Abstract 1472: Enzymatic depletion of adenosine by pegylated, engineered adenosine deaminase 2 (PEG-ADA2): A novel immunotherapeutic approach to treat solid tumors (2016)

Abstract Adenosine is an endogenous immunosuppressant that binds to adenosine receptor checkpoints and protects tissue from immune-mediated rejection. Abnormally high adenosine levels (up to 100-fold greater than other tissues) contribute to a highly immunosuppressive tumor microenvironment (TME). We hypothesized that adenosine deaminase 2 (ADA2), a human enzyme that catalyzes the deamination of adenosine, could be administered at therapeutic levels to deplete high levels of TME adenosine and stimulate anti-tumor immune activity. Recombinant wild-type ADA2 (wtADA2) was cleared extremely rapidly from circulation (t1/2 = 69 min, 7.5 mg/kg iv, n = 9 mice), rendering it unsuitable for therapeutic testing. Therefore, a series of variants was designed to attenuate the heparin binding properties of ADA2 to improve bio-distribution and conjugated with 20K PEG to improve pharmacokinetics (PK). The variant PEG-ADA2-K374D displayed 94% less binding to heparin compared to wtADA2, enzymatic activity comparable to wtADA2, and 33-fold improved PK (t1/2 = 2,256 min); and consistently induced at least 60% (p&lt;0.0001) tumor growth inhibition (TGI) of established subcutaneous syngeneic CT26 tumors (b.i.w., 0.3mg/kg, n = 8). Treatment with PEG-ADA2-K374D resulted in a 5-fold increase in tumor infiltrating CD3+ T-cells (p&lt;0.0001, 6 hours post dose), as assessed by histology. We hypothesized that CD73, which catalyzes the turnover of AMP to adenosine, could be used as a biomarker to identify tumors with elevated adenosine levels. Gene expression studies against a panel of syngeneic tumors revealed that lung KLN205 and pancreatic MH194/PSC4 tumors had high CD73 levels. PEG-ADA2-K374D inhibited the growth of established MH194/PSC4 and KLN205 tumors, with TGI reaching 47% (p&lt;0.0001) and 78% (p&lt;0.0001) after 2 weeks of treatment (b.i.w, 0.3 mg/kg, n = 8). A second series of variants was generated based on structure-based design to have significantly improved kcat/km for adenosine deaminase activity. ADA2-R222Q/S265N had the highest improvement, with a 15-fold greater kcat/km than wtADA2. After pegylation, the circulating half-life of PEG-ADA2-R222Q-S265N in mice was extended from 69 min to 2,790 min (&gt;40-fold increase). This variant induced a maximum TGI of 69% (p&lt;0.05) in the MH194/PSC4 model at 0.003 mg/kg, a 100-fold lower dose than PEG-ADA2-K374D (n = 8). A third variant, PEG-ADA2-E182T lacked detectable enzymatic activity and displayed no tumor growth inhibition, suggesting that ADA2 enzyme activity is required for efficacy. These data suggest that engineered PEG-ADA2 variants induce significant tumor growth inhibition activity in several syngeneic solid tumor models, validating enzymatic depletion of high TME adenosine levels as novel immunotherapeutic approach to treat solid tumors. Citation Format: Lin Wang, Jessica Cowell, Sanna Rosengren, Lei Huang, Xiaoming Li, Qiping Zhao, Jennifer Souratha, Mathieu Marella, Barbara Blouw, Keri Cannon, Chunmei Zhao, Kim Phan, Curtis Thompson, Michael Shepard, Christopher Thanos. Enzymatic depletion of adenosine by pegylated, engineered adenosine deaminase 2 (PEG-ADA2): A novel immunotherapeutic approach to treat solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1472.