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In vitro antibody affinity maturation has generally been achieved by display of mouse or human antibodies on the surface of microorganisms (phage, bacteria, and yeast). However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. An ideal system would select and improve antibodies in a mammalian cell environment where they are naturally made. Here we show that human embryonic kidney 293T cells that are widely used for transient protein expression can be used for cell surface display of single-chain Fv antibodies for affinity maturation. In a proof-of-concept experiment, cells expressing a rare mutant antibody with higher affinity were enriched 240-fold by a single-pass cell sorting from a large excess of cells expressing WT antibody with a slightly lower affinity. Furthermore, we successfully obtained a highly enriched mutant with increased binding affinity for CD22 after a single selection of a combinatory library randomizing an intrinsic antibody hotspot. Important features are that one display selection cycle requires only 1 week, and transfection of cells in a single 100-mm dish produces 10(7) individual clones so that a repertoire of 10(9) is feasible under current experimental conditions.

Isolation of anti-CD22 Fv with high affinity by Fv display on human cells