Prophylactic infusion of allogeneic double-negative T cells as immune modulators to prevent relapse in high-risk AML patients after allo-HSCT: A phase I trial.

Abstract

2532 Background: Our previous study demonstrated that double-negative T cells (DNTs) hold potential for treating relapsed or refractory acute myeloid leukemia (r/r AML) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). In a first-in-human Phase I trial (ChiCTR-IPR-1900022795), we reported a complete response (CR) rate of 50% (5/10) with a favorable safety profile. This Phase I/II study aims to evaluate the safety and efficacy of off-the-shelf allo-DNTs in preventing relapse in AML patients following allo-HSCT. Methods: Six high-risk AML patients undergoing allo-HSCT were enrolled and assigned to two dosage groups: 1×10^8 DNTs/kg and 1.5×10^8 DNTs/kg. Each patient received three infusions at one-month intervals without prior lymphodepleting chemotherapy. The median time from transplantation to the first infusion was 3.1 months. Primary endpoint was the occurrence of adverse events and dose-limiting toxicities, while the secondary endpoint was cumulative incidence of relapse (CIR). GMP-grade DNTs were expanded ex vivo from healthy donor PBMCs and cryopreserved in liquid nitrogen until infusion. Results: As of January 20, 2025, with a median follow-up of 17.55 months post-HSCT, four of six patients (66.7%) remained in MRD-negative CR, with the longest recurrence-free survival exceeding 17 months. The two relapsed patients both carried high-risk genetic mutations (TP53 mutation) and were MRD-positive prior to transplantation. They succumbed at 11.4 and 14.2 months post-HSCT respectively. Donor-derived DNTs were detectable in peripheral blood shortly after each infusion, peaking at 1–4 days and persisting for up to 28 days. In two patients with MRD-negative CR, infused DNTs remained detectable for up to 360 days post-infusion. Elevated levels of IFN-γ, IL-6, and IL-10 post-infusion indicated immune activation. Importantly, no dose-limiting toxicities, neurotoxicity, cytokine release syndrome greater than Grade 2, or graft-versus-host disease were observed. In contrast to the two relapsed patients, MRD-negative CR patients showed expanded levels of CD4+, CD8+, and DNT cells, particularly those with the effector memory T cell phenotype. Both the infused DNTs and the recipient's CD4+ and CD8+ T cells in these patients secreted higher levels of granzymes A and K. To investigate the interaction between CD8+ T cells and allo-DNTs in MRD-negative CR patients, co-culture experiments were conducted. CD8+ T cells exhibited an increase in the secretion of granzyme B and IFN-γ within 3–4 days. Transcriptome sequencing and multi-cytokine analyses revealed strong immune activation. Conclusions: The dual ability of DNTs to suppress GvHD while preserving the graft-versus-leukemia effect, along with its potential for off-the-shelf availability, makes it a transformative therapy in the post-transplant setting. Clinical trial information: NCT05858814 .