Surface, anchored Fc-bait antibody display system
The present invention provides, in part, an antibody display system that simultaneously uses a secretion and a display mode. A bait complexed with a monovalent antibody fragment can be expressed on the surface of the host cell wherein the fragment may be assayed for antigen binding while full antibody is simultaneously secreted from the host cell. Methods of using the system for identifying antibodies that bind specifically to an antigen of interest are also provided. Polypeptides, polynucleotides and host cells useful for making the antibody display system are also provided along with methods of use thereof.
1. A method for determining if an antibody specifically binds to an antigen comprising contacting an antibody display system with said antigen; wherein the antibody display system comprises an isolated host cell wherein the host cell is selected from the group consisting of yeast and filamentous fungi; wherein said isolated host cell comprises
(a) a polynucleotide encoding a bait comprising a heavy Fc immunoglobulin domain fused to a surface anchor polypeptide operably associated with a regulatable promotor;
(b) one or more polynucleotides encoding an immunoglobulin light chain variable region; and
(c) one or more polynucleotides encoding an immunoglobulin heavy chain variable region wherein the heavy chain is VH-CH1-CH2-CH3;
comprising including expression from the regulatable promoter in said host cell; wherein in said isolated host cell the Fc of said bait complexes with an Fc/antigen-binding fragment comprising said immunoglobulin heavy and immunoglobulin light chains on the surface of the host cell; and determining if said Fc/antigen-binding fragment specifically binds to said antigen; wherein the antibody is determined to specifically bind said antigen if the monovalent antibody fragment specifically binds to said antigen and inhibiting expression of the bait from the regulatable promotor.
2. A method for identifying:
(i) an antibody that binds specifically to an antigen; or
(ii) a polynucleotide encoding an immunoglobulin heavy chain of said antibody and/or a polynucleotide encoding an immunoglobulin light chain of said antibody; comprising contacting an antibody display system with said antigen wherein the antibody display system comprises an isolated host cell wherein the host cell is selected from the group consisting of a yeast and filamentous fungi; wherein said isolated host cell comprises
(a) a polynucleotide encoding a bait comprising a heavy Fc immunoglobulin domain fused to a surface anchor polypeptide operably associated with a regulatable promotor;
(b) one or more polynucleotides encoding an immunoglobulin light chain variable region; and
(c) one or more polynucleotides encoding an immunoglobulin heavy chain variable region wherein the heavy chain is VH-CH1-CH2-CH3;
comprising including expression from the regulatable promoter in said host cell; wherein in said isolated host cell the Fc of said bait complexes with an Fc/antigen-binding fragment comprising said immunoglobulin heavy and immunoglobulin light chains on the surface of the host cell; and determining if said Fc/antigen-binding fragment specifically binds to said antigen; wherein the antibody or polynucleotide is identified if said specific binding to said antigen is observed and inhibiting expression of the bait from the regulatable promotor.
3. The method of claim 1 further comprising isolating the identified polynucleotides.
4. The method of claim 1 further comprising determining the affinity of said identified antibody for said antigen.
5. The method of claim 1 further comprising recombinantly expressing the immunoglobulin chains encoded by the polynucleotides and, optionally, isolating an antibody or antigen-binding fragment thereof comprising said immunoglobulins and, optionally, producing a pharmaceutical formulation comprising combining said antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
6. The method of claim 1 , wherein the host cell is Pichia.
7. The method of claim 1 , wherein the host cell is Pichia pastoris.
8. The method of claim 1 , wherein the host cell is S. cerevisiae.
9. The method of claim 2 , wherein the host cell is Pichia.
10. The method of claim 2 , wherein the host cell is Pichia pastoris.
11. The method of claim 2 , wherein the host cell is S. cerevisiae.