Background Combination of Venetoclax, a BCL-2 inhibitor, and 5-Azacytidine, a hypomethylating agent, is the standard of care for patients with newly diagnosed acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy.¹ In addition to their anti-leukemic activity, 5-Azacytidine was shown to improve cancer cell recognition by immune effector cells through induction of stress ligand expression,^2,3 and Venetoclax was described to enhance T-cell and NK-cell–mediated cytotoxicity against AML blasts [3,4]. Among attractive mediators of anti-tumor immunity, g9d2 T-cells are emerging as effector cells that harbor strong cytolytic and pro-inflammatory activities and are associated with good prognosis in cancer patients.^5,6 ICT01 is a first-in-class anti-BTN3A mAb activating g9d2 T-cells and is being evaluated in a Phase 1/2a clinical study in solid tumors and hematologic malignancies (NCT04243499).^7,8

Here, we explore the potential of combining ICT01 with Venetoclax and 5-Azacytidine to increase the anti-leukemic activity of g9d2 T-cells.

Methods Flow cytometry was used to assess the effects of Venetoclax and 5-Azacytidine on ICT01-mediated g9d2 T-cell activation after in vitro culture of healthy donor (HD) or patient derived PBMCs. AML cell lines’ and primary AML blasts’ sensitivity to ICT01-activated g9d2 T-cell killing was monitored after 4 or 24 hours of target:effector co-culture. Targets and/or effectors were pre-treated with Venetoclax, 5-Azacytidine or the combination. Expression of specific stress ligands upon treatment with Venetoclax, 5-Azacytidine or the combination was analyzed by flow cytometry.

Results First, we show that activation of g9d2 T-cells by ICT01 is unaltered in HD-PBMCs treated in vitro with Venetoclax or 5-Azacytidine, in naïve AML patients’ PBMCs or in PBMCs from AML patients under respective treatment. In addition, ICT01-mediated activation of g9d2 T-cells improved resistance to Venetoclax-induced cell death, which is otherwise pronounced in resting g9d2 T-cells. Next, we identified several stress ligands to be upregulated on AML cell lines and primary AML blasts upon treatment with Venetoclax, 5-Azacytidine or the combination. Consequently, pre-treatment of AML (cell lines and primary) with Venetoclax, 5-Azacytidine or the combination improved g9d2 T-cell killing capacities, which confirms previous findings on NK- and T-cells and adds further mechanistic insights. Finally, activation of g9d2 T-cells with ICT01 in combination with Venetoclax and 5-Azacytidine treatment further increased apoptosis of AML cells.

Conclusions These results demonstrate the ability of Venetoclax and 5-Azacytidine to potentiate ICT01-mediated killing of AML cells by γ9δ2 T-cells and support clinical evaluation of this combination as a novel therapeutic approach for AML patients.

References

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