Extended Data Fig. 4: Optimization of antibody display in AHEAD and evolution of anti-GFP and anti-HSA antibodies using the optimized second-generation AHEAD 2.0 system.
From: Rapid generation of potent antibodies by autonomous hypermutation in yeast
a, Architectures for nanobody display in the first-generation AHEAD 1.0 and improved second-generation AHEAD 2.0 systems. b, Selection of a new leader sequence for higher nanobody display. FACS plots showing the progressive enrichment of higher efficiency leader sequences across 3 rounds of selection (left panel). Nanobody display level using app8 compared to the selected app8i1 variant (right panel). n = 6, error bars represent ± s.d. c, Selected FACS plots showing affinity maturation of Nb.b201 through AHEAD cycles. d, Selected FACS plots showing affinity maturation of Lag42 through AHEAD cycles. e, (left) Affinities (EC50) of improved high-affinity anti-HSA nanobodies evolved using AHEAD. Binding of yeast-displayed nanobodies by each concentration of HSA was measured in replicate (n = 3, error bars represent ± s.d.) and EC50s were determined by fitting each binding curve. (right) Affinities (EC50) of improved high-affinity anti-GFP nanobodies evolved using AHEAD. Binding of yeast-displayed nanobodies by each concentration of GFP was measured in replicate (n = 3, error bars represent ± s.d.) and EC50s were determined by fitting each binding curve.
