Directional Engineering of Cyclic β-Hairpin Peptides for PET/CT Imaging of PD-L1

Peptide-based radiopharmaceuticals represent an emerging drug modality for the noninvasive diagnosis and targeted radiotherapy of cancer. However, the <i>de novo</i> discovery of peptide ligands that combine high binding affinity with sufficient <i>in vivo</i> stability remains a formidable challenge. Conformation-oriented rational design has proven to be a robust strategy for identifying peptide binders to protein targets involved in protein-protein interactions (PPIs), particularly those mediated by well-defined secondary structures such as β-sheets or α-helices. Inspired by the highly structured β-sheet binding interface between programmed death ligand 1 (PD-L1) and its receptor PD-1, we applied a β-hairpin conformation-oriented evolution strategy to engineer high-affinity PD-L1 binders. Starting from a <i>de novo</i> discovered PD-L1-targeting peptide, TPP-1, as a β-hairpin prototype, we iteratively optimized the peptide by sequentially introducing various β-turn motifs, a tryptophan zipper (Trpzip) motif, and subsequently amide cyclization to "lock" the β-hairpin conformation. This process yielded TPP-10, a side-chain to tail cyclized peptide with a highly stabilized β-hairpin structure, as confirmed by circular dichroism (CD) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics (MD) simulations. TPP-10 exhibited a significant improvement in binding affinity (<i>K</i>_D) for PD-L1 compared with TPP-1, along with markedly enhanced <i>in vivo</i> stability. We further evaluated these peptides as radioligands using PET imaging with ⁶⁸Ga and ⁶⁴Cu. [⁶⁸Ga]TPP-10 demonstrated significantly increased tumor uptake and retention in mouse models, and this performance improvement was even more pronounced when the longer-lived radionuclide ⁶⁴Cu was employed. Collectively, these results identify TPP-10 as a promising clinical candidate for PD-L1 PET imaging and highlight β-hairpin-oriented peptide engineering as a powerful approach for developing radiopharmaceuticals targeting aberrant PPIs.