Isolating Cells Expressing Secreted Proteins

Abstract

A method of detecting and isolating cells that produce a secreted protein of interest (POI) that has a T cell receptor variable domain, comprising: a) constructing a cell line transiently or stably expressing a cell surface capture molecule, which binds the POI, by transfecting the cell line with a nucleic acid that encodes such cell surface capture molecule; b) transfecting said cell simultaneously or subsequently with a second nucleic acid that encodes a POI wherein such POI is secreted; c) detecting the surface-displayed POI by contacting the cells with a detection molecule, which binds the POI; and d) isolating cells based on the detection molecule.

1 . A method of detecting and isolating a cell that produces a secreted protein of interest, comprising:
 (a) constructing a cell line transiently or stably expressing a cell surface capture molecule that binds a protein of interest, by transfecting the cell line with a first nucleic acid that encodes said cell surface capture molecule;   (b) transfecting the cell line constructed in (a) with a second nucleic acid that encodes a protein of interest that comprises a T cell receptor variable region, wherein the protein of interest is capable of being secreted;   (c) allowing the cell line to express the protein of interest;   (d) detecting the protein of interest displayed on the surface of a cell of the transfected cell line produced in (b) by contacting said transfected cell line produced in (b) with a detection molecule that binds the protein of interest, wherein said contacting is carried out in the presence of a blocking molecule that binds the cell surface capture molecule and prevents binding of said protein of interest to said cell surface capture molecule but does not bind to said detection molecule;   (e) isolating the cell bearing the surface displayed protein of interest detected in step (d).   
     
     
         2 . The method of  claim 1  wherein the protein of interest is a soluble T cell receptor or a TCR-Fc. 
     
     
         3 . The method of  claim 2 , wherein the protein of interest is a TCR-Fc. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid comprising the T cell receptor variable region that encodes the protein of interest is obtained from a DNA library. 
     
     
         5 . The method of  claim 1 , wherein the cell surface capture molecule is selected from an antibody that binds a T cell receptor variable region, an Fc receptor, protein A, protein G, protein L, protein H, and an anti-immunoglobulin ScFv. 
     
     
         6 . The method of  claim 5  wherein the cell surface capture molecule is an Fc receptor. 
     
     
         7 . The method of  claim 5 , wherein the Fc receptor is hFcγR1. 
     
     
         8 . The method of  claim 5 , further comprising adding a membrane anchor to a protein such that it remains anchored in a cell membrane, exposed to the outside of the cell, and functions as a cell surface capture molecule. 
     
     
         9 . The method of  claim 8 , wherein the membrane anchor is a transmembrane anchor or a GPI link. 
     
     
         10 . The method of  claim 1 , wherein the detection molecule(s) are two molecules that bind each other and are differentially labeled. 
     
     
         11 . The method of  claim 1 , wherein a blocking molecule which binds the cell surface capture molecule or protein of interest is added to reduce the binding of the protein of interest to a neighboring cell. 
     
     
         12 . A method of detecting and/or isolating a eukaryotic cell that produces a secreted protein of interest, comprising:
 (a) providing a cell comprising a nucleic acid that encodes a protein of interest comprising a T cell receptor variable region and a nucleic acid that encodes a cell surface capture molecule capable of binding the protein of interest, wherein the nucleic acid encoding the protein of interest or the nucleic acid encoding the cell surface capture molecule, or both, are transfected into the cell;   (b) culturing the cell under conditions in which the protein of interest and cell surface capture molecule are expressed, and a protein of interest-capture molecule complex is formed intracellularly and displayed on the cell surface;   (c) contacting the cell with a detection molecule, which binds to the protein of interest displayed by the cell; and   (d) detecting and/or isolating the cell due to it being bound to the detection molecule.   
     
     
         13 . The method of  claim 12 , wherein the providing step comprises transfecting the nucleic acid encoding the protein of interest and the nucleic acid encoding the cell surface capture molecule into the cell. 
     
     
         14 . The method of  claim 12 , wherein the cell is detected in step (d) by flow cytometry. 
     
     
         15 . The method of  claim 12 , wherein the detection molecule is linked to a solid support or particle. 
     
     
         16 . The method of  claim 12 , performed on a population of cells, wherein the isolating step isolates the cells binding to the detection molecule from the population. 
     
     
         17 . The method  claim 16 , wherein the cells of the population express different levels of the POI, and the isolating step isolates cells based on relative expression level of the protein of interest. 
     
     
         18 . The method of  claim 16 , further comprising contacting the cells of the population with a blocking molecule that binds the cell surface capture molecule or the protein of interest to block diffusion of protein of interest between cells. 
     
     
         19 . The method of  claim 12 , wherein the protein of interest comprises an Fc domain. 
     
     
         20 . The method of  claim 12 , wherein the capture molecule comprises a membrane anchor.