General strategy for the generation of human antibody variable domains with increased aggregation resistance
General strategy for the generation of human antibody variable domains with increased aggregation resistance
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DOI：
10.1073/pnas.1202866109
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发表时间：
2012-07-03
影响因子：
11.1
通讯作者：
Christ, Daniel
中科院分区：
文献类型：
--
作者：
Dudgeon, Kip;Rouet, Romain;Christ, Daniel
稳定的人抗体试剂的可用性对于研究、诊断和治疗应用将具有相当大的优势。不幸的是，介导与抗原相互作用的抗体可变重链和轻链结构域（V-H和V-L）具有聚集的倾向。由于在人可变结构域中观察到的高水平的序列多样性和维持抗原结合的要求，以一般方式增加它们的抗聚集性已被证明是一个困难且持久的问题。在这里，我们概述了这样一种方法。通过使用噬菌体展示，我们鉴定了聚集在抗原结合位点中的特定位置（V-H中为28，30-33，35，V-L中为24，49-53，56）。在这些位置引入天冬氨酸或谷氨酸赋予了来自常见人类生殖系家族（V（H）3和V（kappa）1）的结构域上级的生物物理性质（非聚集、良好表达和热重折叠）。突变的影响是高度位置性的，并且与其他位置的序列多样性无关。此外，突变体V-H和V-L结构域的晶体结构显示出令人惊讶的结构保守程度，表明与V-H/V-L配对和抗原结合的相容性。这使得现有结合剂的改造成为可能，如开发源自乳腺癌治疗剂赫赛汀的稳健的高亲和力抗体片段所强调的。我们的研究结果提供了一个通用的策略，用于生成具有增加的抗聚集性的人抗体可变结构域。
The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V-H and V-L) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30-33, 35 in V-H and 24, 49-53, 56 in V-L). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V(H)3 and V(kappa)1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V-H and V-L domains revealed a surprising degree of structural conservation, indicating compatibility with V-H/V-L pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.