Isolating Cells Expressing Secreted Proteins

Abstract

A method of detecting and isolating cells that produce a secreted protein of interest (POI), for example, an antibody, comprising: a) providing a eukaryotic cell comprising (i) a nucleic acid encoding the POI, and (ii) a nucleic acid encoding a cell surface capture molecule, which comprises a membrane anchor and is capable of binding the POI; (b) culturing the cell under conditions in which the POI and cell surface capture molecule are expressed, and a POI-cell surface capture molecule complex is formed intracellularly and displayed on the cell surface; 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 - 86 . (canceled) 
     
     
         87 . A method for identifying a cell, comprising:
 a. transfecting the cell with (i) a nucleic acid sequence encoding a protein of interest and (ii) a nucleic acid encoding a cell surface capture molecule;   b. culturing the cell under conditions in which the protein of interest and the cell surface capture molecule are expressed and joined intracellularly and capable of being displayed on the cell surface; and   c. contacting the cell with a detection molecule which binds to the protein of interest.   
     
     
         88 . The method of  claim 87 , wherein the protein of interest is selected from the group consisting of an antibody, a Fab, a single chain antibody (ScFv), anything fused to an antibody constant region, ligand, a soluble receptor protein, a growth factor, or a fragment thereof. 
     
     
         89 . The method of  claim 87 , wherein the the cell surface capture molecule comprises a membrane anchor. 
     
     
         90 . The method of  claim 89 , wherein the membrane anchor is a transmembrane anchor. 
     
     
         91 . The method of  claim 90 , wherein the transmembrane anchor remains anchored in the cell membrane, exposed to the outside of the cell, and functions as a cell surface capture molecule. 
     
     
         92 . The method of  claim 89 , wherein the membrane anchor may be native to the cell, recombinantly expressed in the cell, or synthetic to the cell. 
     
     
         93 . The method of  claim 87 , wherein the cell surface capture molecule specifically binds an antibody as the protein of interest. 
     
     
         94 . The method of  claim 93 , wherein the antibody is selected from the group consisting of IgM, IgG, IgA, IgD or IgE, or various subtypes thereof. 
     
     
         95 . The method of  claim 87 , wherein the cell surface capture molecule is selected from the group consisting of a ligand-specific receptor, a receptor-specific ligand, an antibody-binding protein, an antibody, a Fab, a single chain antibody (ScFv), anything fused to a constant region of an antibody, ligand a peptide from a phage display or derivatives that bind the protein of interest, a soluble receptor protein, a growth factor, or a fragment thereof. 
     
     
         96 . The method of  claim 95 , wherein the antibody-binding protein is an Fc receptor, anti-immunoglobulin antibodies, anti-immunoglobulin ScFv, Protein A, Protein L, Protein G, Protein H or functional fragments thereof. 
     
     
         97 . The method of  claim 87 , wherein the cell surface capture molecule is a fusion protein comprising a transmembrane anchor and a protein of interest. 
     
     
         98 . The method of  claim 87  further comprising a method for producing the protein of interest, comprising:
 a. isolating the cell from a plurality of cells by contacting the plurality of cells with a detection molecule which binds to the protein of interest; 
 b. culturing the cell in a culture medium under conditions that allow for expression of a protein of interest, wherein the protein of interest is secreted in the culture medium; and 
 c. obtaining the protein of interest from the culture medium. 
 
     
     
         99 . The method of  claim 98 , wherein the cell has a specific productivity of at least 6 pg/cell/day, 12 pg/cell/day, 17 pg/cell/day, 20 pg/cell/day, 35 pg/cell/day, or 54 pg/cell/day. 
     
     
         100 . The method of  claim 99 , wherein the specific productivity of the cell is detected and measured by flow cytometry. 
     
     
         101 . A composition comprising a protein of interest and a pharmaceutically acceptable excipient, wherein the protein of interest is produced by a method comprising:
 a. transfecting a cell with (i) a nucleic acid sequence encoding the protein of interest and (ii) a nucleic acid encoding a cell surface capture molecule;   b. culturing the cell under conditions in which the protein of interest and the cell surface capture molecule are expressed and joined intracellularly and capable of being displayed on the cell surface; and   c. isolating the cell from a plurality of cells by contacting the plurality of cells with a detection molecule which binds to the protein of interest;   d. culturing the cell in a culture medium under conditions that allow for expression of the protein of interest, wherein the protein of interest is secreted in the culture medium; and   e. obtaining the protein of interest from the culture medium.   
     
     
         102 . The method of  claim 101 , wherein the protein of interest is selected from the group consisting of an antibody, a Fab, a single chain antibody (ScFv), anything fused to an antibody constant region, ligand, a soluble receptor protein, a growth factor, or a fragment thereof. 
     
     
         103 . The method of  claim 101 , wherein the the cell surface capture molecule comprises a membrane anchor. 
     
     
         104 . The method of  claim 103 , wherein the membrane anchor is a transmembrane anchor. 
     
     
         105 . The method of  claim 104 , wherein the transmembrane anchor remains anchored in the cell membrane, exposed to the outside of the cell, and functions as a cell surface capture molecule. 
     
     
         106 . The method of  claim 103 , wherein the membrane anchor may be native to the cell, recombinantly expressed in the cell, or synthetic to the cell. 
     
     
         107 . The method of  claim 101 , wherein the cell surface capture molecule specifically binds an antibody as the protein of interest. 
     
     
         108 . The method of  claim 107 , wherein the antibody is selected from the group consisting of IgM, IgG, IgA, IgD or IgE, or various subtypes thereof. 
     
     
         109 . The method of  claim 101 , wherein the cell surface capture molecule is selected from the group consisting of a ligand-specific receptor, a receptor-specific ligand, an antibody-binding protein, an antibody, a Fab, a single chain antibody (ScFv), anything fused to a constant region of an antibody, ligand a peptide from a phage display or derivatives that bind the protein of interest, a soluble receptor protein, a growth factor, or a fragment thereof. 
     
     
         110 . The method of  claim 109 , wherein the antibody-binding protein is an Fc receptor, anti-immunoglobulin antibodies, anti-immunoglobulin ScFv, Protein A, Protein L, Protein G, Protein H or functional fragments thereof. 
     
     
         111 . The method of  claim 101 , wherein the cell surface capture molecule is a fusion protein comprising a transmembrane anchor and a protein of interest. 
     
     
         112 . The method of  claim 101 , wherein the cell has a specific productivity of at least 6 pg/cell/day, 12 pg/cell/day, 17 pg/cell/day, 20 pg/cell/day, 35 pg/cell/day, or 54 pg/cell/day. 
     
     
         113 . The method of  claim 112 , wherein the specific productivity of the cell is detected and measured by flow cytometry.