Abstract 1023: CD73 inhibition with a novel orally bioavailable small molecule blocks adenosine production and rescues T-cells activation
Authors: Tatiana Zavorotinskaya, Brian Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank Duong, Lori Friedman, Tom Huang, Melissa R. Junttila, Wayne Kong, Todd C. Metzger, Jared T. Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Natalie Yuen
Journal: Cancer Research (2020)

Abstract Adenosine has emerged as a key immunosuppressive metabolite within the tumor microenvironment (TME). A persistently elevated concentration of adenosine in TME can impair immune control of tumor growth by diminishing cytotoxic T-cell responses and function of natural killer cells while augmenting the suppressive activity of myeloid and regulatory T-cells. The ectonucleotidase, CD73 mediates the final dephosphorylation step in the conversion of extracellular ATP to adenosine. Overexpression of CD73 is observed in many solid tumors and correlates with unfavorable clinical outcome. We aimed to overcome adenosine-driven immunosuppression by developing an orally bioavailable, best-in-class, small molecule inhibitor of CD73. Here, we show superior potency of our selective AMP-competitive CD73 inhibitor in blocking adenosine generation by multiple cell types. CD73 inhibitor activity was assessed by directly measuring the generation of adenosine from AMP by LC-MS/MS. In CD73 expressing cells, potent and complete inhibition of CD73 ectonucleotidase activity was observed. To test the functional consequence of CD73 inhibition, we interrogated the effects of CD73 inhibitors on CD8+ T-cell function upon exposure to AMP. CD73 expressed on T-cells is sufficient to drive adenosine generation from AMP resulting in severely suppressed proliferation, diminished production of inflammatory cytokines and reduced expression of activation markers. Our inhibitors successfully counteracted the effects of AMP and completely rescued all aspects of CD8+ T-cell activation. Interestingly, we found that the concentration of AMP in tumors may vary from micromolar to millimolar levels, underscoring the necessity of AMP-competitive CD73 inhibitors to be efficacious in a high AMP environment. We demonstrated that nanomolar concentrations of our CD73 inhibitors can efficiently rescue T-cell function in the presence of millimolar AMP concentrations. We are currently conducting biophysical studies to better understand this unique property of our inhibitors. We next aimed to identify an adenosine responsive gene signature in cytotoxic T-cells. RNA sequencing analysis of primary anti-CD3/28/2 activated CD8+ T-cells from human peripheral blood mononuclear cells revealed a unique set of genes persistently and similarly altered in response to both adenosine and AMP. Finally, we demonstrated that CD73 inhibitors can completely block gene expression changes of this AMP/adenosine-response signature. Discovery of an AMP/adenosine-response gene signature may help identify patients whose tumors harbor elevated adenosine signaling and would benefit from an orally bioavailable small molecule inhibitor of CD73 to reverse immunosuppression. Citation Format: Tatiana Zavorotinskaya, Brian Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank Duong, Lori Friedman, Tom Huang, Melissa R. Junttila, Wayne Kong, Todd C. Metzger, Jared T. Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Natalie Yuen. CD73 inhibition with a novel orally bioavailable small molecule blocks adenosine production and rescues T-cells activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1023.