SELECTION OF INTRACELLULAR IMMUNOGLOBULINS

• US 20090317822A1
• Filed: 07/31/2009
• Published: 12/24/2009
• Est. Priority Date: 03/10/1999
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom), Scuola Internazionale Superiore Di Studi Avanzati
• Inventors: CATTANEO, Antonino, TSE, Wai-Choi Eric, RABBITS, Terence, VISINTIN, Michela
• Granted Patent: US 7,897,347 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 16/08 against material from viruses
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

A general immunoglobulin-target assay system is provided, in which a positive outcome (the generation of a signal) depends only on the intracellular interaction of immunoglobulin with target. This can be accomplished for many immunoglobulins expressed in yeast and/or in mammalian cells and allows the selection of immunoglobulins which are capable of functioning in an intracellular environment.

Claims

1-18. -18. (canceled)

19. A method for preparing an immunoglobulin suitable for use in a procedure for determining binding of an immunoglobulin to a target in an intracellular environment, comprising the steps of:

i) providing a first fusion polypeptide comprising an immunoglobulin fused to a first molecule, wherein the nucleic acid encoding the immunoglobulin is obtained from a phage library encoding a repertoire of immunoglobulin-encoding nucleic acids, wherein the immunoglobulin is unknown and no prior application of phage display is used to isolate immunoglobulins which bind to a target;

ii) providing a second fusion polypeptide comprising said target fused to a second molecule, wherein said first and second molecules are separable domains of a reporter molecule;

iii) expressing said first fusion polypeptide together with said second fusion polypeptide in an intracellular environment, wherein binding of said immunoglobulin with said target brings said first molecule and said second molecule into operative association, such that binding of said immunoglobulin and said target leads to stable interaction of said first and second molecules, thus producing a detectable reporter molecule that generates a signal; and

iv) detecting said signal from said detectable reporter molecule, wherein said detection of a signal is indicative of stable binding activity between said immunoglobulin and said target in the intracellular environment; and

isolating those immunoglobulins that stably bind to the target;

comprising the steps of;

(a) expressing a repertoire of immunoglobulin genes in a selection system and isolating those genes which encode immunoglobulins specific for a desired target;

(b) bringing the isolated genes into operative association with nucleic acids encoding a first molecule, wherein stable interaction of the first molecule with a second molecule generates a signal, in order to produce a fusion polypeptide comprising the immunoglobulin and the first molecule.