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. 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. 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%. 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.