Abstract 5021: LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment
Authors: Festag Julia , Thelemann Tamara , Kashyap Abhishek S. , Klar Richard , Kallert Sandra , Buchi Melanie , Hinterwimmer Lisa , Schell Monika , Raith Stefanie , Michel Sven , Zippelius Alfred , Jaschinski Frank 
Published: August 2019

Abstract
The ectonucleotidases CD39 and CD73 have emerged as promising therapeutic targets to enhance antitumor immune responses, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on cancer cells and support the tumor in escaping immune recognition and destruction. Accordingly, in order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to reduce adenosine concentrations in the tumor microenvironment. As an alternative approach to small molecule inhibitors or antibodies, we developed antisense oligonucleotides (ASOs) using the OligofyerTM tool to suppress the expression of CD39 and CD73. ASOs were synthesized as Gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP. The impact of ASO-mediated target knockdown in vivo on anti-tumor immune responses was analyzed in a syngeneic mouse tumor model. Unformulated CD39- and CD73-specific ASOs achieved potent target knockdown on mRNA and protein level in tumor cell lines and primary human T cells in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability if T cells expressed CD39 or CD73. Notably, ASO-mediated knockdown of CD39 or CD73 reversed this inhibitory effect of ATP. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Treatment of tumor-bearing mice with CD39-specific ASO led to dose dependent reduction of CD39 protein expression e.g. in Tregs, tumor-associated macrophages and myeloid-derived suppressor cells. Strikingly, the frequency of intratumoral Tregs was considerably diminished in CD39 ASO-treated animals. Moreover, tumor growth was strongly reduced by CD39-specific ASO. Anti-tumor efficacy was improved when combining CD39 ASO with anti-PD-1 treatment.
Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors.
Citation Format: Julia Festag, Tamara Thelemann, Abhishek S. Kashyap, Richard Klar, Sandra Kallert, Melanie Buchi, Lisa Hinterwimmer, Monika Schell, Stefanie Raith, Sven Michel, Alfred Zippelius, Frank Jaschinski. LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5021.