Ho Mitchell, Nagata Satoshi, Pastan Ira
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9637-42. doi: 10.1073/pnas.0603653103. Epub 2006 Jun 8.
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.
体外抗体亲和力成熟通常是通过在微生物（噬菌体、细菌和酵母）表面展示小鼠或人抗体来实现的。然而，蛋白质折叠、翻译后修饰和密码子使用等问题仍然限制了可获得的改良抗体的数量。理想的系统应该在抗体自然产生的哺乳动物细胞环境中筛选和改良抗体。在此，我们表明广泛用于瞬时蛋白表达的人胚肾293T细胞可用于单链Fv抗体的细胞表面展示以实现亲和力成熟。在一个概念验证实验中，表达具有较高亲和力的罕见突变抗体的细胞通过单次细胞分选从大量表达亲和力略低的野生型抗体的细胞中富集了240倍。此外，在对一个使内在抗体热点随机化的组合文库进行单次筛选后，我们成功获得了对CD22具有增强结合亲和力的高度富集突变体。重要的是，一个展示筛选周期仅需1周，在一个100毫米培养皿中转染细胞可产生10⁷个单独的克隆，因此在当前实验条件下获得10⁹的文库是可行的。
