title: Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system.
abstract: We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Yin
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Eudean D
authors.initials: ED
authors.lastname: Garces
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Junhao
authors.initials: J
authors.lastname: Yang
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Cuong
authors.initials: C
authors.lastname: Tran
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Steiner
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Roos
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Sunil
authors.initials: S
authors.lastname: Bajad
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Hudak
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Kalyani
authors.initials: K
authors.lastname: Penta
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: James
authors.initials: J
authors.lastname: Zawada
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Sonia
authors.initials: S
authors.lastname: Pollitt
authors.affiliation: Sutro Biopharma, Inc.; South San Francisco, CA USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Murray
mesh_terms: D002474:Cell-Free System / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D053661:Interleukin-13 Receptor alpha1 Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D053759:Interleukin-23 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014158:Transcription, Genetic
chemical_list: C508181:IL13RA1 protein, human; D053661:Interleukin-13 Receptor alpha1 Subunit; D053759:Interleukin-23; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies