Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Sweden.
Bioconjug Chem. 2012 Sep 19;23(9):1802-11. doi: 10.1021/bc3000645. Epub 2012 Aug 29.
Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly(4)Ser)(3) or (Ser(4)Gly)(3), and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser(4)Gly)(3) linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly(4)Ser)(3). It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.
几种 ErbB 受体的共表达已在许多癌症中被发现，并且与肿瘤侵袭性的增加和患者预后的恶化有关。这使得同时针对两种表面受体的双特异性构建成为一种越来越被重视的策略。在这里，我们生成了六种这样的双特异性靶向蛋白，每种蛋白都包含两个分别特异性结合两个人类表皮生长因子受体（EGFR 和 HER2）之一的单体亲和体分子。双特异性构建体的设计具有以下特点：（i）不同亲和体分子的（N-或 C-末端）替代定位，（ii）两种替代肽接头（Gly(4)Ser)(3)或（Ser(4)Gly)(3)，以及（iii）对 HER2 具有不同亲和力（纳摩尔或皮摩尔）的亲和体分子。使用 Biacore 技术和细胞结合测定，证明所有六种构建体都可以同时结合它们的两个目标蛋白。固有单体亲和体分子的 N-末端定位有利于促进与各自靶标的结合。有趣的是，与含有更传统接头（Gly(4)Ser)(3)的构建体相比，含有新型接头（Ser(4)Gly)(3)的双特异性构建体在细胞结合中显示出更高的亲和力。还可以得出结论，双特异性构建体（而不是单体亲和体分子）诱导 SKBR3 细胞中 EGFR 的二聚化和磷酸化，该细胞表达相当高水平的两种受体。还研究了双特异性结合是否会影响细胞生长或使细胞对电离辐射敏感，但没有观察到这种影响。
