Noor Alfiah, Walser Gudrun, Wesseling Matthijs, Giron Philippe, Laffra Albert-Menno, Haddouchi Fatima, De Grève Jacques, Kronenberger Peter
Laboratory of Medical and Molecular Oncology, Vrije Universiteit Brussel, Laarbeeklaan 109, 1090, Brussels, Belgium.
Laboratory of Biotechnology, Erasmushogeschool Brussel, Laarbeeklaan 121, 1090, Brussels, Belgium.
BMC Res Notes. 2018 Oct 22;11(1):751. doi: 10.1186/s13104-018-3852-1.
Our aim was to produce a mono-biotinylated single domain antibody ('nanobody') specific for the epidermal growth factor receptor (EGFR), which is overexpressed in many cancer cells. The binding of the nanobody and its function are tested in cancer cells. The construct could be used to carry variable therapeutic or diagnostic load using biotin-streptavidin bridging.
The EGFR-specific 7D12 nanobody was genetically fused to an IgA hinge linker and to a C-terminal biotin ligase acceptor sequence, allowing mono-biotinylation in E. coli. Expression was in strain BL21-DE3 from a T7 RNA polymerase driven pET22b vector. The biotinylated nanobody, isolated from the periplasm, was purified using streptavidin-mutein affinity chromatography. Final yields were up to 5 mg/l of cell culture. We showed that the construct could bind to EGFR expressing A431 epidermoid carcinoma cells, and to transiently transformed EGFR overexpressing HEK293T cells and not to EGFR negative control cells. The specificity for the EGFR was further demonstrated by immunoprecipitation. To test the functionality, PC9 non-small cell lung cancer cells were treated with mono-biotinylated nanobody or with streptavidin-coupled tetravalent nanobodies. Both were able to block mutant EGFR phosphorylation and slow down growth of PC9 cells. Tetravalent nanobodies were able to downregulate AKT phosphorylation.
我们的目标是制备一种对表皮生长因子受体（EGFR）具有特异性的单生物素化单域抗体（“纳米抗体”），EGFR在许多癌细胞中过度表达。在癌细胞中测试纳米抗体的结合及其功能。该构建体可用于通过生物素 - 链霉亲和素桥接携带可变的治疗或诊断负载。
将EGFR特异性的7D12纳米抗体与IgA铰链连接子和C末端生物素连接酶受体序列进行基因融合，使其在大肠杆菌中能够进行单生物素化。表达由T7 RNA聚合酶驱动的pET22b载体在BL21-DE3菌株中进行。从周质中分离出的生物素化纳米抗体，使用链霉亲和素突变体亲和色谱法进行纯化。最终产量高达每升细胞培养物5毫克。我们表明，该构建体可以与表达EGFR的A431表皮样癌细胞以及瞬时转染过表达EGFR的HEK293T细胞结合，而不与EGFR阴性对照细胞结合。通过免疫沉淀进一步证明了对EGFR的特异性。为了测试其功能，用单生物素化纳米抗体或链霉亲和素偶联的四价纳米抗体处理PC9非小细胞肺癌细胞。两者都能够阻断突变型EGFR的磷酸化并减缓PC9细胞的生长。四价纳米抗体能够下调AKT磷酸化。
