Physicochemical convergence in antibody CDR3-VH repertoires recognizing phosphorothioate-modified oligonucleotides backbone.

Introduction: The development of recombinant antibody binders against phosphorothioate-modified antisense oligonucleotides (PS-ASOs) remains challenging due to the highly polyanionic character and structural flexibility of the phosphorothioate backbone. Here, we applied a next-generation sequencing (NGS)-guided phage display strategy to determine whether selection against PS-modified ASOs induces reproducible repertoire remodeling and the emergence of shared CDR3 physicochemical signatures associated with PS-ASO recognition. Methods: Two independent two-round phage display biopanning strategies against biotinylated PS-ASO targets were coupled to targeted amplicon sequencing of VH and VL FR3-CDR3-FR4 regions on the Illumina MiSeq platform. CDR3 clonotypes were reconstructed using a dedicated bioinformatic pipeline including quality filtering, read merging, in-frame translation, clonotype counting, CPM normalization, enrichment analysis, and physicochemical descriptor profiling. Representative enriched scFv clones were further evaluated by ELISA-based binding and competition assays and by fluorescence microscopy in ASO-treated cells. Results: Deep sequencing revealed a marked reduction in repertoire diversity from Round 1 to Round 2, associated with reproducible clonal dominance across independent selection strategies. These changes were already evident at early stages of selection. A shared enriched set of 113 CDR3-VH clonotypes was identified and displayed a defined physicochemical profile, including increased positive charge, recurrent aromatic residue patterns, constrained CDR3-VH length distribution, higher theoretical pI, and reduced hydrophobicity. Representative functional assays further supported the relevance of this signature: among the selected recombinant scFv clones, 12F2 showed preferential binding to both PS1-ASO and PS2-ASO, with reduced reactivity toward the phosphodiester-backbone oligonucleotide used as control. In ASO-treated cells, 12F2 produced a detectable intracellular signal after PS-ASO transfection, whereas PO-ASO-treated cells showed absent or nearly absent signal. Discussion: These results define an NGS-guided framework for identifying early-stage repertoire focusing and physicochemical signatures associated with recognition of modified nucleic acid backbones. The common property-level features suggest convergent binding solutions compatible with recognition of phosphorothioate-associated molecular features, supporting the rational prioritization of candidate binders against challenging polyanionic targets.