a domain derived from the tenth module of the human fibronectin type III (¹⁰Fn3) comprising an amino acid sequence that

(a) comprises seven beta strands distributed between two antiparallel beta-sheets;

(b) comprises BC, DE and FG loops which connect the beta strands, wherein one, two or three of said loops are mutated;

(c) lacks an integrin binding motif; and

(d) is at least 70% identical to the wild-type human ¹⁰Fn3 domain,

wherein said derivative ¹⁰Fn3 domain binds with a Kd at least as tight as 1 μ

M to an antigen.

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

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

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

53. A pharmaceutical composition comprising the protein of claim 44.

48. An array comprising a library of proteins comprising a domain derived from the tenth module of the human fibronectin type III (¹⁰Fn3) comprising an amino acid sequence that

(a) comprises seven beta strands distributed between two antiparallel beta-sheets;

(b) comprises BC, DE and FG loops which connect the beta strands, wherein one, two or three of said loops are mutated;

(c) lacks an integrin binding motif; and

(d) is at least 70% identical to the wild-type human ¹⁰Fn3 domain.

49. A nucleic acid encoding an isolated protein comprising a domain derived from the tenth module of the human fibronectin type III (¹⁰Fn3) comprising an amino acid sequence that

(a) comprises seven beta strands distributed between two antiparallel beta-sheets;

(b) comprises BC, DE and FG loops which connect the beta strands, wherein one, two or three of said loops are mutated;

(c) lacks an integrin binding motif; and

(d) is at least 70% identical to the wild-type human ¹⁰Fn3 domain.

50. A method of obtaining a protein comprising a domain derived from the tenth module of the wild-type human fibronectin type III (¹⁰Fn3) which binds to an antigen, the method comprising:

(a) providing a wild-type ¹⁰Fn3 domain;

to t

(b) generating mutated derivatives of the wild-type ¹⁰Fn3 domain, thereby producing a library of ¹⁰Fn3 domain derivatives;

(c) contacting the library with the antigen;

(d) selecting from the library at least one derivative protein which binds to the antigen with a Kd at least as tight as 1 μ

M; and

(e) optionally repeating steps (b)-(d).

51. A method of isolating a protein comprising a domain derived from the tenth module of the wild-type human fibronectin type III (¹⁰Fn3) which binds to an antigen, the method comprising:

(a) contacting the antigen with a candidate protein comprising a mutated derivative of the wild-type ¹⁰Fn3 domain which binds with a Kd at least as tight as 1 μ

M the antigen under conditions that allow antigen-protein complex formation; and

(b) obtaining the ¹⁰Fn3 derivative protein from the complex.

52. A method of detecting an antigen in a sample, said method comprising:

(a) contacting said sample with a protein comprising a domain derived from the tenth module of the wild-type human fibronectin type III (¹⁰Fn3) which binds to the antigen with a Kd at least as tight as 1 μ

M the antigen under conditions that allow antigen-protein complex formation; and

(b) detecting the complex;

wherein the detection of the complex indicates the presence of the antigen in the sample.