Li Bing, Fouts Ashley E, Stengel Katharina, Luan Peng, Dillon Michael, Liang Wei-Ching, Feierbach Becket, Kelley Robert F, Hötzel Isidro
Department of Antibody Engineering; Genentech; South San Francisco, CA USA.
Department of Infectious Diseases; Genentech; South San Francisco, CA USA.
MAbs. 2014 Mar-Apr;6(2):437-45. doi: 10.4161/mabs.27875. Epub 2014 Jan 16.
Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD<10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.
从人类供体中分离出的抗体正越来越多地被开发用于抗感染治疗。这些抗体在体内经历亲和力成熟，最大限度地减少了对治疗先导物进行亲和力工程改造的需求。然而，某些治疗应用所需的亲和力可能高于所获得先导物的亲和力，这就需要在体外进一步进行亲和力成熟。为了提高靶向人巨细胞病毒（CMV）的天然人抗体MSL-109的中和效力，我们针对gH/gL糖蛋白复合物对该抗体进行了亲和力成熟。使用一个噬菌体展示文库（其中六个互补决定区（CDR）中的大多数一次仅允许一个氨基酸残基发生变化）来筛选可提高结合亲和力的突变。鉴定出一个T55R突变和重链第53位的多个突变，这些突变单独或组合存在时，对gH/gL具有更高的表观亲和力，并提高了在细菌细胞中表达的Fab片段的CMV中和效力。第53位的这些突变中有三个在重链CDR 2（CDR H2）中引入了糖基化位点，损害了在哺乳动物细胞中表达的抗体的结合。鉴定出一个高亲和力（KD<10 pM）变体，它结合了D53N和T55R突变，同时避免了CDR H2的糖基化。然而，通过噬菌体展示鉴定出的所有提高结合亲和力而不引入糖基化位点的氨基酸取代都需要同时进行两到四个核苷酸突变以避免糖基化。这些结果表明天然人抗体MSL-109已接近局部亲和力最优值。我们表明，通过噬菌体展示进行亲和力成熟可用于识别和绕过由糖基化和密码子使用所导致的抗体体内亲和力成熟的障碍。由于密码子使用和天然人类抗体库编码的氨基酸序列，这些限制在人类抗体中可能相对普遍。
