RNA-antibody fusions and their selection

• US 6,518,018 B1
• Filed: 01/28/1999
• Issued: 02/11/2003
• Est. Priority Date: 01/21/1997
• Status: Expired due to Term

• Current Assignee: Adnexus Therapeutics, Inc. (Bristol-Myers Squibb Company), The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Szostak, Jack W., Roberts, Richard W.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/238,710
• Time in Patent Office: 1,475 Days
• Field of Search: 435/91.21, 435/6, 435/69.1, 536/23.4, 536/23.53, 536/23.1, 530/387.1
• US Class Current: 435/6.1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

Described herein are methods and reagents for the selection of protein molecules that make use of RNA-protein fusions.

Claims

1. A molecule comprising a ribonucleic acid covalently bonded through an amide bond to an antibody, wherein said antibody is encoded by said ribonucleic acid.

3. The molecule of claim 1, wherein said amide bond is resistant to cleavage by a ribosome.

4. The molecule of any of claims 1, 2, or 3, wherein said antibody is a single chain antibody.

5. The molecule of any of claims 1, 2, or 3, wherein said antibody is a human antibody.

6. The molecule of any of claims 1, 2, or 3, wherein the variable region of said antibody is derived from a human antibody.

7. The molecule of any of claims 1, 2, or 3, wherein the variable region of said antibody comprises a randomized region.

8. The molecule of claim 1 or 2, wherein said molecule further comprises a peptide acceptor positioned between said ribonucleic acid and said antibody.

2. A molecule comprising a ribonucleic acid covalently bonded to an antibody, said antibody being entirely encoded by said ribonucleic acid.

9. An RNA-antibody fusion selected by the method comprising the steps of:

a) providing a population of candidate RNA molecules, each of which comprises a translation initiation sequence and a start codon operably linked to a candidate antibody coding sequence and each of which is operably linked to a peptide acceptor at the 3′

end of said candidate antibody coding sequence;

b) in vitro or in situ translating said candidate antibody coding sequences to produce a population of candidate RNA-antibody fusions;

c) contacting said population of candidate RNA-antibody fusions with a target antigen; and

d) selecting an RNA-antibody fusion that binds to said target antigen.

10. An RNA-antibody fusion selected by the method comprising the steps of:

a) providing a population of candidate RNA molecules, each of which comprises a translation initiation sequence and a start codon operably linked to a candidate antibody coding sequence and each of which is operably linked to a peptide acceptor at the 3′

end of said candidate antibody coding sequence;

b) in vitro translating said candidate antibody coding sequences to produce a population of candidate RNA-antibody fusions;

c) contacting said population of candidate RNA-antibody fusions with a target antigen; and

d) selecting an RNA-antibody fusion that binds to said target antigen.

11. An RNA-antibody fusion comprising an antibody portion having an altered binding affinity or specificity for a target antigen relative to the binding affinity or specificity of a reference antibody, said RNA-antibody fusion selected by the method comprising the steps of:

a) producing a population of candidate RNA molecules, each having a candidate antibody coding sequence which differs from said reference antibody coding sequence, said RNA molecules each comprising a translation initiation sequence and a start codon operably linked to said candidate antibody coding sequence and each being operably linked to a peptide acceptor at the 3′

end;

b) in vitro or in situ translating said candidate antibody coding sequences to produce a population of candidate RNA-antibody fusions;

c) contacting said population of candidate RNA-antibody fusions with said target antigen; and

d) selecting an RNA-antibody fusion, the antibody portion of which has a binding affinity or specificity for said target antigen that is altered relative to the binding affinity or specificity of said reference antibody for said target antigen.

12. An RNA-antibody fusion comprising an antibody portion having an altered binding affinity or specificity for a target antigen relative to the binding affinity or specificity of a reference antibody, said RNA-antibody fusion selected by the method comprising the steps of:

a) producing a population of candidate RNA molecules, each having a candidate antibody coding sequence which differs from said reference antibody coding sequence, said RNA molecules each comprising a translation initiation sequence and a start codon operably linked to said candidate antibody coding sequence and each being operably linked to a peptide acceptor at the 3′

end;

b) in vitro translating said candidate antibody coding sequences to produce a population of candidate RNA-antibody fusions;

c) contacting said population of candidate RNA-antibody fusions with said target antigen; and

d) selecting an RNA-antibody fusion, the antibody portion of which has a binding affinity or specificity for said target antigen that is altered relative to the binding affinity or specificity of said reference antibody for said target antigen.