Artificial antibody polypeptides

• US 6,673,901 B2
• Filed: 06/12/1998
• Issued: 01/06/2004
• Est. Priority Date: 06/12/1997
• Status: Expired due to Term

• Current Assignee: Novartis International Pharmaceutical Limited (Novartis Ag)
• Original Assignee: Research Corporation Technologies
• Inventors: Koide, Shohei
• Primary Examiner(s): HELMS, LARRY RONALD
• Application Number:
  US09/096,749
• Publication Number:
  US 20020019517A1
• Time in Patent Office: 2,034 Days
• Field of Search: 530/300, 530/380, 530/350, 530/381, 530/387.1, 424/130.1, 514/2
• US Class Current: 530/380
• 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 said monobody, and a variegated nucleic acid library encoding said monobody, are provided by the invention. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform said 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. 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 (SEQ ID NO;

110, FIG.

     2) loop region sequence by deletion of two to twelve 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 loop region comprises at least two amino acids and binds to a specific binding partner (SBP) to form a polypeptide;

SBP complex.

2. The monobody of claim 1, wherein at least one loop region binds to a specific binding partner (SBP) to form a polypeptide:

SBP complex having a dissociation constant of less than 10<sup>−

6</sup> moles/liter.

3. The monobody of claim 1, wherein a loop region comprises amino acid residues:

i) from 15 to 16 inclusive in an AB loop;

ii) from 22 to 30 inclusive in a BC loop;

iii) from 39 to 45 inclusive in a CD loop;

iv) from 51 to 55 inclusive in a DE loop;

v) from 60 to 66 inclusive in an EF loop;

or

vi) from 76 to 87 inclusive in an FG loop.

4. The monobody of claim 1, wherein the monobody loop region sequence varies from the wild-type Fn3 loop region sequence by the replacement of at least two amino acids in the loop region or the deletion of two to twelve amino acids in the loop region.

5. The monobody of claim 1, wherein the monobody loop region sequence varies from the wild-type Fn3 loop region sequence by the insertion of from 3 to 25 amino acids.

6. A composition comprising a fibronectin type III (Fn3) polypeptide monobody of claim 1.

7. 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 (SEQ ID NO;

110, FIG.

     2) loop region sequence by deletion of two to twelve amino acids in the loop region sequence and insertion of at least two to 25 amino acids in the loop region sequence, and

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

SBP complex.