Nucleic acids encoding artificial antibody polypeptides

• US 6,462,189 B1
• Filed: 08/11/2000
• Issued: 10/08/2002
• Est. Priority Date: 06/12/1997
• Status: Expired due to Term

• Current Assignee: Novartis Ag
• Original Assignee: Research Corporation Technologies
• Inventors: Koide, Shohei
• Primary Examiner(s): Huff, Sheela
• Assistant Examiner(s): HELMS, LARRY RONALD
• Application Number:
  US09/638,202
• Time in Patent Office: 788 Days
• Field of Search: 536/23.1, 536/23.4, 536/23.53, 536/24.1, 536/23.5, 530/300, 530/350, 435/325, 435/243, 435/320.1, 435/365, 435/252.8
• US Class Current: 536/23.5
• 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 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• 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: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• 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 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors

Abstract

A fibronectin type III (Fn3) polypeptide monobody, a nucleic acid molecule encoding the monobody, and a variegated nucleic acid library encoding the monobody, are provided by the invention. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods. Further provided is a method of identifying the amino acid sequence of a polypeptide molecule capable of binding to a specific binding partner (SBP) so as to form a polypeptide:SSP complex, and a method of identifying the amino acid sequence of a polypeptide molecule capable of catalyzing a chemical reaction with a catalyzed rate constant, k_cat, and an uncatalyzed rate constant, k_uncat, such that the ratio of k_cat/k_uncat is greater than 10.

Claims

1. An isolated nucleic acid molecule encoding a fibronectin type III (Fn3) polypeptide monobody comprising at least two Fn3 β

-strand domain sequences with a loop region sequence linked between each Fn3 β

-strand domain sequence,

wherein at least one monobody loop region sequence varies as compared to the

wild-type loop region sequence by deletion of at least two amino acids in the loop region sequence, insertion of at least two to 25 amino acids, or replacement of at least two amino acids in the loop region sequence, and

wherein the polypeptide monobody binds to a specific binding partner (SBP) to form a polypeptide;

SBP complex.

2. An expression vector comprising an expression cassette operably linked to the nucleic acid molecule of claim 1.

3. The expression vector of claim 2, wherein the expression vector is an M13 phage-based plasmid.

4. A host cell comprising the vector of claim 2.