Antibody-based diagnostic and therapeutic reagents armed with effector molecules such as dyes and drugs offer hope in the battle against cancer. Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled, and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a single-pot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye700 and Alexa-Fluor647. This dual-labeled antibody bound to EGFR⁺ ovarian cancer cell lines and tissue samples with high specificity, and its phototherapeutic efficacy was confirmed by the selective killing of EGFR⁺ cells in vitro.

中文翻译：

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重组抗体同时且独立的双重位点特异性自我标记。

配备有效应分子（例如染料和药物）的基于抗体的诊断和治疗试剂为抗击癌症提供了希望。已经开发了几种位点特异性缀合方法来为抗体配备这种效应分子，但是它们倾向于昂贵并且涉及多个反应步骤。两个不同的效应子分子与单个抗体的缀合也仍然是主要挑战。在这里，我们描述了使用自标记SNAP和CLIP蛋白标签的单罐反应中具有两个效应分子的抗体的双位点特异性缀合的简单，受控和鲁棒的方法。我们通过与IRDye700和Alexa-Fluor647同时标记表皮生长因子受体（EGFR）特异性单链抗体片段（scFv-425）验证了该方法的原理。⁺卵巢癌细胞系和组织标本具有很高的特异性，并通过选择性杀死EGFR ⁺细胞来证实其光疗功效。