Purpose: NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8+ T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D–NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated in vitro and in vivo the safety and cytotoxic capacity against osteosarcoma cells of CD45RA− memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR). Experimental Design: CD45RA− cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. In vitro cytotoxicity of NKG2D-CAR+ CD45RA− T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the in vivo orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rgnull mice. Results: Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA− cells. Genetic stability was preserved in transduced cells. In vitro, NKG2D-CAR+ memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR+ memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective. Conclusions: Our results demonstrate NKG2D-4-1BB-CD3z CAR–redirected memory T cells target NKG2DL-expressing osteosarcoma cells in vivo and in vitro and could be a promising immunotherapeutic approach for patients with osteosarcoma. Clin Cancer Res; 23(19); 5824–35. ©2017 AACR.

Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors. NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3ζ signaling domain. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients' own T cells, disease progression, and death prior to return of engineered cells. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. host reactions may occur. To avoid such adverse effects, we used CD45RA⁻ memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. CD45RA⁺ fraction was depleted from healthy donors' non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. A total of 10⁸ CD45RA⁻ cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. Then, we used NKG2D-CD8TM-4-1BB-CD3ζ lentiviral vector for cell transduction (MOI = 2). NKG2D CAR T cells expanded between 10 and 13 days. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency of Medicines and Medical Devices (AEMPS) for the manufacture of investigational advanced therapy medicinal products (ATMPs). We performed four validations. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. CAR T cell final products met release criteria, except for one showing myc overexpression and another with viral copy number higher than five. Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies.

The CD45RA T cell depletion (TCD) method has been used to deplete naive T cells, preventing graft-versus-host disease (GVHD) but preserving memory cells, providing immediate functional T cells with anti-infection, antileukemia, and antirejection effects. We describe a series of 25 consecutive high-risk patients with leukemia who received haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with CD45RA TCD. Each patient received 2 cell products: 1 created by CD34 positive selection and the other through CD45RA depletion from the CD34 negative fraction by a CliniMACS device. CD45RA-depleted haplo-HSCT was well tolerated, with rapid engraftment and low risk of severe acute GVHD and chronic GVHD. Although this treatment achieved a good control of viral reactivations, such as cytomegalovirus and adenovirus, we observed an unexpectedly high rate of limbic encephalitis due to human herpesvirus-6 (HHV-6; 8 cases). Characteristically, the infection appeared early in almost all patients, just after the engraftment. Although no patient died from encephalitis, 1 patient showed neuropsychological sequelae, and another experienced secondary graft failure just after the HHV-6 reactivation.

Key Points: IL-7R expression is a functional biomarker of T-ALL cells with leukemia-initiating potential and plays a crucial role in T-ALL pathogenesis. Targeting IL-7R–mediated signaling hampers leukemia-initiating activity and progression of human T-ALL.