Abstract 5583: Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells

Extracellular adenosinecan be generated from ATP and/or ADP through the concerted action of the ectoenzymes CD39 and CD73. Adenosinecan bind different type-1 purinergic receptorseliciting potent cytoprotectiveand immunosuppressive effects. We previously focused on chronic lymphocytic leukemia(CLL), a disease characterized by the progressive expansion of a mature population of B lymphocytes, showing that a subset of patients with a CD73 + clone possess a poor prognosis. This patient subset can actively generate adenosine, which favors leukemic cell survival, synthesis of immunosuppressive cytokines, while inhibiting T lymphocyte proliferation. ADA2 is a human enzyme that catalyzes the conversion of adenosineinto inosine. We hypothesized that depletion of high adenosinelevels from the tumor microenvironment through the administration of exogenous ADA2 could be therapeutically relevant to limit tumor protection and immunosuppression. Several engineered PEG-ADA2 variants possessed improved enzyme activity and pharmacokinetics compared to wild-type ADA2. Inhibition of adenosine receptoractivation was evaluated by measuring intracellular cAMP concentrations in T lymphocytes purified from healthy donors. Adenosinedepletion was observed with increasing concentrations of PEG-ADA2-R222Q/S265N and significantly reduced cAMP levels. PEG-ADA2 variants were then tested on CD73 + CLL lymphocytes to determine their effects on extracellular adenosineand inosine. Treatment of leukemic B cells with PEG-ADA2-R222Q/S265N and PEG-ADA2-R222Q/S265N/K374D resulted in complete removal of high levels of adenosine, as measured by HPLC. Conversely, PEG-ADA2-E182T, which possesses significantly attenuated catalytic activity, was less effective in reducing adenosine. The functional effects of adenosinedepletion on CLL cell survival were analyzed following treatment with the chemotherapeutic, DNA-damaging agent etoposide that robustly induces apoptosis within 16 hours. Addition of exogenous adenosineto cultures of purified CD73 + CLL cells significantly rescued cells from etoposide-induced apoptosis. However, when these primary leukemic cells were pretreated with PEG-ADA2-R222Q/S265N or PEG-ADA2-R222Q/S265N/K374D, the cell viability rate was significantly decreased, abrogating the cytoprotectiveeffects of adenosine. On the contrary, PEG-ADA2-E182T had a minimal effect, suggesting enzymatic depletion of adenosineis critical to observe these effects. Similar effects were observed by the PEG-ADA2 variants on a CLL cell line expressing CD73 + . These preliminary results suggest that enzymatic depletion of extracellular adenosinefollowing treatment with PEG-ADA2 is a relevant approach to counteract the cytoprotectiveeffects of adenosine, warranting further development of PEG-ADA2 as a possible approach to treat CLL. Citation Format: Sara Serra, Cinzia Bologna, Luz Londono, Lin Wang, Michael Shepard, Sanna Rosengren, Christopher Thanos, Silvia Deaglio. Pegylated adenosine deaminase2 (PEG-ADA2) abrogates the cytoprotectiveeffects of adenosineagainst chronic lymphocytic leukemiacells [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 5583. doi:10.1158/1538-7445.AM2017-5583