FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates

Despite the recent success of antibody–drug
conjugates (ADCs)
in cancer therapy, a detailed understanding of their entry, trafficking,
and metabolism in cancer cells is limited. To gain further insight
into the activation mechanism of ADCs, we incorporated fluorescence
resonance energy transfer (FRET) reporter groups into the linker connecting
the antibody to the drug and studied various aspects of intracellular
ADC processing mechanisms. When comparing the trafficking of the antibody–FRET
drug conjugates in various different model cells, we found that the
cellular background plays an important role in how the antigen-mediated
antibody is processed. Certain tumor cells showed limited cytosolic
transport of the payload despite efficient linker cleavage. Our FRET
assay provides a facile and robust assessment of intracellular ADC
activation that may have significant implications for the future development
of ADCs.

尽管抗体偶联药物（antibody–drug conjugates，ADCs）在癌症治疗领域近年取得了显著进展，但学界对其进入癌细胞、胞内转运及代谢的详细机制仍缺乏充分认知。为进一步阐明ADC的激活机制，我们将荧光共振能量转移（fluorescence resonance energy transfer，FRET）报告基团引入连接抗体与药物的连接子中，对细胞内ADC的加工过程开展了多维度研究。在对比不同模型细胞中抗体-FRET药物偶联物的胞内转运情况时，我们发现细胞背景对抗原介导的抗体加工过程具有重要影响：部分肿瘤细胞虽可实现连接子的有效裂解，但其载药的胞质转运效率却十分有限。本研究建立的FRET检测方法可便捷且可靠地评估细胞内ADC的激活状态，这对ADC的未来开发具有重要的指导意义。