Abstract B46: Small-molecule inhibitors of ecto-nucleotidase CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumor models
Authors: Tan Joanne BL , Chen Ada , Leleti Manmohan , Becker Annette , Lindsey Erick , Kalisiak Jaroslaw , Powers Jay P. , Young Steve , Schindler Ulrike , Jaen Juan C. 
Published: March 2017

Abstract
Introduction: The intra-tumoral generation of adenosine (ADO), a potent inhibitor of T-cell activation, depends on the coordinated and sequential cleavage of extracellular adenosine triphosphate (ATP) by the ecto-nucleotidases CD39 (which produces adenosine monophosphate, AMP) and CD73 (which hydrolyzes AMP to form ADO). For this reason, a number of anti-CD73 antibodies are being advanced into clinical trials; however, to date there have been few reports of potent, selective, small-molecule CD73 inhibitors, such as those described here.
Methods: Ecto-nucleotidase activity was calculated using the Malachite green assay after 50-min incubation with 25µM AMP, in the presence of varying concentrations of test compound(s). The following systems were used. Endogenous expression: hCD73/SKOV-3 cells; hCD73/CD8 T cells. Stable over-expression: hCD73/CHO. Transient expression: mCD73/CHO; NTPDase2/CHO; NTPDase3/CHO; NTPDase8/CHO. Human CD8 T cells enriched from buffy coats or leukopaks were pre-treated with varying concentrations of CD73 inhibitors prior to addition of 50 μM AMP + 10 μM EHNA and activated with T cell Activation/Expansion kit (Miltenyi). In some experiments, exogenous recombinant human IL12p70 (1-10 ng/mL) was added to the culture. Activation (CD25) and effector functions (Granzyme B and IFNγ) were measured by flow cytometry. CT26 cells were implanted into the shaved right flank of 7-8 week old Balb/c mice and measured three times a week starting at 7 days. Mice were enrolled into 4 cohorts and dosed according to the following conditions when tumour volume reached ~100 mm3.
Group 1: 1% HPMC (sc/QD) + 2A3 (10 mg/kg; IP/Q3D)
Group 2: A000830 (30 mg/kg; sc/QD) + 2A3 (10 mg/kg; IP/Q3D)
Group 3: 1% HPMC (sc/QD) + RMP1-14 (10 mg/kg; IP/Q3D)
Group 4: A000830 (30 mg/kg; sc/QD) + RMP1-14 (10 mg/kg; IP/Q3D)
For interim analysis, single cell suspension was generated from tissues, blocked (clone 2.4G2), and stained with antibodies. For intracellular FOXP3 staining, samples were fixed and stained using FOXP3/Transcription Factor Staining Buffer Set. Non-specific blocking was performed with 20% normal rat serum prior to addition of anti-mouse FOXP3 antibody.
Results: We have designed a series of potent and specific small-molecule inhibitors of human and mouse CD73, represented by A000830 and A001190 with the following IC50 values in overexpression systems:
A000830: Mouse IC50 (1 nM); Human IC50 (3 nM)
A001190: Mouse IC50 (n.d.); Human IC50 (0.03 nM)
A000830 and A001190 also blocked AMP hydrolysis by freshly isolated human CD8 T cells at potencies comparable to the over-expression systems. In in vitro models of AMP/ADO-driven inhibition of human CD8+ T-cell activation, A000830 and A001190 showed robust rescue of CD25 expression and granzyme B production. Complete rescue of IFNγ; production was achieved by adding exogenous IL12. In vivo, A000830 was well-tolerated in mice, resulting in sustained plasma concentrations above IC90. Therapeutic dosing of A000830 to these mice in combination with an α-PD1 antibody resulted in robust CT26 tumor growth inhibition, greater than either treatment alone.
Conclusions: Cumulatively, these data provide the initial characterization of a novel class of potent and selective small-molecule CD73 inhibitors that effectively block the generation of ADO from extracellular ATP, reverse the ADO-driven inhibition of human T-cell activation, and display promising anti-tumor activity when dosed in combination with PD-1 blockade.
Citation Format: Joanne BL Tan, Ada Chen, Manmohan Leleti, Annette Becker, Erick Lindsey, Jaroslaw Kalisiak, Jay P. Powers, Steve Young, Ulrike Schindler, Juan C. Jaen. Small-molecule inhibitors of ecto-nucleotidase CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumor models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B46.