Long-term outcomes of double-negative T cell therapy in AML patients with relapse post allo-HSCT

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the principal curative approaches in the treatment of acute myeloid leukemia (AML); however, relapse is the main cause of treatment failure following transplantation. CD3+CD4-CD8- double-negative T cells (DNTs) are unconventional mature T cells with potent anti-leukemia effects with “off-the-shelf” potential. Here, we present the long-term outcomes up to five years from a Phase 1 clinical trial. A total of 10 patients with relapsed AML after allo-HSCT were enrolled and received three planned doses of allo-DNTs given 1 week apart. No signs of graft-versus-host disease (GVHD) or severe neurotoxicity were observed in these patients, although all patients exhibited symptoms of grade 1 or 2 cytokine release syndrome. Of 10 patients with relapsed or refractory AML who received DNTs administration, 4 are disease-free at their last follow-up, 5 years after infusion (5-year overall survival rate was 40%). In addition, single-cell RNA sequencing of bone marrow mononuclear cells reveals a favorable cellular signature for CD8+T cells and natural killer (NK) cells activation and anti-tumor for these 4 patients. Moreover, using Bio-Plex Pro Human Cytokine Screening, we identified significantly elevated levels of IL-7 and IL-15 in the bone marrow microenvironment of the 4 patients who achieved complete response, compared to the relapsed group. Importantly, donor-derived DNT cells could still be detected in 2 patients even at 5 years after DNT therapy. Using flow cytometry, we identified that DNTs from these 2 patients were mainly CD45RA+CD45RO-, with higher expression of CD69 and lower expression of NKG2A, compared with the other 2 patients without donor-derived DNT cells persisting. Taken together, our findings establish third-party DNT therapy as a viable therapeutic candidate for patients exhibiting unfavorable prognoses and limited treatment alternatives, offering long-term outcomes.