Background

Monoclonal antibodies (mAbs) have proved to be a valuable tool for the treatment of different cancer types. However, clinical use of an increasing number of mAbs, have also highlighted limitations with monotherapy for cancers, in particular for such with more complex mechanisms, requiring action on additional molecules or pathways, or for cancers quickly acquiring resistance following monotherapy. An example for the latter is the mAb trastuzumab, FDA approved for treatment of metastatic gastric carcinoma. To circumvent this, researchers have reported synergistic, anti-proliferative effects by combination targeting of HER2 and EGFR by trastuzumab and the EGFR-targeting mAb Cetuximab overcoming trastuzumab resistance.

Methods

Maintaining the proven functionality of trastuzumab, we have designed bi-specific antibody molecules, called AffiMabs, by fusing an EGFR-targeting Affibody molecule to trastuzumab’s heavy or light chains. Having confirmed binding to EGFR and Her2 and cytotoxicity of our AffiMabs, we analyzed apoptosis rate, receptor surface levels, phosphorylation levels of receptors and associated signaling pathways as well as differentially expressed genes on transcriptome level with the aim to elucidate the mode of action of our AffiMabs.

Results

The AffiMabs are able to simultaneously bind HER2 and EGFR and show increased cytotoxic effect compared to the original trastuzumab therapeutic molecule and, more importantly, even to the combination of trastuzumab and EGFR-targeting Affibody molecule. Analyzing the mode of action, we could show that bi-specific AffiMabs lead to reduced surface receptor levels and a downregulation of cell cycle associated genes on transcriptome level.

Conclusion

Our study shows that transcriptome analysis can be used to validate the choice of receptor targets and guide the design of novel multi-specific molecules. The inherent modularity of the AffiMab format renders it readily applicable to other receptor targets.

中文翻译：

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双特异性抗体分子比两分子组合更有效地抑制肿瘤细胞增殖

背景

单克隆抗体 (mAb) 已被证明是治疗不同癌症类型的宝贵工具。然而，越来越多的 mAb 的临床使用也凸显了癌症单一疗法的局限性，特别是对于那些机制更复杂、需要对额外分子或途径起作用的癌症，或者对于在单一疗法后迅速获得耐药性的癌症。后者的一个例子是 mAb 曲妥珠单抗，FDA 批准用于治疗转移性胃癌。为了避免这种情况，研究人员报告了通过曲妥珠单抗联合靶向 HER2 和 EGFR 以及靶向 EGFR 的单克隆抗体西妥昔单抗克服曲妥珠单抗耐药性的协同抗增殖作用。

方法

通过将靶向 EGFR 的 Affibody 分子与曲妥珠单抗的重链或轻链融合，我们设计了称为 AffiMabs 的双特异性抗体分子，以保持曲妥珠单抗已证实的功能。在确认了我们的 AffiMabs 与 EGFR 和 Her2 的结合以及细胞毒性后，我们分析了细胞凋亡率、受体表面水平、受体的磷酸化水平和相关信号通路以及转录组水平上的差异表达基因，目的是阐明我们的作用模式AffiMabs。

结果

与原始曲妥珠单抗治疗分子相比，AffiMab 能够同时结合 HER2 和 EGFR，并显示出更高的细胞毒性作用，更重要的是，甚至与曲妥珠单抗和靶向 EGFR 的 Affibody 分子的组合相比。分析作用模式，我们可以证明双特异性 AffiMabs 导致表面受体水平降低和转录组水平上细胞周期相关基因的下调。

结论

我们的研究表明，转录组分析可用于验证受体靶标的选择并指导新型多特异性分子的设计。AffiMab 格式的固有模块化使其易于适用于其他受体目标。