Division of Molecular Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden.
Biotechnol Appl Biochem. 2009 Aug 21;54(2):121-31. doi: 10.1042/BA20090096.
HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G4S)3 [(Gly4-Ser)3]-encoding gene fragment. The encoded 30 kDa affibody construct (ZHER2)2-(G4S)3-(ZEGFR)2, with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.
HER2（人表皮生长因子受体-2；ErbB2）和 EGFR（表皮生长因子受体）在各种形式的癌症中过表达，并且在许多研究中已经报道了这两种受体的共表达。同时靶向 HER2 和 EGFR 已被讨论为一种策略，可以潜在地提高某些癌症的分子成像和治疗的效率和选择性。为了生成能够双特异性靶向 HER2 和 EGFR 的分子，通过（G4S）3 [（Gly4-Ser）3]编码基因片段，将编码单价 HER2 结合亲和体分子的基因片段基因融合在单价 EGFR 结合亲和体分子中。编码的 30 kDa 亲和体构建体（ZHER2）2-（G4S）3-（ZEGFR）2，具有与 HER2 和 EGFR 进行 bs（双特异性）结合的潜力，在大肠杆菌中表达，并根据其结合能力进行了表征。使用生物传感器技术和流式细胞分析，后者使用 HER2 和 EGFR 过表达细胞，分别证明了其保留分别结合 HER2 和 EGFR 的能力。此外，在以下方面证明了同时与 HER2 和 EGFR 结合：（i）在使用 bs 亲和体分子结合固定化 EGFR 和可溶性 HER2 的实时生物特异性相互作用分析中采用三明治格式；（ii）免疫荧光显微镜，其中 bs 亲和体分子结合 EGFR 过表达细胞和可溶性 HER2；和（iii）细胞-细胞相互作用分析，其中 bs 亲和体分子结合 HER2 过表达 SKBR-3 细胞和 EGFR 过表达 A-431 细胞。据我们所知，这是第一个报道的具有同时靶向 HER2 和 EGFR 的潜力的 bs 亲和力蛋白。讨论了这种和类似的构建体的潜在未来用途，这些构建体能够 bs 靶向受体，以提高成像和治疗的效率和选择性。
