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.

This proof-of-concept single-arm open-label phase I clinical trial (NCT02481934) studied the safety and efficacy of multiple infusions of activated and expanded natural killer (NKAE) cells in combination with anti-myeloma drugs in multiple myeloma patients. It included five patients with relapsed or refractory MM who had received two to seven prior lines of therapy; NK cells were expanded for 3 weeks with K562-mb15-41BBL cells. Patients received four cycles of pharmacological treatment with two infusions of 7.5 × 10⁶ NKAEs/kg per cycle. NKAE generation, expansion, and NK monitoring was assessed using flow cytometry. Eighteen clinical-grade NKAE cell GMP-grade products were generated to obtain 627 × 10⁶ NKAEs (range: 315–919 × 10⁶) for the first infusion and 943 × 10⁶ (range: 471–1481 × 10⁶) for the second infusion with 90% (±7%) purity. Neutropenia grade II occurred in two patients and was related to chemotherapy. Of the five patients, four showed disease stabilization before the end of NKAE treatment, and two showed a 50% reduction in bone marrow infiltration and a long-term (>1 y) response. The NKAE cells had a highly cytotoxic phenotype and high cytotoxicity in vitro. Infused NKAE cells were detected in bone marrow and peripheral blood after infusions. Ex vivo expansion of autologous NK cells is feasible, NKAE cells are clinically active and the multiple infusions are well tolerated in patients with relapsed or refractory myeloma.