Preclinical Evaluation of Combination Allogeneic Gamma Delta (γδ) T Cell Therapy with Azacitidine and Venetoclax for T-Cell Acute Lymphoblastic Leukemia

Outcomes for relapsed/refractory (R/R) T-cell acute lymphoblastic leukemia (T-ALL) remain poor, with long-term survival less than 30%. While allogeneic hematopoietic stem cell transplantation (HSCT) offers a chance for cure, many patients are unable to achieve the necessary disease remission prior to transplantation, limiting its use. Thus, new treatment strategies are urgently needed. Though there has been great success in the implementation of chimeric antigen receptor (CAR) T cell therapy for the treatment of B-cell malignancies, similar strategies have been challenging to adapt to T-ALL due to a lack of known T-ALL specific cell-surface antigens that distinguish malignant from healthy T cells, resulting in CAR T-cell fratricide. Alternative cellular therapy platforms such as the use of allogeneic gamma delta (γδ) T cells offer a solution as a cytotoxic alternative to the traditional alpha beta (⍺β) T cell.

γδ T cells form a unique class of T cell that possess the innate ability to recognize both foreign pathogens and cellular stress antigens and, importantly, can be administered across major histocompatibility complex (MHC)-barriers, permitting their use in the “off-the-shelf” setting. Indeed, our institution has developed a GMP-compliant manufacturing strategy to expand γδ T cells from healthy donor peripheral blood at clinically relevant doses, with our cellular product currently under clinical investigation in the context of neuroblastoma in combination with chemo-immunotherapy [ClinicalTrials.gov Identifier: NCT05400603]. In the leukemia setting, we have previously shown that the proteasome inhibitor bortezomib enhances γδ T cell-mediated killing of T-ALL through the upregulation of NKG2DL stress antigens (Story JY., et al. 2021), although in vivo studies proved to be inconclusive. This present study investigates the hypothesis that the hypomethylating agent azacitidine in combination with the Bcl-2 inhibitor venetoclax, a chemotherapy regimen commonly used for R/R acute leukemias, can enhance γδ T cell-mediated killing of T-ALL through similar mechanisms.

Herein, we found that ex vivo expanded γδ T cells induce significant cell death against three T-ALL cell lines - Jurkat E6-1, MOLT-4, and CCRF-CEM, at increasing effector-to-target (E:T) ratios in a four-hour flow cytometry based co-culture assay examining apoptotic and cell death markers. Azacitidine and venetoclax were both highly cytotoxic against the three T-ALL cell lines, with increased cell death observed when combined with γδ T cells. To investigate mechanisms of enhanced killing, we performed a detailed multi-parametric flow cytometry analysis of T-ALL cell lines to study the effects of azacitidine and venetoclax on cell surface expression of NKG2D ligands, DNAM-1 ligands, Fas, TRAIL receptors, and BTN3A1 - all surface molecules involved in γδ T-cell mediated killing. In a T-ALL xenograft model using the Jurkat E6-1 cell line, we found that one cycle of azacitidine and venetoclax treatment significantly lowered peripheral disease burden compared to vehicle-treated mice (Student's T Test, p=0.0010) as determined by flow cytometric analysis of PBMCs. Furthermore, the addition of γδ T cells to the chemotherapy regimen resulted in an increase in median survival of nine days (37 vs. 28 days), and a greater significance in survival advantage compared to vehicle-treated mice (Mantel-Cox, p=0.007) in contrast to treatment with just chemotherapy alone (Mantel-Cox, p=0.0117). Interestingly, when mice were stratified based on sex, we see a significant survival advantage when female mice are treated with γδ T cells in combination with chemotherapy compared to chemotherapy alone (Mantel-Cox, p=0.0246). This is in contrast to male mice, where we only observe a significant survival advantage when mice are treated with γδ T cells in combination with chemotherapy compared to vehicle-treated mice (Mantel-Cox, p=0.0066), and do not see such survival advantage when males are treated with chemotherapy alone.

Together, these studies demonstrate that the combination of azacitidine, venetoclax and γδ T cells results in increased killing of T-ALL cell lines both in vitro and in vivo. Ongoing studies will further explore prior observed sex-based differences in survival advantage of our xenograft T-ALL model, in addition to completion of safety studies necessary to begin clinical investigation.

No relevant conflicts of interest to declare.