Abstract 1606: Targeting human T regulatory cells with novel Interleukin 2 alpha - IL2 complex-specific RNA aptamer
Authors: Suresh Veeramani, Sue E. Blackwell, William H. Thiel, Paloma H. Giangrande, George J. Weiner
Journal: Cancer Research (2017)

Abstract Background: RNA aptamers are small RNA molecules that bind antigens like antibodies and are currently being explored as alternatives to antibodies for diagnosis and therapy. A potential merit of aptamers is that they can be generated against native cellular antigens, such as those with unique post-translational modifications or receptor-ligand complexes, for which antibody generation can be difficult. Here, we report the use of a cell-based systematic enrichment approach (SELEX) to develop a novel Treg-binding RNA aptamer specific to IL2Rα-IL2 receptor-ligand complex. Methods and Results: A. Generation of Treg-binding aptamers: We designed a cell-based SELEX strategy to generate RNA aptamers specific to human T regulatory (Treg) cells. The starting library consisted of random RNA aptamers with a structural diversity of ~1012. Aptamers against common T cell antigens were pre-cleared using CD4+CD25- Teff cells. Treg-binding aptamers were then positively selected using CD4+CD25+ Tregs from the same donor. After amplification of Treg binders by RT-PCR, the whole selection was repeated eight times, each time with T cells from a different donor. At the end, SELEX-enriched aptamer pools predominantly bound to Tregs, but not to Teff cells, which were then sequenced. The most prevalent Treg-binder, Tr-1, showed 63,875-fold enrichment by the end of SELEX (from 0.36 copies in the starting library to 22,995 copies in the eighth round per million total reads). B. Tr-1 binds to IL2Rα- IL2 complex We tested if Tr-1 recognized human IL2Rα by measuring Tr-1 binding to recombinant IL2Rα protein using RT-qPCR. Results indicated that Tr-1 bound to IL2Rα and showed ~3-fold higher binding to IL2Rα when IL2 was added indicating that Tr-1 recognizes either the receptor-ligand complex or a conformational change in IL2-bound IL2Rα. Binding of Tr-1 to IL2Rα did not significantly alter its affinity to IL2. C. Tr-1 inhibits Treg induction Transformed B cells can induce Treg development in the tumor microenvironment. Experimentally, this was evaluated by quantifying Tregs generated from autologous CD4+ T cells co-cultured with Epstein-Barr virus-transformed B (EBV-B) cells. Addition of Tr-1 resulted in ~30% reduction in EBV-B-induced Tregs (p=0.017). Conclusion: We used a Treg cell-based SELEX strategy to derive a novel Treg-binding RNA aptamer (Tr-1) that preferentially binds to the IL2Rα-IL2 complex. Tr-1 reduced Treg induction by transformed B cells suggesting its potential as a therapeutic agent to reduce tumor-induced immunosuppression. Ongoing studies are further exploring its use in Treg inhibition and in targeting receptor-ligand complexes in cancer. While aptamers recognizing cellular receptors exist, to our knowledge, this is the first report of an aptamer recognizing a receptor-ligand complex. Our approach to generating aptamers against receptor-ligand complexes could have huge scientific impact. Citation Format: Suresh Veeramani, Sue E. Blackwell, William H. Thiel, Paloma H. Giangrande, George J. Weiner. Targeting human T regulatory cells with novel Interleukin 2 alpha - IL2 complex-specific RNA aptamer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1606. doi:10.1158/1538-7445.AM2017-1606