Reconstituted polypeptides

• US 8,258,265 B2
• Filed: 08/30/2007
• Issued: 09/04/2012
• Est. Priority Date: 06/06/2002
• Status: Active Grant

• Current Assignee: Research Corporation Technologies
• Original Assignee: Research Corporation Technologies
• Inventors: Koide, Shohei
• Primary Examiner(s): BRISTOL, LYNN ANNE
• Application Number:
  US11/848,135
• Publication Number:
  US 20090253899A1
• Time in Patent Office: 1,832 Days
• Field of Search: 530/387.1, 424/133.1
• US Class Current: 530/387.1
• 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 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

Abstract

The present invention provides modified fibronectin type III (Fn3) molecules, and nucleic acid molecules encoding the modified Fn3 molecules. Also provided are methods of preparing these molecules, and kits to perform the methods.

Claims

1. A fibronectin monobody polypeptide comprising a first fibronectin type III (Fn3) monobody fragment having at least two to five Fn3 beta-strand domains in the Fn3 wild-type arrangement with a loop region linked between each beta-strand domain, and at least one additional complementary fibronectin monobody fragment comprising at least two to five Fn3 beta strand domains in the Fn3 wild-type arrangement with a loop region linked between each beta-strand domain, wherein the loop regions correspond to wild-type Fn3 loop regions, or comprise an AB, BC, CD, DE, or FG loop region sequence that is randomized or varies from a corresponding wild-type Fn3 loop region by deletion of one to all except one amino acid in the loop region, insertion of one to 25 amino acids, and/or replacement of at least one amino acid in the loop region.

2. The monobody of claim 1, wherein the first fibronectin type III (Fn3) monobody fragment is a tenth fibronectin type III fragment (Fn3¹⁰) and the at least one additional fibronectin monobody fragment is a fibronectin type III (Fn3) fragment.

3. The monobody of claim 1, wherein at least one loop region of the first or the at least one additional monobody fragment is capable of binding to a specific binding partner (SBP) to form a monobody:

SBP complex.

4. The monobody of claim 1, wherein at least one loop region of the first fibronectin type III (Fn3) monobody fragment or of at least one additional complementary fibronectin monobody fragment 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.

5. The monobody of claim 1, wherein at least one loop region of the first fibronectin type III (Fn3) monobody fragment or of at least one additional complementary fibronectin monobody fragment comprises an AB, BC, CD, DE, or FG loop region sequence that varies from a corresponding wild-type Fn3 loop region by deletion of one to all except one amino acid in the loop region, insertion of one to 25 amino acids, and/or replacement of at least one amino acid in the loop region.

6. The monobody of claim 1, wherein at least one loop region of the first fibronectin type III (Fn3) monobody fragment or of at least one additional complementary fibronectin monobody fragment comprises an AB, BC, CD, DE, or FG loop region sequence that varies from a corresponding wild-type Fn3 loop region by deletion of one to all except one amino acid and/or replacement of at least one amino acid.

7. The monobody of claim 1, wherein at least one loop region of the first fibronectin type III (Fn3) monobody fragment or of at least one additional complementary fibronectin monobody fragment comprises an AB, BC, CD, DE, or FG loop region sequence that varies from a corresponding wild-type Fn3 loop region by insertion of one to 25 amino acids.

8. The monobody of claim 1, wherein the first fragment comprises a first cysteine and the at least one additional fragment comprises a second cysteine, and wherein the first cysteine and the second cysteine form a disulfide bond.

9. The monobody of claim 8, wherein the first cysteine is located in a loop region.

10. The monobody of claim 8, wherein the second cysteine is located in a loop region.

11. The monobody of claim 8, wherein the first cysteine is located in a beta-strand region.

12. The monobody of claim 8, wherein the second cysteine is located in a beta-strand region.

13. The monobody of claim 2, wherein the first fibronectin type III (Fn3) monobody fragment is a tenth fibronectin type III fragment)(Fn3¹⁰) and the at least one additional fibronectin monobody fragment is a Fn3¹⁰ fragment.