General strategy for antibody library screening

Patent metadata

Field	Value
Publication number	US-9733240-B2
Application number	US-201214361358-A
Country	US
Kind code	B2
Filing date	Dec 14, 2012
Priority date	Dec 16, 2011
Publication date	Aug 15, 2017
Grant date	Aug 15, 2017

Abstract

A generally applicable method for the selective covalent attachment of a reporter molecule to a replicating entity that allows one to obtain specific binders from a single round of library screening is disclosed. For example, selective biotinylation of phage particles and yeast cells displaying a binder to any given target can be achieved via application of a coupled enzyme reaction that includes a peroxidase, an oxidase and a catalase.

First claim

The invention claimed is:

1. A method for the isolation of specifically labelled replicating entities, said method comprising
a) providing in a reaction mixture a collection of replicating entities displaying variants of a receptor molecule on their surface and having either a first or a second enzyme linked to said surface, the reaction mixture of step a) comprising a third enzyme capable of decomposing excess product of said first and/or second enzyme, the first enzyme being a peroxidase, the second enzyme being an oxidase, and the third enzyme being a catalase, the substrate of the second enzyme being glucose and the product being H 2 O 2 , the cosubstrate of said first enzyme being a tyramide conjugated to a marker molecule,
b) adding to the mixture of step a) ligand molecules that are either linked to said first enzyme if said entities are linked to said second enzyme, or said ligands are linked to said second enzyme if said entities are linked to said first enzyme,
c) adding to the reaction mixture a substrate for the second enzyme, the substrate being utilized by said second enzyme in an enzymatic reaction to produce a product that is utilized by said first enzyme to convert a cosubstrate of said first enzyme into a reactive marker molecule that physically links to the respective replicating entity, thereby specifically labelling the replication entity, and
d) isolating the specifically labelled replicating entities.

2. Method according to claim 1 , wherein the replicating entity is selected from the group consisting of a phage, a phagemid, or a cell.

3. Method according to claim 2 , wherein the replicating entity is a yeast cell.

4. Method according to claim 3 , wherein the yeast cell is Saccharomyces cerevisiae.

5. Method according to claim 1 , the tyramide being linked to the marker molecule by a cleavable linker.

6. Method according to claim 5 , wherein the cleavable linker is a disulfide bond or a peptide sequence that can be cleaved by a protease.

7. Method according to claim 1 , the marker molecule being biotin, (2,4)-dinitrophenyl, fluorescein or digoxygenin derivative.

8. Method according to claim 6 , wherein the disulfide bond or a peptide sequence that can be cleaved by a protease is a Biotintyramide with cleavable disulfide bond of the following structure:

9. Method according to claim 1 , the marker molecule being biotin, and the isolation step d) being accomplished by affinity chromatography on an avidin or streptavidin matrix.

10. Method according to claim 1 , the marker molecule being fluorescein or a fluorescent avidin or strepatvidin bound biotin and the isolation step e) being accomplished by fluorescence activated cell sorting.

11. Method according to claim 1 , wherein the marker molecule is biotin and the isolation comprises magnetic cell sorting in of streptavidin of avidin coated paramagnetic particles bound to the biotin labelled entity.

12. Method according to claim 1 wherein the receptor molecule is an antibody.

13. Method according to claim 12 wherein the antibody is Cetuximab.

Assignees

• Merck Patent Gmbh
• Merck Patents Gmbh

Inventors

• Becker Stefan
• Heiseler Tim
• Maass Alexander
• Kolmar Harald

Classifications

• G01N33/54353 Primary

  with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393) · CPC title

• C12N15/1086

  Preparation or screening of expression libraries, e.g. reporter assays · CPC title

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• C40B30/08

  by measuring catalytic activity · CPC title