5-Azacytidine and Venetoclax Combination Boost Vγ9Vδ2 T Cell Effector Functions

Introduction

Key role of Vγ9Vδ2 T cells has recently emerged in anti-tumor immunity. However, efficacy results of clinical studies remain partial and attempts are being made to improve Vγ9Vδ2 T cells-based immunotherapy.

Although Vγ9Vδ2 T cells mediate its effector functions against a wide range of tumor cells, we have identified acute myeloid leukemia (AML) as a prime target for the cytotoxic potential of Vγ9Vδ2 T cells in preclinical studies (Gertner-Dardenne et al., 2012; Benyamine et al., 2017). In addition, Vγ9Vδ2 T cells are quite resistant to senescence in comparison to other cytotoxic effectors, and they are able to recognize target cells inherently of MHC recognition. These unique features make them ideal target in the elderly AML population. However, few data are available on the impact of Venetoclax or 5-azacytidine on immune cells functions including Vγ9Vδ2 T cells.

Recent important discovery on Vγ9Vδ2 T cell biology have been made in the last two decades. The antitumor activity of Vγ9Vδ2 is in fact triggered by pAg produced trough the mevalonate pathway binding to the intracellular domain of butyrophillin 3A1. Importantly, targeting BTN3A using an activating monoclonal Ab improved Vγ9Vδ2 T cells functions in vitro and in vivo AML models (Benyamine et al., 2017). A humanized form of the activating BTN3A molecule called ICT01, is being evaluated in the multicentric Phase 1/2a EVICTION trial in solid tumors and hematologic malignancies (NCT04243499).

Aim of this study was to evaluate the preclinical relevance of the combination of two major products used in hematological malignancies 5-Azacytidine and Venetoclax, with an autologous Vγ9Vδ2 T cells-based immunotherapy targeting BTN3A molecules.

Patients and methods

PBMCs from healthy volunteers (HV) were delivered by the French blood bank (EFS). Fresh expanded or thawed Vγ9Vδ2 T cells were tested for their viability using Cell titer glo assay. Cell lines derived from acute myeloid leukemia (GF-D8, HL-60, Kasumi-3, MOLM-13, SKM-1, THP-1, KG-1, U93, NB-4) and PBMCs from newly diagnosed adults AML patients were evaluated for their apoptosis after coculture with allogenic Vγ9Vδ2 T cells. Patients were enrolled within the HematoBio cohort (PI Pr. N. Vey). Extensive phenotyping of patient cells was performed by K-Cytof on PBMCs of 21 AML patients.

Results

First, we found that relapsed AML patients exhibit important alterations with a distinct phenotypical shift by comparison to HV and AML patients in complete remission.

We show that pretreatment of AML cell lines and of primary AML blasts with 5-Azacytidine and Venetoclax combination enhanced their Vγ9Vδ2 T cells-mediated apoptosis, both in control condition and after treatment with ICT01. We observed distinct susceptibility profiles after Venetoclax or 5-Azacytidine depending on the target. Interestingly, after 5-Azacytidine we found a dose-dependent increase of BTN2A surface expression on AML cell lines (n=9) and on primary AML blasts (n=19).

We next evaluated impact of 5-Azacytidine and Venetoclax on expanded Vγ9Vδ2 T cells. We observed that pretreatment of Vγ9Vδ2 T cells with Venetoclax had a low impact on viability of expanded Vγ9Vδ2 T cells as compared to AML cell lines, and low dose of Venetoclax (100-400nM) even empowered their cytotoxic functions against AML cell lines and primary AML blasts, including after ICT01 treatment.

Discussion

Vγ9Vδ2 T cell alterations found in AML at diagnosis confirm their prognostic importance and the need to study impact of the current therapies on these cells.

Our results highlighted that Vγ9Vδ2 T cell functions could be enhanced after Venetoclax and 5-Azacytidine and that this combination could potentiate Vγ9Vδ2 T cells-based immunotherapy based on BTN3A targeting.

Our data thus provide a rationale for combinatorial strategy of 5-Azacytidine and Venetoclax, with a Vγ9Vδ2 T cells-based immunotherapy.

Wieduwild:Imcheck Therapeutics: Current Employment, Honoraria. Garulli:Imcheck Therapeutics: Current Employment, Honoraria. Bourass:Imcheck Therapeutics: Current Employment, Honoraria. De Gassart:Imcheck Therapeutics: Current Employment, Honoraria. Vey:Roche: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria; BMS: Honoraria; Amgen: Honoraria. Madakamutil:Imcheck Therapeutics: Current Employment, Honoraria. Olive:Emergence Therapeutics: Consultancy, Current equity holder in publicly-traded company; Alderaan Biotechnology: Consultancy, Current equity holder in publicly-traded company; Imcheck Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.