Cell-based methods for coupling protein interactions and binding molecule selection

摘要

The invention relates to cell-based methods for diversifying, expressing and selecting binding molecules, e.g., antibodies, and target molecules to which they bind, all of which are expressed in the same cell. The target molecule can be a member of a ligand binding pair comprising a cell-surface expressed ligand binding receptor molecule and its cognate ligand, which interact within the cell. The methods provide retaining either the antibody or its target in a cell organelle as the site of binding and interaction. By performance of the methods, the binding or non-binding of the antibody to its target molecule within the cell produces a cell phenotype that is detectable at the cell surface via high throughput assays, e.g., flow cytometry. The methods are particularly useful for generating, recovering and providing antibodies that have optimal target molecule binding properties or activities for potential therapeutic use. Methods for generating diversity in such antibodies are also provided.

主权项

1. A method of selecting a binding molecule which blocks or disrupts an interaction between a cell surface expressed receptor protein and its cognate ligand within a cell, comprising:
(a) expressing in the cell a receptor protein which is expressible on the surface of the cell; (b) expressing in the cell a cognate ligand of the receptor protein, said cognate ligand being molecularly tagged with a peptide for retaining the ligand in an intracellular organelle under conditions allowing for the retention of the ligand in the organelle and the interaction of the receptor protein and the cognate ligand in the organelle; wherein the intracellular organelle is selected from the group consisting of endoplasmic reticulum (ER), Golgi, cellular plasma membrane; mitochondrion; nucleus; and peroxisome; (c) introducing into the cell a binding molecule, or a binding fragment or portion thereof, which specifically binds either the receptor protein or the ligand protein retained in the intracellular organelle; and (d) detecting the level of the receptor protein expressed on the cell surface; wherein, if the binding molecule, or a binding fragment or portion thereof, binds to either the receptor protein or the ligand and blocks or disrupts their interaction in the organelle, the receptor protein is expressed and detectable on the cell surface, and wherein the binding molecule, or a binding fragment or portion thereof, which blocks or disrupts the receptor protein and ligand interaction is selectable.