Affinity maturation of antibodies: optimized methods to generate high-quality ScFv libraries and isolate IgG candidates by high-throughput screening.
As a growing number of therapeutic antibodies are developed, robust methods to efficiently improve the affinity and/or specificity of antibody candidates are needed. Here we describe our powerful platform that combines scFv affinity maturation and IgG high-throughput screening. After creating diversity with our random mutagenesis technology (MutaGen™), the scFv libraries are fully cleaned using a fusion system introducing the beta-lactamase gene to select in-frame and stop codon free variants on the basis of ampicillin resistance. The high-quality scFv libraries thereby constructed are then selected on the target in vitro using phage display technology. Contrary to standard procedures, instead of producing a limited number of affinity matured scFv as IgG molecules, we developed a cloning system to directly transfer the entire pool of selected scFv into an IgG expression vector permitting rapid IgG small-scale production (96 wells) in mammalian cells. Our integrated process allows us to generate high-quality scFv libraries and test numerous IgG variants, increasing the chances to select the best therapeutic antibody candidate.
MILLEGEN SA, Immeuble BIOSTEP-Bâtiment A, Labege, France.
Laurence
L
Renaut
Céline
C
Monnet
Olivier
O
Dubreuil
Ouafa
O
Zaki
Fabien
F
Crozet
Khalil
K
Bouayadi
Hakim
H
Kharrat
Philippe
P
Mondon
D000667:Ampicillin / Q000494:pharmacology; D000915:Antibody Affinity / Q000187:drug effects / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D016366:Open Reading Frames / Q000235:genetics; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000096:biosynthesis*
D007074:Immunoglobulin G; D019151:Peptide Library; D057127:Single-Chain Antibodies; D000667:Ampicillin