Related Experiment Video
Updated: Sep 18, 2025
Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries
Published on: January 17, 2015
Development of vectors for reformatting scFv fragments derived from phage display libraries into native IgG1
Marton Fogarasi1, Corina Roman2, Simona Dima3
1Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, United Kingdom; Center of Excellence in Translational Medicine, Fundeni Clinical Institute, 022328 Bucharest, Romania.
Monoclonal antibodies are a crucial class of therapeutic agents utilized for the treatment of various disorders. Typically, fully human therapeutic antibodies are created using the antibody fragment phage display from naive libraries and are selected against the antigen in a format known as the single-chain Fv (scFv) fragment. To be employed in immunotherapy, the scFv fragment needs to be reformatted into the complete IgG structure with a native-like conformation and expressed in mammalian cells. In this context, we have established an expression system for reformatting scFv fragments into complete IgG1 molecules, enabling both antibody chains to be cloned within the same vector. These constructs are based on the IgGγ1 heavy chain, with the light chain belonging to either the lambda or kappa isotype. For in vivo imaging, we have designed vectors with specific mutations in the IgG1 lower hinge region and CH2 domain to impede the antibody's effector function, ensuring that immune cells are not activated. The efficiency of this expression system was evaluated by producing two antibodies: one with the lambda light chain and the other with the kappa light chain. Their expression levels were assessed in HEK-293 and CHO-K1 cell lines. Structurally, the resulting antibodies are properly assembled and folded into their quaternary structure, while functionally, these immunoglobulins specifically recognize the antigens and inhibit cancer cell invasion.
Monoclonal antibodies are a crucial class of therapeutic agents utilized for the treatment of various disorders. Typically, fully human therapeutic antibodies are created using the antibody fragment phage display from naive libraries and are selected against the antigen in a format known as the single-chain Fv (scFv) fragment. To be employed in immunotherapy, the scFv fragment needs to be reformatted into the complete IgG structure with a native-like conformation and expressed in mammalian cells. In this context, we have established an expression system for reformatting scFv fragments into complete IgG1 molecules, enabling both antibody chains to be cloned within the same vector. These constructs are based on the IgGγ1 heavy chain, with the light chain belonging to either the lambda or kappa isotype. For in vivo imaging, we have designed vectors with specific mutations in the IgG1 lower hinge region and CH2 domain to impede the antibody's effector function, ensuring that immune cells are not activated. The efficiency of this expression system was evaluated by producing two antibodies: one with the lambda light chain and the other with the kappa light chain. Their expression levels were assessed in HEK-293 and CHO-K1 cell lines. Structurally, the resulting antibodies are properly assembled and folded into their quaternary structure, while functionally, these immunoglobulins specifically recognize the antigens and inhibit cancer cell invasion.
