Abstract
Liquid biopsies using cell-free DNA (cfDNA) enable non-invasive detection and characterization of disease. Advances in sequencing methods have significantly improved the performance of liquid biopsies. Yet, despite these advances, sensitivity remains a fundamental challenge. In oncology, circulating tumor DNA (ctDNA) screening tests only detect 20-40% of stage I tumors and tests for minimal residual disease have only 25-50% sensitivity after surgery. The major barrier to better sensitivity is the intrinsic low level of ctDNA in plasma. Physical absence of tumor DNA molecules in a blood draw from a patient with low disease burden will result in a negative test, no matter the sensitivity of the ex vivo detection platform. To overcome this barrier, here we report a first-in-class intravenous DNA-binding priming agent that is given 2 hours prior to a blood draw to recover more ctDNA, boosting the detection of tumor mutations in plasma by 19-fold and increasing sensitivity from 6% to 84%. Given the rapid clearance of cfDNA from circulation, we reasoned that a priming agent that could bind and protect cfDNA from clearance could increase the tumor DNA recovered from plasma. We selected monoclonal antibodies (mAbs) as the class of molecules to use as cfDNA protectors given their persistence in circulation and ease of engineering. We identify a mAb that binds double-stranded DNA (dsDNA) and find on electrophoretic mobility shift assays that it binds both free and histone-bound dsDNA, the constituent components of cfDNA. We then demonstrate that this mAb can delay the clearance of dsDNA from plasma in vivo through co-injection of the mAb with free- and histone-bound dsDNA in mice. We further identify interactions with Fc-gamma-receptors as a key mediator of early clearance of dsDNA bound to the priming mAb. To address this early clearance and limit potential immune interactions, we engineer the mAb to abrogate its Fc effector function. The engineered variant decreases clearance of injected dsDNA by over 150-fold at one hour post-injection compared to dsDNA alone. We next evaluate the effect of our priming mAb on cancer detection. We use a targeted panel against 1,822 mutations in the MC26 murine colon carcinoma cell line to detect tumor mutations in the plasma of tumor bearing mice. The priming mAb results in 19-fold higher recovery of tumor DNA molecules compared to a control mAb. This improved recovery leads to detection of 77% of targeted sites in plasma compared to only 15% in the control group. In sensitivity analyses, higher recovery of mutant molecules improves sensitivity for cancer detection from 6% to 84% at 0.001% tumor fraction. In summary, we demonstrate an approach to overcome a key barrier in liquid biopsies. We envision that similar to contrast agents in clinical imaging, priming agents could significantly boost the diagnostic sensitivity of liquid biopsies and enable further applications across biomedicine.
Citation Format: Shervin Tabrizi, Carmen Martin-Alonso, Kan Xiong, Timothy Blewett, Sainetra Sridhar, Zhenyi An, Sahil Patel, Sergio Rodriguez-Aponte, Christopher Naranjo, Douglas Shea, Todd Golub, Sangeeta N. Bhatia, Viktor A. Adalsteinsson, J. Christopher Love. A DNA-binding priming agent protects cell-free DNA and improves the sensitivity of liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3371.
