title: A mammalian expression system for high throughput antibody screening.
abstract: We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100μg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune, LLC, One MedImmune Way, Gaithersburg, MD 20878, United States.
authors.forename: Linda
authors.initials: L
authors.lastname: Xu
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Jin
authors.forename: G Jonah
authors.initials: GJ
authors.lastname: Rainey
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
authors.forename: Changshou
authors.initials: C
authors.lastname: Gao
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics; D000906:Antibodies / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005822:Genetic Vectors; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D019309:Epstein-Barr Virus Nuclear Antigens; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C417029:EBV-encoded nuclear antigen 1
keywords: Adenovirus expression system; CMV; EBNA-1; Epstein–Barr virus nuclear antigen 1; Fab; HTP; High throughput screening; IRES; Mammalian expression; Phage display; VH; cytomegalovirus.; fragment antigen-binding; heavy chain variable regions; high throughput; internal ribosome entry site; scFv; single chain Fv