Hugo N, Weidenhaupt M, Beukes M, Xu B, Janson J-C, Vernet T, Altschuh D
UMR 7100, 'Biotechnologie des Interactions Macromoléculaires', Ecole Supérieure de Biotechnologie de Strasbourg (ESBS), Parc d'Innovation, boulevard Sébastien Brant, BP 10413, F-67412 Illkirch Cedex, France.
Protein Eng. 2003 May;16(5):381-6. doi: 10.1093/protein/gzg042.
Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates.
对具有快速动力学特性的抗体进行预测性工程改造可为生物技术应用提供试剂，如化合物的连续监测或亲和色谱法。基于对小鼠种系抗体可变结构域的协方差分析，我们选择L34位置（Kabat编号）进行突变研究。该位置位于VL/VH界面，在互补决定区的底部，但与抗原的接触有限，因此使其成为轻微改变抗原结合特性的一个有吸引力的位置。我们在两种不同的抗体中L34位置引入了丝氨酸：分别识别烟草花叶病毒外壳蛋白衍生肽段和癌蛋白E6的Fab（抗原结合片段）57P（Asn34Ser）和scFv（单链可变片段）1F4（Gln34Ser）。两种突变抗体表现出相似的特性：（i）活性片段在大肠杆菌中的表达水平显著提高；（ii）热稳定性增强；（iii）解离速率参数（k(off)）对于scFv1F4和Fab57P分别增加了2倍和至少57倍，而它们的结合速率参数（k(on)）保持不变。两种抗体片段L34的丙氨酸和苏氨酸突变体不具备这些特性。在具有不同特异性的两种抗体中观察到类似效应的首次证明，可能为设计具有增强稳定性和快速解离速率的分子开辟道路。
