PROJECT SUMMARY/ABSTRACT Robust diagnostics are critical to healthcare. Analysis of circulating nucleic acids such as cell-free DNA (cfDNA) is a rapidly expanding class of diagnostics in several fields of medicine, including cancer and infectious diseases. In oncology, detection of circulating tumor DNA (ctDNA) often precedes appearance of disease on imaging, raising the possibility that it can be used for early detection of cancer. But despite significant improvements in the accuracy and cost of next-generation sequencing, routine use of cfDNA in most clinical settings remains limited by low sensitivity. The cause of low sensitivity is the intrinsic low level of ctDNA in plasma. ctDNA fragments bearing tumor-specific features are often absent from a blood draw due to stochastic partitioning at low ctDNA concentrations. This physical absence of ctDNA fragments cannot be overcome with more accurate sequencing or better computational approaches after a sample has been collected. We have developed a novel approach to directly address this limitation at time of sample collection. Our preliminary results show that an engineered DNA-binding monoclonal antibody (mAb) can protect cfDNA in plasma and increase recovery of ctDNA by 20-fold. This proposal expands on this finding to understand what factors are important for improving recovery of ctDNA using mAbs, to engineer mAbs that can recover even more ctDNA, and to extend this concept to target epigenetic features of cfDNA. Our hypothesis is that engineering the binding affinity, binding specificity, and pharmacokinetics of mAbs can further improve the recovery of ctDNA, and that mAbs that target methylated cfDNA can help enrich ctDNA from highly methylated regions of the tumor genome. In Aim 1, we will test a custom-designed panel of engineered mAbs with different binding relationships to cfDNA and different Fc modifications to determine what factors influence their performance. In Aim 2, we will extend this approach to epigenetic markers, focusing on cfDNA methylation. Taking advantage of the fact that CpG islands are hypermethylated in many tumors, we will develop agents that can enrich DNA with methylated CpGs, and hence enrich hypermethylated tumor DNA at CpG islands. The candidate is a radiation oncologist and physician-scientist at Massachusetts General Hospital (MGH) and a post-doctoral scientist at Massachusetts Institute of Technology (MIT) and the Broad Institute, where the work outlined in this proposal will be conducted. A diverse team of mentors (Dr. J. Christopher Love, Dr. Sangeeta Bhatia, Dr. Viktor Adalsteinsson) and advisors (Dr. Theodore Hong, Dr. David Miyamoto), spanning three world-class institutions (MIT, Broad Institute, MGH), will support the candidate in successful completion of the research goals and training plan outlined in this proposal. The proposed work includes a career development plan that will enable the candidate to gain the necessary technical, scientific, and leade...