The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

Aptamers are synthetic oligonucleotides that bind to their specific receptors with high specificity, offering immense potential for the development of molecular tools. Using recently introduced Ligand-Guided Selection (LIGS), a variant of the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) we previously identified anti-CD19 and anti-CD20 aptamers with specificity and affinity. Given their high expression levels, B-cell markers CD19 and CD20 are widely utilized in the diagnosis of B-cell-related malignancies and autoimmune diseases. Here, we report the design and functional characterization of bivalent aptamer assemblies targeting CD19 and CD20 expressed in B-cell lymphomas. Using a strategic approach, we synthesized dimeric constructs of these aptamers with polyethylene glycol (PEG) linkers of varying lengths to tether the two aptamer units. The bivalent aptamers demonstrated enhanced binding affinity and specificity, with an optimal linker length of ∼3.96 nm. Functional studies revealed that dimeric CD19 aptamers selectively internalized in CD21-negative B-cells, while CD20 aptamers exhibited improved antigen binding without triggering calcium release. These findings highlight the potential of bivalent aptamers in engineering cost-effective, stable, and precise therapeutic agents for B-cell-related malignancies, such as diffuse large B-cell lymphoma (DLBCL). This work advances the development of aptamer-based synthetic therapeutics with promising clinical applications.