Selection of intracellular immunoglobulins

• US 7,897,347 B2
• Filed: 07/31/2009
• Issued: 03/01/2011
• Est. Priority Date: 03/10/1999
• Status: Expired due to Fees

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom), Scuola Internazionale Superiore Di Studi Avanzati
• Inventors: Tse, Wai-Choi Eric, Cattaneo, Antonino, Visintin, Michela, Rabbits, Terence
• Primary Examiner(s): BRISTOL, LYNN ANNE
• Application Number:
  US12/533,614
• Publication Number:
  US 20090317822A1
• Time in Patent Office: 578 Days
• Field of Search: 435/6
• US Class Current: 435/6.14
• 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. A method for preparing an immunoglobulin suitable 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 a 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;

the isolating step 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) wherein the isolated immunoglobin genes specific for a desired target are cloned in-frame with a nucleic acid encoding the first molecule and expressed,

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.

2. The method of claim 1, wherein the reporter molecule is selected from the group consisting of a transcription factor, an enzyme and a bioluminescent molecule.

3. The method of claim 2 wherein the reporter molecule is an enzyme and the method is performed in the presence of a substrate for the enzyme.

4. The method of claim 1, wherein the first molecule is the activation domain of VP16and the second molecule is the DNA-binding domain of LexA.

5. The method of claim 1, wherein the detecting step is selected from the group consisting of a change in an optical property and the activation of a reporter gene.

6. The method of claim 5, wherein the detecting step allows the sorting of cells.

7. The method of claim 1, wherein the immunoglobulin is selected from the group consisting of an intact immunoglobulin, a Fv, a scFv, a Fab and a F(ab′

)₂.

8. The method of claim 1, wherein the immunoglobulin is provided by expressing an immunoglobulin-encoding nucleic acid within a cell.