Surface, anchored fc-bait antibody display system
AssigneeMerck Sharp & Dohme Corp.
InventorsDongxing Zha, Hussam Hisham Shaheen
Life of this patent
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Abstract
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.
Claims24
An antibody display system comprising:
- (a) an isolated host cell;
- (b) a bait comprising a heavy Fc immunoglobulin domain fused to a surface anchor polypeptide;
- (c) one or more polynucleotides encoding an immunoglobulin light chain variable region;
- and
- (d) one or more polynucleotides encoding an immunoglobulin heavy chain variable region.
An isolated bait polypeptide comprising an Fc immunoglobulin domain or functional fragment thereof fused, optionally by a peptide linker, to a surface anchor polypeptide or functional fragment thereof;
- which bait polypeptide is optionally amino-terminally fused to a signal peptide.
A method for determining if an antibody or antigen-binding fragment thereof specifically binds to an antigen comprising contacting an antibody display system with said antigen;
- wherein the antibody display system comprises:
- (a) an isolated eukaryotic host cell comprising a polynucleotide encoding an immunoglobulin light chain;
- and a polynucleotide encoding an immunoglobulin heavy chain;
- and
- (b) a bait comprising an Fc immunoglobulin domain or functional fragment thereof fused to a surface anchor polypeptide or functional fragment thereof on the surface of said eukaryotic host cell;
- wherein 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.
A method for identifying:
- (i) an antibody or antigen-binding fragment thereof that binds specifically to an antigen;
- or
- (ii) a polynucleotide encoding an immunoglobulin heavy chain of said antibody or fragment and/or a polynucleotide encoding an immunoglobulin light chain of said antibody or fragment;
- comprising contacting an antibody display system with said antigen wherein the antibody display system comprises:
- (a) an isolated eukaryotic host cell comprising a polynucleotide encoding an immunoglobulin light chain;
- and a polynucleotide encoding an immunoglobulin heavy chain;
- and
- (b) a bait comprising an Fc immunoglobulin domain or functional fragment thereof fused to a surface anchor polypeptide or functional fragment thereof on the surface of said eukaryotic host cell;
- wherein 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 fragment or polynucleotide is identified if said specific binding to said antigen is observed.
A method for making an antibody display system comprising:
- (a) an isolated eukaryotic host cell;
- (b) a bait comprising a human Fc immunoglobulin domain fused to a surface anchor polypeptide;
- (c) one or more polynucleotides encoding an immunoglobulin light chain variable region;
- (d) one or more polynucleotides encoding an immunoglobulin heavy chain variable region;
- comprising introducing, into said eukaryotic host cell, a polynucleotide encoding said bait, said one or more polynucleotides encoding an immunoglobulin light chain variable region;
- and said one or more polynucleotides encoding an immunoglobulin heavy chain variable region.
A method for making an antibody or antigen-binding fragment thereof comprising introducing, into an isolated eukaryotic host cell comprising a bait that includes a human Fc immunoglobulin domain or functional fragment thereof fused to a surface anchor polypeptide or functional fragment thereof, one or more polynucleotides encoding an immunoglobulin light chain variable region;
- and/or one or more polynucleotides encoding an immunoglobulin heavy chain variable region;
- and culturing the host cell under condition whereby the polynucleotides encoding the immunoglobulin chains are expressed and an antibody or antigen-binding fragment thereof is formed from said chains;
- wherein said bait is operably associated with a regulatable promoter and bait expression is inhibited when said immunoglobulin chains are expressed.
A method for making an antibody or antigen binding fragment thereof comprising culturing an isolated eukaryotic host cell in a growth medium under conditions allowing expression of an immunoglobulin light chain and an immunoglobulin heavy chain of said antibody or fragment;
- wherein the eukaryotic host cell comprises:
- (i) a polynucleotide encoding said immunoglobulin light chain;
- and a polynucleotide encoding said immunoglobulin heavy chain of said antibody or fragment;
- and
- (ii) a bait comprising an Fc immunoglobulin domain or functional fragment thereof fused to a surface anchor polypeptide or functional fragment thereof on the surface of said eukaryotic host cell;
- wherein 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 wherein the expression of the bait is optionally inhibited;
- wherein said antibody or fragment is optionally secreted from said eukaryotic host cell;
- optionally comprising isolating said antibody or fragment from said eukaryotic host cell and medium.
A method for determining the effect of a sugar on an antibody or antigen-binding fragment thereof which specifically binds to an antigen comprising contacting an antibody display system with said antigen;
- wherein the antibody display system comprises:
- (a) an isolated eukaryotic controlled glycosylation host cell comprising a polynucleotide encoding an immunoglobulin light chain;
- and a polynucleotide encoding an immunoglobulin heavy chain;
- and
- (b) a bait comprising an Fc immunoglobulin domain or functional fragment thereof fused to a surface anchor polypeptide or functional fragment thereof on the surface of said eukaryotic host cell;
- wherein 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;
- wherein said heavy or light chain comprises said sugar;
- determining if said Fc/antigen-binding fragment specifically binds to said antigen;
- determining the binding affinity of the antibody or antigen-binding fragment thereof comprising said sugar for the antigen;
- and
- comparing the affinity of the antibody or antigen-binding fragment thereof with affinity of an otherwise identical antibody or antigen-binding fragment thereof which lacks said sugar;
- wherein the sugar is determined to increase affinity for the antigen if the affinity of the antibody or antigen-binding fragment thereof comprising said sugar is higher than the affinity of the antibody or antigen-binding fragment thereof which lacks the sugar, and/or wherein the sugar is determined to decrease affinity for the antigen if the affinity of antibody or antigen-binding fragment thereof comprising said sugar is lower than the affinity of the antibody or antigen-binding fragment thereof which lacks the sugar.
Dependencies are inferred from the claim text.