Peptide-displayed phage libraries are billion-clone collections of diverse chimeric bacteriophage particles, decorated by genetically fused peptides built from a random combination of natural amino acids. Studying the molecular evolution of peptide-displayed libraries in mammalian model systems, using in vivo phage display techniques, can provide invaluable knowledge about the underlying physiology of the vasculature system, allow recognition of organ- and tissue-specific networks of protein–protein interactions, and provide ligands for targeted diagnostics and therapeutics. Recently, we discovered that landscape phage libraries, a specific type of multivalent peptide phage display library, expose on their surface comprehensive collections of elementary binding units (EBUs), which can form short linear motifs (SLiMs) that interact with functional domains of physiologically relevant proteins. Because of their unique structural and functional features, landscape phages can use an alternative mechanism of directed molecular evolution, i.e., combinatorial avidity selection. These discoveries fueled our interest in revisiting the in vivo evolution of phage displayed libraries using another format of display, i.e., landscape phages. In this study, we monitored the evolution of a landscape phage library in a mouse model with and without an implanted human breast cancer tumor xenograft. As expected, the multivalent architecture of landscape phage displayed proteins provided strong tissue selectivity and resulted in a huge diversity of tissue penetrating, chimeric phage particles. We identified several types of EBU interactions that evolved during the course of tissue distribution, which included interactions of EBUs with all tissue types, those EBUs that interacted selectively with specific organs or tissues with shared gene expression profiles or functionalities, and other EBUs that interacted in a tissue-selective manner. We demonstrated that landscape phage libraries are a rich collection of unique nanobioparticles that can be used to identify functional organ and tissue-binding elements after the evolution of a phage display library in vivo.
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