Hayes, O. G. | bioRxiv Science

bioRxiv2026

Customizable bead-based platform for EV surface protein immunocapture and detection in plasma

Extracellular vesicles (EVs) are increasingly recognized as valuable biomarkers for liquid biopsy because they carry molecular signatures that reflect their cells of origin. However, EV isolation remains a challenge, and existing EV immunocapture platforms are constrained by nonspecific binding, limited flexibility, and insufficient sensitivity. Here, we present a customizable bead-based platform for the isolation and detection of specific EV populations directly from plasma. The platform combines bead-immobilized capture antibodies with fluorescent detection antibodies, enabling sensitive and multiplexed EV profiling. Optimization of bead conjugation chemistry improved antibody immobilization and reduced nonspecific background signals, enhancing assay specificity and performance. Using breast cancer-derived EVs expressing fluorescent protein, we demonstrate the detection of tumor-derived EVs in plasma at concentrations as low as 1E9 EVs/ml. Furthermore, the assay distinguished HER2- and TROP2-high from low EV populations, demonstrating its ability to detect clinically relevant differences in biomarker expression. The workflow is compatible with direct EV analysis in the unprocessed plasma samples, thereby reducing sample preparation requirements and assay complexity. These findings establish a sensitive, flexible, and scalable platform for EV biomarker analysis. The approach overcomes several limitations of current commercial bead-based technologies and may facilitate the development of customizable multiplexed EV-based assays for cancer detection, patient stratification, and disease monitoring.

cancer biology↗

bioRxiv2026

Enhancing the efficacy of siRNA Antibody Oligonucleotide Conjugates (AOCs) through chemical design

Antibody-oligonucleotide conjugates (AOCs) offer a promising solution to delivery challenges of therapeutic oligonucleotides. However, the relationship between their complex chemical architectures and biological activity remains poorly understood, limiting the development of important structure-function relationships. For siRNA-containing AOCs, increasing the siRNA-to-antibody ratio beyond one (drug-to-antibody ratio, DAR>1) reduces potency, attributed to altered pharmacokinetics arising from increased negative charge density. Here, we investigated whether simple chemical modifications could improve AOC efficacy and mitigate limitations associated with higher DAR. Introduction of a single C16 lipid modification to the siRNA significantly enhanced target gene silencing compared to the unmodified AOC. Extending this modification to a DAR2 architecture, in which two siRNAs are conjugated per antibody, restored the loss of activity associated with increasing DAR from 1 to 2. Notably, at an equivalent siRNA dose (1 mg/kg), the DAR2-C16 AOC requires half the amount of antibody while achieving knockdown comparable to the DAR1-C16 AOC. We also developed a charge-balancing ionizable linker (CBIL) designed to partially compensate for the negative charge of siRNA. Incorporation of the CBIL enhanced target gene silencing in heart and skeletal muscle without a corresponding increase in hepatic activity, resulting in a shift toward greater extrahepatic activity relative to liver. Together, these findings demonstrate that chemical modification of both the siRNA payload and antibody-siRNA linker can be used to tune AOC potency and tissue activity, while enabling higher payload loading without compromising efficacy. These results establish chemical design as an important strategy for expanding the architecture and therapeutic potential of AOCs.

biochemistry↗