INTRODUCTION:

Liquid biopsies including the analysis of cell-free DNA (cfDNA) from blood can be used to diagnose, monitor, or molecularly profile disease. Despite the fast adoption of liquid biopsies in oncology, prenatal testing, infectious disease, and organ transplant monitoring, higher sensitivity is needed in many important clinical applications. In oncology, efforts to improve the sensitivity for detecting circulating tumor DNA (ctDNA) have mostly focused on ex vivo sequencing and analysis methods. However, an intrinsic challenge is the scarcity of ctDNA in vivo, which leaves little ctDNA to be collected and analyzed.

RATIONALE:

We hypothesized that transiently attenuating cfDNA clearance in vivo would augment the levels of ctDNA in circulation and increase the amount recovered from a blood draw. The two natural mechanisms for clearing cfDNA are uptake by liver-resident macrophages and degradation by circulating nucleases. In this work, we sought to develop two intravenous priming agents given 1 to 2 hours before a blood draw that act on these mechanisms and enhance ctDNA recovery. Our priming agents comprise (i) nanoparticles that act on the cells responsible for cfDNA clearance and (ii) DNA-binding monoclonal antibodies (mAbs) that protect cfDNA.

RESULTS:

We first investigated the nanoparticle priming strategy and identified a succinyl phosphoethanolamine–based liposomal agent that inhibited cfDNA uptake in vitro and transiently increased the recovery of cfDNA from blood in healthy mice. We confirmed that liposomes rapidly accumulated in the liver and that liver resident macrophages were necessary for cfDNA half-life extension. As an orthogonal strategy, we showed that DNA-binding mAbs interacted with elements of cfDNA and protected double-stranded DNA from nuclease digestion. Engineering the mAb to abrogate Fc-γ-receptor (FcγR) binding increased its persistence Circulation and the recovery of cfDNA from blood compared with that of the native mAb and an isotype control mAb in healthy mice. Using a bespoke ctDNA assay tracking 1822 tumor-specific single-nucleotide variants (SNVs) in plasma samples from mouse preclinical cancer models, we demonstrated that our two orthogonal priming strategies increase the recovery of ctDNA by >10-fold, enable more complete tumor molecular profiling from ctDNA, and increase the sensitivity for detection of small tumors from <10% to >75%.

CONCLUSION:

By modulating cfDNA clearance in vivo, priming agents improved the sensitivity and robustness of ctDNA testing in tumor-bearing mice. Just as intravenous contrast agents have profoundly improved clinical imaging, we envision that priming agents will improve the sensitivity and utility of liquid biopsies across clinical applications. Additionally, the concept of delivering priming agents that transiently attenuate analyte clearance in vivo and boost diagnostic sensitivity may inform similar approaches to enhance the testing for other scarce biomarkers in oncology and beyond.

Graphical Abstract

Priming agents reduce the clearance of cfDNA and enhance the sensitivity of liquid biopsies. Priming agents transiently attenuate natural clearance mechanisms for cfDNA and consist of nanoparticles that act on the cells responsible for cfDNA clearance (top left) or DNA-binding antibodies that protect cfDNA from cellular uptake and enzymatic digestion (bottom left). In preclinical models, priming agents increased the half-life of cfDNA, enhanced recovery of ctDNA, and improved tumor molecular profiling from ctDNA and sensitivity of ctDNA testing (middle). We envision that priming agents could be administered 1 to 2 hours prior to a blood draw in the clinic to improve the recovery of ctDNA and boost the sensitivity of many types of liquid biopsy tests (right).

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Summary