A Homodimeric Aptamer Variant Generated from Ligand-Guided Selection Activates the T Cell Receptor Cluster of Differentiation 3 Complex

Abstract

Recently, immunotherapeutic modalities with engineered cells and monoclonal antibodies have been effective in treating several malignancies. Nucleic acid aptamers can serve as alternative molecules to design immunotherapeutic agents with high functional diversity. Here we report a synthetic prototype consisting of DNA aptamers that can activate the T cell receptor cluster of differentiation 3 (TCR-CD3) complex in cultured T cells. We show that the activation potential is similar to that of a monoclonal antibody (mAb) against TCR-CD3, suggesting potential for aptamers in developing efficacious synthetic immunomodulators. The synthetic prototype of anti-TCR-CD3ε, as described here, was designed using aptamer ZUCH-1 against TCR-CD3ε, generated by ligand-guided selection (LIGS). Aptamer ZUCH-1 was truncated and modified with nuclease-resistant RNA analogs to enhance stability. Several dimeric analogs with truncated and modified variants were designed with variable linker lengths to investigate the activation potential of each construct. Among them, a dimeric aptamer with dimensions approximately similar to those of an antibody showed the highest T cell activation, suggesting the importance of optimizing linker lengths in engineering functional aptamers. The observed activation potential of dimeric aptamers shows the vast potential of aptamers in designing synthetically versatile immunomodulators with tunable pharmacokinetic properties, expanding immunotherapeutic designs by using nucleic acid-based ligands such as aptamers.

Utility of Multivalent Aptamers to Develop Nanoscale DNA Devices against Surface Receptors

“…This suggests that the design of functional dimeric aptamer scaffolds is predominantly governed by the space between the aptamers and directly related to the linker length, particularly against TCR-CD3ε. 23 Summary of multivalent aptamers and their functions is listed in Table 1 .…”

Dimeric and Multimeric DNA Aptamers for Highly Effective Protein Recognition

“…In detail, three shorter aptamers, named OSJ-T1, OSJ-T2, and OSJ-T3—composed of 48, 49, and 39 nucleotides, respectively—were designed. These ODNs exhibited similar binding affinity compared to the parent aptamer, with OSJ-T3 ( Figure 9 b) as the best analogue in the series showing a K d value of 2.1 nM (Table 1) [ 195 ].…”

“…Further modifications of OSJ-T3 provided OSJ-T3-LNA-OMe ( Figure 9 c), with the 1st and 2nd nucleotides from the 5′-end replaced with LNA residues, and the 40th and 41st nucleotides from the 3′-extremity substituted by 2′-OMe RNA monomers. The double modification in the original aptamer sequence proved to be very effective in terms of binding affinity, providing a K d value of 1.7 nM (Table 1) [ 195 ].…”

“…Three of the four designed dimers ( Figure 9 d) exhibited improved binding affinity compared to their monomeric counterpart. The only exception was the longer dimer OSJ-dimer-8S, which showed no improvement in binding ability as a consequence of the dimerization (Table 1) [ 195 ].…”

“…LNA and 2′-OMe RNA nucleobases are marked in red and blue, respectively. Figures were adapted from Freage et al [ 195 ] with permission.…”