Renard Emma, Collado Camps Estel, Canovas Coline, Kip Annemarie, Gotthardt Martin, Rijpkema Mark, Denat Franck, Goncalves Victor, van Lith Sanne A M
Institute de Chimie Moléculaire de l'Université de Bourgogne ICMUB UMR CNRS 6302, Université Bourgogne Franche-Comté, 21000 Dijon, France.
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboudumc, 6525 GA Nijmegen, The Netherlands.
Cancers (Basel). 2021 Jan 23;13(3):428. doi: 10.3390/cancers13030428.
Variable domains of heavy chain only antibodies (VHHs) are valuable agents for application in tumor theranostics upon conjugation to both a diagnostic probe and a therapeutic compound. Here, we optimized site-specific conjugation of the chelator DTPA and the photosensitizer IRDye700DX to anti-epidermal growth factor receptor (EGFR) VHH 7D12, for applications in nuclear imaging and photodynamic therapy. 7D12 was site-specifically equipped with bimodal probe DTPA-tetrazine-IRDye700DX using the dichlorotetrazine conjugation platform. Binding, internalization and light-induced toxicity of DTPA-IRDye700DX-7D12 were determined using EGFR-overexpressing A431 cells. Finally, ex vivo biodistribution of DTPA-IRDye700DX-7D12 in A431 tumor-bearing mice was performed, and tumor homing was visualized with SPECT and fluorescence imaging. DTPA-IRDye700DX-7D12 was retrieved with a protein recovery of 43%, and a degree of labeling of 0.56. Spectral properties of the IRDye700DX were retained upon conjugation. In-labeled DTPA-IRDye700DX-7D12 bound specifically to A431 cells, and they were effectively killed upon illumination. DTPA-IRDye700DX-7D12 homed to A431 xenografts in vivo, and this could be visualized with both SPECT and fluorescence imaging. In conclusion, the dichlorotetrazine platform offers a feasible method for site-specific dual-labeling of VHH 7D12, retaining binding affinity and therapeutic efficacy. The flexibility of the described approach makes it easy to vary the nature of the probes for other combinations of diagnostic and therapeutic compounds.
仅重链抗体（VHHs）的可变结构域是与诊断探针和治疗化合物结合后用于肿瘤诊疗的有价值的试剂。在此，我们优化了螯合剂二乙烯三胺五乙酸（DTPA）和光敏剂IRDye700DX与抗表皮生长因子受体（EGFR）VHH 7D12的位点特异性结合，用于核成像和光动力疗法。使用二氯四嗪结合平台，将双模态探针DTPA-四嗪-IRDye700DX位点特异性地连接到7D12上。使用过表达EGFR的A431细胞测定DTPA-IRDye700DX-7D12的结合、内化和光诱导毒性。最后，在荷A431肿瘤的小鼠中进行了DTPA-IRDye700DX-7D12的体内生物分布研究，并通过单光子发射计算机断层扫描（SPECT）和荧光成像观察肿瘤归巢情况。回收的DTPA-IRDye700DX-7D12的蛋白质回收率为43%，标记度为0.56。结合后IRDye700DX的光谱特性得以保留。放射性标记的DTPA-IRDye700DX-7D12特异性结合A431细胞，光照后可有效杀死这些细胞。DTPA-IRDye700DX-7D12在体内归巢至A431异种移植物，这可通过SPECT和荧光成像观察到。总之，二氯四嗪平台为VHH 7D12的位点特异性双标记提供了一种可行的方法，保留了结合亲和力和治疗效果。所述方法的灵活性使得易于改变用于其他诊断和治疗化合物组合的探针性质。
