Disulfide-Directed Multicylic Peptides for Targeting and Radionuclide Imaging of HER2 Expressing Tumors

Human epidermal growth factor receptor 2 (HER2) is a critical therapeutic target in various cancers, but its accurate noninvasive quantification remains challenging. Existing positron emission tomography (PET) tracers, such as antibodies and peptides, are limited by suboptimal clearance, insufficient tumor uptake and retention, and poor proteolytic stability. Here, we introduce a new class of disulfide-directed multicyclic peptides (DDMPs) that combine a conformationally rigid and stable scaffold with excellent HER2-binding properties. Using yeast display and directed evolution, we identified optimized DDMPs with single-digit nanomolar affinity for HER2. Alanine scanning mutagenesis revealed conserved residues that were amenable to functional modification. After gallium-68 (⁶⁸Ga) radiolabeling, these DDMPs demonstrated significantly enhanced tumor accumulation, prolonged retention, and superior sensitivity in detecting HER2-expressing tumors compared to those of existing peptide tracers. Our work establishes DDMPs as a structurally distinct class of peptide tracers with superior performance, representing a significant advancement toward the clinical translation of precision PET imaging for HER2-positive cancers.