Bispecific DNA-Peptide Probes for Targeting and Modulating Oncogenic Receptor Pairs

点击翻译按钮获取中文摘要

By way of chemical modification of oligonucleotides and Watson-Crick self-assembly, protein receptor ligands can be programmed to form precisely controlled arrangements in space. While this property has been exploited extensively for probing of homomultivalent interactions in solution, on cells and viruses, little attention has been paid to the exploration of heteromultivalent interactions. In this research proposal, we will explore DNA-programmed bispecific binders. (Cyclo)Peptides and peptoids will be conjugated with oligonucleotides and presented on self-assembled DNA scaffolds in up to 220 Å distance. It is the aim to provide, by using a rational approach, avidity and selectivity enhanced binders in order to recognize the unique fingerprint of cancers cells. In subproject 1 we will display the N-methylated cyclopentapeptide cilengitide and the peptoid GU40C4 or cyclopeptides L1 und BD1 on dsDNA assemblies and explore the recognition of VEGFR2 and αVβ3-integrin on HUVECs and other (VEGFR2+/ αVβ3+)-cancer cell lines. Detailed spatial screening will reveal complexes that bind target cells with improved avidity and selectivity against single positive (VEGFR2+ or αVβ3+) cells. For further avidity enhancements we will concatenate optimized bispecific binder on long templates. Moreover, DNA scaffolds provide the opportunity to detect/recognize target cells with enhanced sensitivity by enabling the recruitment of several fluorescence dyes via repeated hybridization of labeled oligonucleotides. Co-expression of VEGFR2 and αVβ3 allows their reciprocal activation which drives neovascularization in growing tumors. Referring to own preliminary work, we will assess the distance-receptor activity-profile and examine the distance-dependent switch from agonistic to antagonistic action of bispecific binders. A comparison between distance-affinity and distance-activity relationships will corroborate hints to proximity-controlled receptor cross-talk and afford the criteria for the design of therapeutically active agents. The second oncogenic receptor pair comprises the EGFR and the MET receptor, which are found physically associated in non-small lung cancers. This receptor pair provides the opportunity to study targeted internalization of armed bispecifics. Cyclopeptides will be conjugated with oligonucleotides and arranged on dsDNA. We will explore, whether the ability to disrupt the reciprocal receptor acitivation correlates with the affinity for EGFR+/MET+-cells. Distance-affinity-, distance-receptor acitivity and distance-internalization-profiles will be compared for the first time. The knowledge obtained will be useful for the design of molecules that enable the in vivo-assembly of bispecific complexes that enable the selective targeting and internalization of cytotoxic auristatin-DNA-conjugates in cancer cells.