title: In vitro Fab display: a cell-free system for IgG discovery.
abstract: Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.
authors.affiliation: Sutro Biopharma, Inc., 310 Utah Ave Suite 150, South San Francisco, CA 94080, USA.
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Stafford
authors.forename: Marissa L
authors.initials: ML
authors.lastname: Matsumoto
authors.forename: Gang
authors.initials: G
authors.lastname: Yin
authors.forename: Qi
authors.initials: Q
authors.lastname: Cai
authors.forename: Juan Jose
authors.initials: JJ
authors.lastname: Fung
authors.forename: Heather
authors.initials: H
authors.lastname: Stephenson
authors.forename: Avinash
authors.initials: A
authors.lastname: Gill
authors.forename: Monica
authors.initials: M
authors.lastname: You
authors.forename: Shwu-Hwa
authors.initials: SH
authors.lastname: Lin
authors.forename: Willie D
authors.initials: WD
authors.lastname: Wang
authors.forename: Mary Rose
authors.initials: MR
authors.lastname: Masikat
authors.forename: Xiaofan
authors.initials: X
authors.lastname: Li
authors.forename: Kalyani
authors.initials: K
authors.lastname: Penta
authors.forename: Alex R
authors.initials: AR
authors.lastname: Steiner
authors.forename: Ramesh
authors.initials: R
authors.lastname: Baliga
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Murray
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Thanos
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Hallam
authors.forename: Aaron K
authors.initials: AK
authors.lastname: Sato
mesh_terms: D000906:Antibodies / Q000235:genetics; D061067:Antibodies, Monoclonal, Humanized / Q000235:genetics; D002272:Carcinoembryonic Antigen / Q000378:metabolism; D061505:Cell Surface Display Techniques / Q000379:methods*; D002474:Cell-Free System; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics; D019151:Peptide Library; D000068878:Trastuzumab; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D002272:Carcinoembryonic Antigen; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D042461:Vascular Endothelial Growth Factor A; D000068878:Trastuzumab
keywords: Fab selections; antibody reformatting; cell-free protein synthesis; ribosome display