title: An improved single-chain Fab platform for efficient display and recombinant expression.
abstract: Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3 mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81 °C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94158, USA; Recombinant Antibody Network, University of California, San Francisco, CA, 94158, USA.
authors.forename: James T
authors.initials: JT
authors.lastname: Koerber
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94158, USA; Recombinant Antibody Network, University of California, San Francisco, CA, 94158, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hornsby
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94158, USA; Recombinant Antibody Network, University of California, San Francisco, CA, 94158, USA. Electronic address: jim.wells@ucsf.edu.
authors.forename: James A
authors.initials: JA
authors.lastname: Wells
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics; D003198:Computer Simulation; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D008322:Mammals / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics
chemical_list: D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: antibody; bacterial and mammalian expression; high-throughput screening; phage display; signal peptide