Epitope-captured antibody display

• US 7,741,062 B2
• Filed: 04/07/2009
• Issued: 06/22/2010
• 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: Cwirla, Steven E., Dower, William J.
• Primary Examiner(s): CHEU, CHANGHWA J
• Application Number:
  US12/420,001
• Publication Number:
  US 20090280999A1
• Time in Patent Office: 441 Days
• Field of Search: None
• 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. An array of polypeptides, comprising

(a) a support;

and

(b) a plurality of replicable genetic package/antibody reagents (package/antibody reagents) immobilized at different locations on the support;

wherein;

each of the replicable genetic package/antibody reagents comprises;

(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 has a plurality of binding sites with at least one first binding site having specific binding affinity for the displayed polypeptide and at least one second binding site which is unbound; and

the replicable genetic package of each package/antibody reagent is immobilized on the support.

2. The array of claim 1, wherein there are at least 10³ locations/cm² on the support, each location having at least one of the plurality of package/antibody reagents immobilized on the location, and wherein the displayed polypeptides in at least some of the locations differ in amino acid sequence from the displayed polypeptides in other locations.

3. The array of claim 2, wherein there are at least 10⁶ locations/cm².

4. The array of claim 3, wherein there are at least 10⁸ locations/cm².

5. The array of claim 4, wherein there are at least 10¹⁰ locations/cm².

6. The array of claim 2, wherein there are at least 100 different displayed polypeptides immobilized to the support.

7. The array of claim 6, wherein there are at least 1000 different displayed polypeptides immobilized to the support.

8. The array of claim 7, wherein there are at least 10,000 different displayed polypeptides immobilized to the support.

9. The array of claim 8, wherein there are at least 100,000 different displayed polypeptides immobilized to the support.

10. The array of claim 1, wherein the at least one displayed polypeptide at each location differs in amino acid sequence from the at least one displayed polypeptide at each of the other locations.

11. The array of claim 1, wherein:

each of the replicable genetic packages are phage having a heterologous nucleic acid segment that encodes a polypeptide displayed on the phage;

the antibodies are bound to the polypeptides displayed on the phage;

the phage are immobilized to the support; and

the array is prepared by;

(i) plating the phage on a layer of cells to form bacterial microcolonies or micro-plaques;

(ii) replicating the microcolonies or micro-plaques onto the support, whereby the phage displaying the polypeptides become immobilized to the support; and

(iii) contacting the displayed polypeptides with a population of antibodies under conditions such that the antibodies form complexes with the displayed polypeptides for which the antibodies have specific binding affinity, whereby the array is formed.

12. The array of claim 1, wherein a second polypeptide is complexed with the at least one second binding site of at least some of the captured antibodies of the replicable genetic package/antibody reagents.

13. The array of claim 12, wherein the second polypeptide is a functional polypeptide.

14. The array of claim 1, wherein each of the replicable genetic packages is selected from the group consisting of a virus, a bacteriophage, a bacterium, a polysome, and a spore.

15. The array of claim 1, wherein each of the replicable genetic packages is a bacteriophage.

16. The array of claim 1, wherein the captured antibodies comprise a monovalent antibody displayed in a multivalent format.

17. The array of claim 16, wherein the monovalent antibody is a scFv polypeptide.

18. The array of claim 1, wherein the package/antibody reagents are labeled.

19. The array of claim 18, wherein the replicable genetic package is a bacteriophage and the label is attached to the bacteriophage.

20. The array of claim 1, wherein the captured antibody is a diabody, a tribody or a tetrabody.

21. The array of claim 1, wherein at least some of the package/antibody reagents display a single copy of the displayed polypeptide, and the displayed polypeptide is bound to a single copy of the captured antibody.

22. The array of claim 1, wherein the displayed polypeptide is bound to a plurality of different captured antibodies.

23. The array of claim 1, wherein at least some of the replicable genetic packages display multiple copies of the displayed polypeptide, and one or more captured antibodies having specific affinity for the displayed polypeptide are bound to each copy of the displayed polypeptide.

24. The array of claim 23, wherein at least some of the captured antibodies have different amino acid sequences and have specific binding affinity for the same epitope.

25. The array of claim 24, wherein at least some of the captured antibodies have different amino acid sequences and have specific binding affinity for different epitopes.

26. The array of claim 1, 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.

27. The array of claim 26, 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.