Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Schematic of approach to design, express, and characterize a panel of immunologically-silenced mAbs against cfDNA components (dsDNA, mononucleosomes, histones). Priming agent mAbs were generated using VH and VL amino acid sequences of mAbs that bind cfDNA components fused to a murine IgG2a backbone with L234A/L235A/P329G silencing mutations. B) EMSA of mAbs binding to free dsDNA or MNs, and quantification of binding interaction with biolayer interferometry. Free dsDNA binding was determined by the presence of shifted bands in the DNA lane. MN-only binding was determined by shift or disappearance of the MN band in the MN lane and no evidence of a shifted band in the DNA lane. For mAbs that bound free dsDNA or MN, the binding interaction was subsequently quantified with BLI. C) Measurement of binding kinetics (k on and k off ) and avidity (apparent K d ) to free dsDNA (left) and MN (right) of intact mAbs. D) Comparison of K d of dsDNA binders and MN binders to free dsDNA and MN. E) Interaction of MN binders with individual components of mononucleosomes and different dsDNA topologies, including individual histones, H2A/H2B dimer, H3/H4 tetramer, H2A/H2B/H3/H4 octamer, bent dsDNA (supercoiled pUC18), linear dsDNA (linearized pUC18, lin. pUC18), and intact MN (histone octamer + 147bp dsDNA). NB - no binding.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Generated, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Degradation of free dsDNA and dsDNA in mononucleosomes in presence of 0.2U/mL of DNase I and mAb priming agents. IgG2a is an unrelated control antibody. Each dot represents the mean of two replicates. B) Experimental approach for testing impact of priming agents on clearance of free dsDNA and MN. C) Percentage of W601 DNA remaining in plasma 1 hour after injection of W601 either in free dsDNA form (left) or as MN (right), with or without dsDNA binding and MN binding priming agents. D) Percentage of cfDNA isolated from mouse plasma by various priming agents via immunoprecipitation using mAb-coupled magnetic beads, adjusted for background binding to beads alone. E) Percentage of cfDNA, W601 free dsDNA, W601 MN, and W601 MN with mild DNase I treatment isolated from mouse plasma using each MN-only mAb priming agent. F) cfDNA fragment length distribution in mice (n=8) with dashed lines at 167bp and 147bp (left) and percent of fragments <=147 bp and <=167 bp (right). * p < 0.05, ** p < 0.01, *** p < 0.001, ns - not significant.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Control, Clinical Proteomics, Injection, Binding Assay, Isolation, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: Antibody-based priming agents bind cfDNA in the bloodstream and protect it from clearance, enabling more to be collected in a subsequent blood draw. This study identified the key molecular determinants of priming activity. The optimal target binder was dsDNA, rather than mononucleosomes, and the priming activity was correlated with strength of binding to dsDNA, with the best priming agents having K d dsDNA < 10nM. The best dsDNA-binding priming agents had different magnitudes of DNase protection ability and impact on cfDNA fragmentation. In particular, one agent, DNA1, best preserved the endogenous fragmentation profile in cfDNA and protected short, informative cfDNA fragments at transcription factor binding sites from clearance. The Fc domain was found to be dispensable for the priming effect, suggesting that agents with more rapid clearance can still elicit a priming effect. Finally, we leveraged some of the principles identified in this study to engineer new single chain molecules that can similarly elicit a priming effect in tumor bearing mice, extending the space of priming agents to non-immunoglobulin dsDNA binding domains.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Activity Assay, Binding Assay