Allogeneic Human Double Negative T Cells as a Novel Immunotherapy for Acute Myeloid Leukemia and Its Underlying Mechanisms

Abstract

To explore the potential of expanded healthy donor-derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients. Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry-based killing assays and xenograft models against AML patient blasts and healthy donor-derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays. Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples , and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor-unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands' expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1-blocking antibodies. This study supports healthy donor-derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. .

Developing Allogeneic Double-Negative T Cells as a Novel Off-the-Shelf Adoptive Cellular Therapy for Cancer

“…DNT expansions were done as previously described (20) under GMP conditions with some modifications. Briefly, CD4 þ and CD8 þ T-cell-depleted peripheral blood mononuclear cells (PBMC) were cultured on anti-CD3 antibody-coated plates (GMP grade OKT3; Miltenyi Biotec) for 3 days in AIM-V (Thermo Fisher) or GT-551 (Takara Bio) media with 250 IU/mL of IL2 (Proleukin, Novartis Pharmaceuticals); soluble anti-CD3 antibody and IL2 were added to the cultures.…”

“…DNTs (2 Â 10 7 ) were injected intravenously on days 3, 6, and 10 after cancer cell injection. MV4-11-infused mice were sacrificed 2 weeks after the last DNT injection, and the engraftment of MV4-11 in the bone marrow was determined using flow cytometry as described previously (20). EBV-LCL-infused mice were euthanized when their body weight decreased by 20%.…”

“…Expansion of a cellular product under GMP conditions is required before its clinic application. Our previous studies were conducted with DNTs expanded using research grade reagents containing xenogeneic components (20,21). Use of animalderived supplements restricts the use of final products for patient treatment due to risks associated with the xenogeneic additives (25).…”

“…Double-negative T cells (DNT) are mature T cells that comprise 3% to 5% of peripheral T cells and is defined by expression of CD3 in the absence of CD4, CD8, and iNKT cell markers (19)(20)(21)(22). Recently, we demonstrated that healthy donor (HD) derived allogeneic DNTs target acute myeloid leukemia (AML) in vitro and in patient-derived xenograft models (20) and have synergistic anticancer activities with conventional chemotherapies (19). Importantly, allogeneic DNTs are not toxic toward normal cells and infusion of DNTs into mice does not cause xenogeneic GvHD (20).…”

“…Recently, we demonstrated that healthy donor (HD) derived allogeneic DNTs target acute myeloid leukemia (AML) in vitro and in patient-derived xenograft models (20) and have synergistic anticancer activities with conventional chemotherapies (19). Importantly, allogeneic DNTs are not toxic toward normal cells and infusion of DNTs into mice does not cause xenogeneic GvHD (20). Although these characteristics support the use of allogeneic DNTs as an off-the-shelf product, other required features such as expansion capacity and storage of expanded cells under clinically compliant conditions, as well as their ability to avoid HvG reaction and in vivo persistence have not been addressed previously.…”