Epitope-captured antibody display

• US 6,777,239 B2
• Filed: 04/17/2002
• Issued: 08/17/2004
• 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:
  US10/125,062
• Publication Number:
  US 20020192690A1
• Time in Patent Office: 853 Days
• Field of Search: 435/6, 435/7.1, 435/7.8, 435/91.4, 435/471, 435/489, 536/23.1, 536/23.4, 536/23.5
• US Class Current: 436/6
• 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 method of analyzing an amino acid sequence of polypeptides by determining whether they share an epitope, the method comprising:

(a) providing a population of replicable genetic package/antibody reagents, each replicable genetic package/antibody reagent (package/antibody reagent) comprising (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 having a plurality of binding sites, each site having specific affinity for the first polypeptide, with at least one of the sites available for binding, the first polypeptide and the captured antibody complexed with it varying between at least some of the package/antibody reagents;

(b) contacting the population of package/antibody reagents with a second polypeptide, whereby package/antibody reagents bearing captured antibodies having specific affinity for the second polypeptide bind to the second polypeptide;

(c) identifying at least one package/antibody reagent that binds to the second polypeptide; and

(d) determining the nucleotide sequence of the nucleic acid segment of the at least one package/antibody reagent, whereby the amino acid sequence corresponding to the nucleotide sequence can be deduced and is an indication of the amino acid sequence of an epitope shared by the first and second polypeptide.

2. The method of claim 1, wherein the providing step (a) comprises

(i) cloning cDNA molecules prepared from a cell or tissue into a vector to create the replicable genetic packages; and

(ii) incubating a population of antibodies with the replicable genetic packages to form the population of package/antibody reagents.

3. The method of claim 2, wherein the preparing step (a) further comprises preparing a population of immunogens and generating the population of antibodies with the population of immunogens.

4. The method of claim 3, wherein the population of immunogens comprise some or all of the proteins expressed in the cell or tissue.

5. The method of claim 4, wherein the at least some of the proteins are part of a fusion protein.

6. The method of claim 5, wherein the fusion protein includes glutathione-S-transferase (GST).

7. The method of claim 3, wherein the population of immunogens comprises a display library, the members of the display library comprising a replicable genetic package that displays one of the first polypeptides displayed by the package/antibody reagents.

8. The method of claim 7, wherein the polypeptides displayed on members of the display library are peptides having random amino acid sequences.

9. The method of claim 8, wherein the replicable genetic packages which display the peptides differ in type from the replicable genetic packages of the package/antibody reagents.

10. The method of claim 8, wherein the peptides displayed on members of the display library are up to 100 amino acids in length.

11. The method of claim 10, wherein the peptides displayed on members of the display library are 6 to 20 amino acids in length.

12. The method of claim 11, wherein the peptides displayed on members of the display library are 6 to 10 amino acids in length.

13. The method of claim 1, wherein

(i) the second polypeptide is one of multiple different polypeptides within a sample;

(ii) the contacting step (b) comprises contacting the population of package/antibody reagents with the sample; and

(iii) the identifying step (c) comprises identifying package/antibody reagents that have formed complexes with the different polypeptides.

14. The method of claim 1, wherein the second polypeptide is within an electrophoretic separation matrix, and the contacting step (b) comprises excising a section from the separation matrix that contains the second polypeptide, eluting the second polypeptide from the excised section and contacting the eluted second polypeptide with the package/antibody reagents.

15. The method of claim 1, wherein

(i) the second polypeptide is a polypeptide located within an electrophoretic separation matrix;

(ii) the contacting step (b) comprises contacting the separation matrix or a replica thereof with the package/antibody reagents; and

(iii) the identifying step (c) comprises isolating the at least one package/antibody reagent from the separation matrix or the replica thereof.

16. The method of claim 1, wherein

(i) the second polypeptide is one of a plurality of polypeptides at different locations within a two-dimensional electrophoretic separation matrix;

(ii) the contacting step (b) comprises contacting the two-dimensional electrophoretic separation matrix or a replica thereof with the package/antibody reagents;

(iii) the identifying step (c) comprises isolating package/antibody reagents that have formed complexes with the different polypeptides; and

(iv) the determining step (d) comprises determining the sequence of the segment of the nucleic acid of each of the isolated package/antibody reagents.

17. The method of claim 16, wherein

(i) the plurality of polypeptides within the separation matrix comprise a plurality of polypeptides that have different primary sequences and a plurality of polypeptides that have the same primary sequence but which are differentially modified;

and

(ii) the determining step (d) comprises determining which of the plurality of proteins within the separation matrix have the same primary sequence but are differentially modified.

18. The method of claim 1, wherein the second polypeptide is obtained from or contained in a tissue sample.

19. The method of claim 18, wherein the tissue sample is a slice from a tissue.

20. The method of claim 1, wherein the second polypeptide is obtained from or contained in a subcellular compartment.

21. The method of claim 1, wherein the replicable genetic package of the package/antibody complex is selected from the group consisting of a virus, a bacteriophage, a bacterium, a polysome and a spore.

22. The method of claim 1, wherein the replicable genetic package is a bacteriophage.

23. The method of claim 1, wherein the captured antibodies comprise monovalent antibodies displayed in a multivalent format.

24. The method of claim 23, wherein the monovalent antibodies are scFv polypeptides.

25. The method of claim 24, wherein the scFv polypeptides are displayed in a multivalent format on phage.

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

27. The method of claim 1, wherein at least some of the members of the population display a single copy of the first polypeptide which is complexed with a single copy of the captured antibody.

28. The method of claim 1, wherein the first polypeptide is complexed with a plurality of copies of the same captured antibody.

29. The method of claim 1, wherein the first polypeptide is complexed with multiple different antibodies.

30. The method of claim 1, wherein at least some of the members of the population display multiple copies of the first polypeptide, each copy of the first polypeptide complexed with one or more captured antibodies having specific affinity for the first polypeptide, whereby such members display a plurality of antibodies.

31. The method of claim 30, wherein at least some of the plurality of antibodies have different protein sequences but specific affinity for the same epitope.

32. The method of claim 30, wherein at least some of the plurality of antibodies have different protein sequences and have specific affinity for different epitopes.

33. The method of claim 1, wherein at least some members of the population form an aggregate, comprising a plurality of members linked via the captured antibody that is complexed to the first polypeptide on each of the plurality of members.

34. The method of claim 1, wherein

(i) the second polypeptide with which the population of package/antibody complexes is contacted is a receptor expressed on a surface of a cell, whereby the at least one package/antibody complex binds to the receptor and is subsequently transported into and/or through the cell;

and

(ii) the identifying step comprises detecting the transport of the at least one package/antibody reagent into and/or through the cell.

35. The method of claim 34, wherein the cell is a polarized cell and the identifying step comprises detecting transport through the cell.

36. The method of claim 34, wherein the cell is a non-polarized cell and the identifying step comprises detecting transport into the cell.