Surface anchored light chain bait antibody display system

A method for determining if an antibody or antigen-binding fragment thereof, secreted from a host cell, 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 one or more polynucleotides encoding an immunoglobulin light chain;
• and one or more polynucleotides encoding an immunoglobulin heavy chain that complexes with the light immunoglobulin chain or a functional fragment thereof of a bait;
• and
• (b) a bait comprising a light immunoglobulin chain or functional fragment thereof fused to a surface anchor polypeptide or functional fragment thereof;
• and determining if a bait/antigen-binding fragment or bait/antibody comprising said bait and heavy and light chain immunoglobulins specifically binds, at the cell surface, to said antigen;
• wherein the secreted antibody or antigen-binding fragment thereof is determined to specifically bind said antigen if said bait/antigen-binding fragment or bait/antibody specifically binds, at the cell surface, to said antigen.

A method for making an antibody display system comprising:

• (a) providing an isolated eukaryotic host cell,
• (b) introducing, into said host cell a bait comprising a light chain immunoglobulin domain fused to a surface anchor polypeptide;
• and
• (i) one or more polynucleotides encoding an immunoglobulin light chain;
• and
• (ii) one or more polynucleotides encoding an immunoglobulin heavy chain.

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 controlled glycosylation eukaryotic host cell comprising a polynucleotide encoding an immunoglobulin light chain;
• and a polynucleotide encoding an immunoglobulin heavy chain;
• and
• (b) a bait comprising a light chain immunoglobulin or functional fragment thereof fused to a surface anchor polypeptide or functional fragment thereof on the surface of said host cell;
• wherein the light chain immunoglobulin of said bait complexes with said immunoglobulin heavy chain and immunoglobulin light chain on the surface of the host cell;
• wherein said heavy or light chain comprises said sugar;
• determining if said bait/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.

A method for determining the whether an mutation in an immunoglobulin heavy chain Fc region increases or decreases binding of said chain to an Fc receptor or to a lectin comprising contacting an antibody display system with said Fc receptor or lectin;

• 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 which comprises the mutation(s);
• and
• (b) a bait comprising a light chain immunoglobulin or functional fragment thereof fused to a surface anchor polypeptide or functional fragment thereof on the surface of said eukaryotic host cell;
• wherein the light chain immunoglobulin of said bait complexes with the heavy chain immunoglobulin, to form a bait/antigen-binding fragment or bait/antibody complex, on the surface of the host cell;
• and determining if said bait/antigen-binding fragment or bait/antibody specifically binds to said Fc receptor or lectin;
• and determining the binding affinity of the antibody or antigen-binding fragment thereof for the Fc receptor or lectin;
• 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 mutation;
• wherein the mutation is determined to increase affinity of the heavy chain for the Fc receptor or lectin if the affinity of the antibody or antigen-binding fragment thereof comprising said mutation is higher than the affinity of the antibody or antigen-binding fragment thereof which lacks the mutation, and/or wherein the mutation is determined to decrease affinity for the Fc receptor or lectin if the affinity of antibody or antigen-binding fragment thereof comprising said mutation is lower than the affinity of the antibody or antigen-binding fragment thereof which lacks the mutation.