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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

2017; American Association for Cancer Research; Volume: 5; Issue: 3_Supplement Linguagem: Inglês

10.1158/2326-6074.tumimm16-b46

2326-6074

Joanne B.L. Tan, Ada Chen, Manmohan R. Leleti, Annette Becker, Erick A. Lindsey, Jarosław Kalisiak, Jay P. Powers, Steve Young, Ulrike Schindler, Juan C. Jaén,

Nanoplatforms for cancer theranostics

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.