Epitope-captured antibody display

• US 7,534,574 B2
• Filed: 01/08/2007
• Issued: 05/19/2009
• Est. Priority Date: 04/17/2001
• Status: Expired due to Fees

• Current Assignee: XenoPort, Inc. (Arbor Pharmaceuticals LLC)
• Original Assignee: XenoPort, Inc. (Arbor Pharmaceuticals LLC)
• Inventors: Dower, William J., Cwirla, Steven E.
• Primary Examiner(s): Le; Long V
• Assistant Examiner(s): CHEU, CHANGHWA J
• Application Number:
  US11/621,022
• Publication Number:
  US 20070269798A1
• Time in Patent Office: 862 Days
• Field of Search: 435/7.1, 435/7.2, 435/7.91, 435/7.92, 436/518
• US Class Current: 435/7.1
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• 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 2035/00158 Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2600/00 Assays involving molecular imprinted polymers/polymers created around a molecular template
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6857 Antibody fragments

Abstract

Reagents and methods for detecting target proteins in a sample are provided. The reagents include a replicable genetic package, a protein displayed on an exterior surface of the package that is expressed from a heterologous nucleic acid borne by the package, and one or more antibodies complexed with the expressed protein and which have an open binding site for a target protein. Thus, a segment of the nucleic acid encodes for an epitope that is shared by the expressed polypeptide and the target protein. The reagents can be utilized individually or as part of a library or an array to bind target proteins within protein samples to form one or more complexes. By determining the sequence of the segment of the heterologous nucleic acid of a package within a complex, one can identify the target protein since the segment encodes for an epitope that is shared by the expressed and target proteins.

Claims

1. A replicable genetic package/antibody reagent (package/antibody reagent) comprising:

(i) a replicable genetic package having a heterologous nucleic acid segment that encodes a polypeptide displayed on the replicable genetic package; and

(ii) a captured antibody bound to the displayed polypeptide, wherein the captured antibody comprises a plurality of binding sites with a first binding site having specific binding affinity for the displayed polypeptide and is bound to the displayed polypeptide and a second binding site having specific affinity for the displayed polypeptide and is unbound.

21. The replicable genetic package/antibody reagent of claim 1, wherein the package/antibody reagent is immobilized to a support.

2. A collection of replicable genetic package/antibody reagents (package/antibody reagent), wherein each package/antibody reagent comprises:

(i) a replicable genetic package having a heterologous nucleic acid segment that encodes a first polypeptide displayed on the replicable genetic package; and

(ii) a captured antibody bound to the first displayed polypeptide, wherein the captured antibody comprises a plurality of binding sites with a first binding site having specific binding affinity for the first displayed polypeptide and is bound to the first displayed polypeptide and a second binding site having specific affinity for the first displayed polypeptide and is unbound;

wherein at least some of the package/antibody reagents comprise the same first polypeptide bound to a different captured antibody and/or a different first polypeptide bound to a different captured antibody.

3. The collection of claim 2, wherein the replicable genetic package is selected from the group consisting of a virus, a bacteriophage, a bacterium, a polysome and a spore.

4. The collection of claim 3, wherein the replicable genetic package is a bacteriophage.

5. The collection of claim 2, wherein the captured antibody comprises a monovalent antibody displayed in a multivalent format.

6. The collection of claim 2, wherein the package/antibody reagents are labeled.

7. The collection of claim 6, wherein the replicable genetic package is a bacteriophage and the label is attached to the bacteriophage.

8. The collection of claim 5, wherein the monovalent antibody is a scFv polypeptide.

9. The collection of claim 2, wherein the captured antibody is a antibody, a tribody or a tetrabody.

10. The collection of claim 2, wherein at least some of the package/antibody reagents display a single copy of the first polypeptide, and the first polypeptide is bound to a single copy of the captured antibody.

11. The collection of claim 2, wherein the first polypeptide is bound to a plurality of different captured antibodies.

12. The collection of claim 2, wherein at least some of the replicable genetic packages display multiple copies of the first polypeptide, and one or more captured antibodies having specific affinity for the first polypeptide are bound to each copy of the first polypeptide.

13. The collection of claim 12, wherein at least some of the captured antibodies have different protein sequences and have specific binding affinity for the same epitope.

14. The collection of claim 12, wherein at least some of the captured antibodies have different protein sequences and have specific binding affinity for a different epitope.

15. The collection of claim 2, wherein the package/antibody reagents are immobilized to a support to form an array.

16. The collection of claim 15, comprising at least 100 different package/antibody reagents, wherein each different package/antibody reagent comprises a captured antibody having specific binding affinity for a different epitope.

17. The collection of claim 16, comprising at least 1000 different package/antibody reagents, wherein each different package/antibody reagent comprises a captured antibody having specific binding affinity for a different epitope.

18. The collection of claim 15, wherein the package/antibody reagent comprises captured antibodies having specific binding affinity for at least 80% of the expressed proteins of a cell or tissue.

19. The collection of claim 15, wherein at least some of the captured antibodies are further bound to a target polypeptide at the at least one second binding site.

20. The collection of claim 19, wherein the target polypeptide is a functional polypeptide.