School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, UK.
The Henry Wellcome Laboratory of Cell Imaging, University of East Anglia, Norwich, NR4 7TJ, UK.
Angew Chem Int Ed Engl. 2019 Dec 9;58(50):17986-17993. doi: 10.1002/anie.201908655. Epub 2019 Oct 31.
Antibodies have found applications in several fields, including, medicine, diagnostics, and nanotechnology, yet methods to modulate antibody-antigen binding using an external agent remain limited. Here, we have developed photoactive antibody fragments by genetic site-specific replacement of single tyrosine residues with photocaged tyrosine, in an antibody fragment, 7D12. A simple and robust assay is adopted to evaluate the light-mediated binding of 7D12 mutants to its target, epidermal growth factor receptor (EGFR), on the surface of cancer cells. Presence of photocaged tyrosine reduces 7D12-EGFR binding affinity by over 20-fold in two out of three 7D12 mutants studied, and binding is restored upon exposure to 365 nm light. Molecular dynamics simulations explain the difference in effect of photocaging on 7D12-EGFR interaction among the mutants. Finally, we demonstrate the application of photoactive antibodies in delivering fluorophores to EGFR-positive live cancer cells in a light-dependent manner.
抗体在多个领域都有应用，包括医学、诊断学和纳米技术，但是利用外部试剂来调节抗体-抗原结合的方法仍然有限。在这里，我们通过遗传的方法，在抗体片段 7D12 中，将单个酪氨酸残基定点替换为光笼酪氨酸，从而得到了光活性抗体片段。我们采用了一种简单而强大的测定方法来评估 7D12 突变体与癌细胞表面其靶标表皮生长因子受体 (EGFR) 的光介导结合。在研究的三个 7D12 突变体中的两个中，光笼酪氨酸的存在使 7D12-EGFR 的结合亲和力降低了 20 多倍，并且在暴露于 365nm 光时结合恢复。分子动力学模拟解释了光笼对突变体中 7D12-EGFR 相互作用影响的差异。最后，我们证明了光活性抗体在以光依赖的方式将荧光团递送至 EGFR 阳性活癌细胞中的应用。
