Efficacy and Safety of Allogeneic Double Negative T Cell As a Cellular Therapy for AML and Its Underlying Mechanism
Authors: Lee Jong Bok , Chen Weihsu Claire , Streck Elena , Kang Sohyeong , Minden Mark D. , Dick John E. , Zhang Li 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is the most common form of adult acute leukemia that is associated with a low long-term survival rate. While chemotherapy achieves remission in more than 70% of the treated AML patients, most relapse due to residual chemotherapy-resistant AML populations. Allogeneic hematopoietic stem cell transplantation reduces the relapse rate and demonstrates the efficacy of a cell-mediated treatment for the chemotherapy-resistant disease. However, its wide application is limited by donor availability and associated toxicity such as graft-versus-host disease (GvHD). Hence, there is a need for a new treatment approach that targets chemotherapy-resistant AML blasts with minimal side effects. The goals of this study were to characterize allogeneic CD3+ CD4-CD8- double negative T cells (DNTs) as a potential new therapy for AML patients, and to dissect its underlying mechanisms.
Using a flow cytometry-based in vitro killing assay, we demonstrated that the allogeneic DNTs expanded from healthy volunteers were cytotoxic against 23/29 primary AML patient blasts in a dose-dependent manner. Thirteen blast samples were obtained from chemotherapy refractory or relapsing AML patients and, of those, 9 were susceptible to DNTs. Further, the average level of specific killing mediated by DNTs against chemotherapy-susceptible and -resistant blasts were comparable (19.30% ± 3.34% and 15.91% ± 3.63%, respectively). The anti-leukemia activity of DNTs was further validated in AML-NSG xenograft models. A single infusion of DNTs into mice pre-engrafted with primary AML blasts from chemoresponsive, chemorefractory, and relapsed patients significantly reduced the leukemia burden. While a single injection of DNTs resulted in a significant reduction of leukemia burden from 30% to 12.8%, it was further reduced to 2.6% after three injections of DNTs. Furthermore, the survival of NSG mice administered with a lethal dose of AML cell line, MV4-11, in DNT-treated group was significantly superior to the untreated group (57% vs. 0% survival on day 34, respectively). Although residual blasts were observed from the DNT -treated group, their susceptibility to DNT cell-mediated cytotoxicity remained comparable to blasts obtained from the PBS-treated group and primary AML blasts initially used for engraftment, suggesting that AMLs do not develop resistance to DNT-mediated cytotoxicity. In contrast to its potent cytolytic activity against leukemic cells, DNTs did not target allogeneic peripheral blood mononuclear cells (PBMC) and hematopoietic stem/progenitor cells (HSPC) in vitro. Administration of allogeneic DNTs into NSG mice engrafted with human HSPC had no effect on the engraftment level of human hematopoietic cells and their differentiation into different lineages. Further, in contrast to human PBMC, infusion of human DNTs did not cause xenogeneic GvHD in mice, collectively demonstrating the selective cytotoxic activity of allogeneic DNTs against leukemic cells. Using blocking assays, we showed that HLA-class I, NKG2D, and DNAM-1 were involved in DNT cell-mediated cytotoxicity against AML, whereas HLA-class II and T cell receptor did not play a significant role. We detected high levels of IFNγ release by DNTs upon encounter of susceptible AML targets. While IFNγ treatment alone did not induce AML cell death, neutralizing IFNγ reduced and pretreating AML cells with recombinant IFNγ increased their susceptibility to DNT cell killing. IFNγ treatment induced higher expressions of NKG2D and DNAM-1 ligands and blocking of NKG2D and DNAM-1 partially abrogated the effect of IFNγ on the AML-DNT interaction. Collectively, these studies demonstrated the safety and efficacy of allogeneic DNT therapy as a potential treatment for AML patients, including those with chemotherapy-resistant leukemia, and revealed the important molecules for the anti-leukemic activity of DNTs.

Disclosures
No relevant conflicts of interest to declare.