Combining Allogeneic Gamma Delta T Cells with a Phosphoantigen Prodrug Promotes Apoptosis of Acute Myeloid Leukemia Stem and Progenitor Cells

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.