Bispecific DNA‐Peptide Probes for Targeting Receptor Pairs on Live Cells

Chemical modification and nucleic acid self‐assembly can be used to make protein receptor ligands form specific arrangements. While this property has been extensively exploited for probing of homomultivalent interactions, there has been comparatively little attention paid to the exploration of heteromultivalent interactions. In this study, we investigated the use of readily assemblable DNA duplexes for programming bispecific targeting of specific cell types. In contrast to previous bispecific agents, we leverage the potential of peptide‐based high‐affinity binders of cell surface proteins used in diagnostics/therapeutics. Systematic spatial screening revealed the optimal distance between two (cyclo)peptides required for selectively recognizing cells expressing unique combinations of receptors. The VGFR2/αVβ3 receptor system on HUVECs was tolerant to changes of the distance between two cyclopeptides (L and cyclo(‐RGDf(N‐Me)K‐)) and required that the distance exceeded the equivalent of 20 nucleotides distance. A different distance‐affinity landscape was observed for recognition of EGFR and MET on A549 cells (through GE11 and bicyclic peptide GE‐137). The DNA‐programmed bispecific binders demonstrated specificity and efficient internalization into target cells. Auristatin‐loaded DNA enabled a selective targeting of cytotoxic payload. Of note, the distance‐optimized bispecific DNA‐peptide probes have much lower molecular weight than previously used agents based on DNA nanostructures or antibodies.