Baca M, Scanlan T S, Stephenson R C, Wells J A
Department of Protein Engineering, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA.
Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10063-8. doi: 10.1073/pnas.94.19.10063.
Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. "Humanized" 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH --> Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.
催化抗体在催化种类繁多的天然和非天然化学转化方面显示出巨大潜力。然而，很少有催化抗体的效率能接近天然酶。原则上，诸如噬菌体展示等随机诱变程序可用于提高现有抗体的催化活性；然而，这些研究因抗体的重组表达困难而受阻。在此，我们将源自小鼠的催化抗体17E8的抗原结合环嫁接到人抗体框架上，以克服与该抗体的重组表达和噬菌体展示相关的困难。“人源化”的17E8保留了与小鼠抗体相似的催化和半抗原结合特性，而噬菌体上展示的功能性Fab水平比小鼠可变区/人恒定区嵌合Fab高200倍。该构建体用于制备组合文库。对这些文库进行亲和淘选，结果选出了对膦酸酯过渡态类似物的结合亲和力提高了2至8倍的变体。令人惊讶的是，没有一个亲和力成熟的变体比亲本抗体具有更高的催化活性，有些变体的活性甚至显著降低。相比之下，鉴定出一个结合较弱但催化活性高2倍的变体，将该变体中的单个取代（Tyr-100aH→Asn）引入亲本抗体导致催化效率提高了5倍。因此，噬菌体展示方法可轻易用于优化催化抗体与过渡态类似物的结合，并且与有限的催化筛选结合使用时可鉴定出具有更高催化效率的变体。
