Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.

中文翻译：

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pH依赖性抗体结合的基于结构的工程设计，可选择性靶向固体肿瘤微环境。

单克隆抗体作为主流抗癌药的最新发展要求进一步优化其在人体中使用的安全性。对正常组织的有效靶向和/或效应子活性是一个明显的毒性问题。通过利用实体瘤相对于正常组织的胞外酸度，可以实现特异性肿瘤靶向的优化。在这里，我们应用了一种基于结构的计算方法来设计具有抗性的酸性人类微环境中的抗人表皮生长因子受体2（Her2）抗体。我们使用了亲和力成熟的平台，其中实现了双pH组氨酸扫描诱变以优化pH选择性。对与重组Her2胞外域结合的一小部分设计的测试导致鉴定了具有所需pH依赖性结合行为的抗原结合片段（Fab）变体。相对于亲本bH1-Fab，对酸性pH的结合选择性提高了25倍。在表达完整Her2的细胞上进行的体外实验证实，设计为IgG1 / k全尺寸抗体的变异体具有很高的亲和力，并以与标准抗Her2抗体曲妥珠单抗（Herceptin®）相当的水平抑制肿瘤球体的生长。酸性pH值，而在生理pH值下这些作用明显降低。相反，赫赛汀和亲本bH1抗体均显示出强的细胞结合和生长抑制作用，而与pH无关。