Novel IL-2Ralpha (CD25)-Binding RNA Aptamer to Target T Regulatory Cells
Authors: Veeramani Suresh , Blackwell Sue E , Thiel William H , Giangrande Paloma H , Weiner George J 
Published: November 2019

Abstract
Background:
RNA aptamers are short RNA molecules that bind to antigens and ligands in a manner analogous to antibodies. RNA aptamers are being evaluated as clinical therapeutic agents based on their advantages and flexibility as cell targeting agents. Here, we report development and evaluation of a novel human IL-2Ralpha (CD25)-binding RNA aptamer that can be used to target T regulatory (Treg) cells.
Methods:
A. RNA aptamers:
A whole cell SELEX strategy was designed to enrich for RNA aptamers that specifically bind to CD4+CD25high Tregs cells, but not to lineage-related CD4+CD25-T effector (Teff) cells. Selection began with a library of random RNA aptamers (SEL2-N20) with an expected structural diversity of ~1012. The aptamer pool was pre-cleared of aptamers that bound to common T cell antigens using Teff cells before aptamers were positively selected that bound to Tregs from the same donor. This process was repeated for eight successive enrichment rounds, each round using T cells from different donor. Enriched aptamers were sequenced and the top enriched aptamer (Tr-1) was characterized for its antigen specificity.
B. Target antigen identification:
The specificity of Tr-1 aptamer and its structural mutants was evaluated by testing its binding to recombinant CD25 by RT-qPCR.
C. Targeting Tregs with chimeric Tr-1:
Chimeric Treg-targeting reagents were created by linking Tr-1 aptamers with cytotoxic Saporin (Tr-1-Sap) or with Treg-inhibiting Foxp3 silencing RNA (Tr-1-Foxp3 siRNA). Unfractionated CD4+ T cells or Tregs were treated with Tr-1-Sap or Tr-1-Foxp3 siRNA. Reduction in Treg percentage (for Tr-1-Sap treatment) or Foxp3 mRNA levels (for Tr-1-siRNA treatment) was determined by flow cytometry or RT-qPCR, respectively.
Results:
A. Treg-binding RNA aptamers:
A large panel of Treg-binding RNA aptamers was identified using whole cell SELEX and were synthesized. Most of the selected aptamers, particularly Tr-1, bound to CD25high Treg cells to a greater degree than to CD25low Teff cells (Figure 1).
B. Target antigen identification:
Tr-1 bound to human CD25 (IL-2Ralpha) protein while a control aptamer (C-248) did not. Structural mutants of Tr-1 that had lost Treg binding ability showed significantly reduced binding to CD25 protein (Figure 2).
C. Treg targeting with chimeric Tr-1:
Treg-targeting agents created with Tr-1 aptamers successfully delivered toxic Saporin and Foxp3 siRNA into Tregs.
a. CD4+ T cells treated with Tr-1-Sap had a decrease in the percentage of CD25+ Treg population as determined by flow cytometry.
b. Enriched human Tregs treated with Tr-1-Foxp3 siRNA chimera showed reduction in their Foxp3 mRNA levels.
Conclusion and significance:
RNA aptamers that target human Tregs were identified. The most predominant Treg-binding aptamer, Tr-1, binds to human IL-2Ralpha/CD25, a clinically-targeted molecule expressed by Tregs. Chimeric reagents based on Tr-1 aptamer effectively targeted Tregs signifying their potential use as novel immunomodulators. Ongoing studies are further exploring the significance of Tr-1 aptamer as a diagnostic agent and as a therapeutic modulator of Treg activity.

Disclosures
No relevant conflicts of interest to declare.