Cheng Jackie, Liang Meina, Carvalho Miguel F, Tigue Natalie, Faggioni Raffaella, Roskos Lorin K, Vainshtein Inna
Integrated Bioanalysis, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, South San Francisco, CA 94080, USA.
Antibody Discovery & Protein Engineering, BioPharmaceuticals R&D, AstraZeneca, Granta Park, Cambridge CB21 6GH, UK.
Antibodies (Basel). 2020 Sep 18;9(3):49. doi: 10.3390/antib9030049.
Amplification and overexpression of HER2 (human epidermal growth factor receptor 2), an ErbB2 receptor tyrosine kinase, have been implicated in human cancer and metastasis. A bispecific tetravalent anti-HER2 antibody (anti-HER2-Bs), targeting two non-overlapping epitopes on HER2 in domain IV (trastuzumab) and domain II (39S), has been reported to induce rapid internalization and efficient degradation of HER2 receptors. In this study, we investigated the molecular mechanism of this antibody-induced rapid HER2 internalization and intracellular trafficking. Using quantitative fluorescent imaging, we compared the internalization kinetics of anti-HER2-Bs and its parental arm antibodies, alone or in combinations and under various internalization-promoting conditions. The results demonstrated that concurrent engagement of both epitopes was necessary for rapid anti-HER2-Bs internalization. Cellular uptake of anti-HER2-Bs and parental arm antibodies occurred via clathrin-dependent endocytosis; however, inside the cells antibodies directed different trafficking pathways. Trastuzumab dissociated from HER2 in 2 h, enabling the receptor to recycle, whereas anti-HER2-Bs stayed associated with the receptor throughout the entire endocytic pathway, promoting receptor ubiquitination, trafficking to the lysosomes, and efficient degradation. Consistent with routing HER2 to degradation, anti-HER2-Bs significantly reduced HER2 shedding and altered its exosomal export. Collectively, these results enable a better understanding of the mechanism of action of anti-Her2-Bs and can guide the rational design of anti-HER2 therapeutics as well as other bispecific molecules.
HER2（人表皮生长因子受体2）作为一种ErbB2受体酪氨酸激酶，其扩增和过表达与人类癌症及转移有关。据报道，一种双特异性四价抗HER2抗体（抗HER2-Bs）可靶向HER2第四结构域（曲妥珠单抗）和第二结构域（39S）上两个不重叠的表位，能诱导HER2受体快速内化并有效降解。在本研究中，我们探究了这种抗体诱导HER2快速内化及细胞内运输的分子机制。通过定量荧光成像，我们比较了抗HER2-Bs及其亲本单臂抗体在单独、联合使用时以及在各种促进内化条件下的内化动力学。结果表明，两个表位的同时结合对于抗HER2-Bs的快速内化是必要的。抗HER2-Bs和亲本单臂抗体通过网格蛋白介导的内吞作用进入细胞；然而，在细胞内，这些抗体遵循不同的运输途径。曲妥珠单抗在2小时内从HER2上解离，使受体能够再循环，而抗HER2-Bs在整个内吞途径中都与受体结合，促进受体泛素化，运输至溶酶体并有效降解。与将HER2导向降解一致，抗HER2-Bs显著减少了HER2的脱落并改变了其外泌体输出。总的来说，这些结果有助于更好地理解抗HER2-Bs的作用机制，并可为抗HER2疗法以及其他双特异性分子的合理设计提供指导。
