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

IntroductionThe 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.MethodsTwo 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.ResultsDeep 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.