Bimodal liquid biopsy for cancer immunotherapy based on peptide engineering and nanoscale analysis.

Despite its high potential, PD-L1 expressed by tumors has not been successfully utilized as a biomarker for estimating treatment responses to immunotherapy. Circulating tumor cells (CTCs) and tumor-derived exosomes that express PD-L1 can potentially be used as biomarkers; however, currently available assays lack clinically significant sensitivity and specificity. Here, a novel peptide-based capture surface is developed to effectively isolate PD-L1-expressing CTCs and exosomes from human blood. For the effective targeting of PD-L1, this study integrates peptide engineering strategies to enhance the binding strength and specificity of a β-hairpin peptide derived from PD-1 (pPD-1). Specifically, this study examines the effect of poly(ethylene glycol) spacers, the secondary peptide structure, and modification of peptide sequences (e.g., removal of biologically redundant amino acid residues) on capture efficiency. The optimized pPD-1 configuration captures PD-L1-expressing tumor cells and tumor-derived exosomes with 1.5-fold (p = 0.016) and 1.2-fold (p = 0.037) higher efficiencies, respectively, than their whole antibody counterpart (aPD-L1). This enhanced efficiency is translated into more clinically significant detection of CTCs (1.9-fold increase; p = 0.035) and exosomes (1.5-fold increase; p = 0.047) from patients' baseline samples, demonstrating stronger correlation with patients' treatment responses. Additionally, we confirmed that the clinical accuracy of our system can be further improved by co-analyzing the two biomarkers (bimodal CTC/exosome analysis). These data demonstrate that pPD-1-based capture is a promising approach for capturing PD-L1-expressing CTCs and exosomes, which can be used as a reliable biomarker for cancer immunotherapy.