A Shorter Route to Antibody Binders via Quantitative in vitro Bead-Display Screening and Consensus Analysis.
Affinity panning of large libraries is a powerful tool to identify protein binders. However, panning rounds are followed by the tedious re-screening of the clones obtained to evaluate binders precisely. In a first application of Bead Surface Display (BeSD) we show successful in vitro affinity selections based on flow cytometric analysis that allows fine quantitative discrimination between binders. Subsequent consensus analysis of the resulting sequences enables identification of clones that bind tighter than those arising directly from the experimental selection output. This is demonstrated by evolution of an anti-Fas receptor single-chain variable fragment (scFv) that was improved 98-fold vs the parental clone. Four rounds of quantitative screening by fluorescence-activated cell sorting of an error-prone library based on fine discrimination between binders in BeSD were followed by analysis of 200 full-length output sequences that suggested a new consensus design with a Kd∼140 pM. This approach shortens the time and effort to obtain high affinity reagents and its cell-free nature transcends limitations inherent in previous in vivo display systems.
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.|Antibody Discovery and Protein Engineering, MedImmune Ltd, Milstein Building, Granta Park, Cambridge, CB21 6GH, UK.
Sylwia A
SA
Mankowska
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Pietro
P
Gatti-Lafranconi
Antibody Discovery and Protein Engineering, MedImmune Ltd, Milstein Building, Granta Park, Cambridge, CB21 6GH, UK.
Matthieu
M
Chodorge
Antibody Discovery and Protein Engineering, MedImmune Ltd, Milstein Building, Granta Park, Cambridge, CB21 6GH, UK.
Sudharsan
S
Sridharan
Antibody Discovery and Protein Engineering, MedImmune Ltd, Milstein Building, Granta Park, Cambridge, CB21 6GH, UK.
Ralph R
RR
Minter
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Florian
F
Hollfelder
D061505:Cell Surface Display Techniques; D005434:Flow Cytometry; D006801:Humans; D019151:Peptide Library; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000378:metabolism*
D019151:Peptide Library; D011506:Proteins; D057127:Single-Chain Antibodies