Generation and Evaluation of Bispecific Affibody Molecules for Simultaneous Targeting of EGFR and HER2
Generation and Evaluation of Bispecific Affibody Molecules for Simultaneous Targeting of EGFR and HER2
复制标题
DOI：
10.1021/bc3000645
复制
发表时间：
2012-09-01
影响因子：
4.7
通讯作者：
Gedda, Lars
中科院分区：
文献类型：
--
作者：
Ekerljung, Lina;Wallberg, Helena;Gedda, Lars
几种ErbB受体的共同表达在许多癌症中被发现，并与肿瘤侵袭性增加和患者预后不良有关。这使得利用双特异性构建物同时靶向两个表面受体成为一种越来越受欢迎的策略。在这里，我们已经产生了六个这样的双特异性靶向蛋白，每个蛋白都包含两个单体亲和体分子，分别与两个人表皮生长因子受体EGFR和HER2中的任何一个特异结合。所设计的双功能构建体具有(I)不同亲和体分子的交替定位(N-末端或C-末端)，(Ii)两个可选的多肽连接物(Gly(4)Ser)(3)或(Ser(4)Gly)(3)，以及(Iii)对HER2具有不同亲和力的亲和力不同的亲和体分子(纳摩尔或皮摩尔)。使用Biacore技术和细胞结合分析，证明所有六个构建物都可以同时与它们的两个目标蛋白结合。固有单体分子的N-末端定位有利于促进与相应靶点的结合。有趣的是，与含有更传统的连接体(Gly(4)Ser)(3)的构建体相比，包含新的(Ser(4)Gly)(3)连接子的双特异性构建体在细胞结合中显示出更高的亲和力。可以进一步得出结论，双特异性构建体(但不是单体亲和体分子)诱导SKBR3细胞中二聚体的形成和EGFR的磷酸化，这两种受体都表达相当高的水平。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，但没有观察到这样的影响。
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.