NKG2D is a C-type lectin-like transmembrane activating receptor present on the surface of natural killer (NK) cells, NKT cells, CD8+γδT cells and certain subsets of CD4+T cells. In humans, there are two families of NKG2D ligands (NKG2DL): the MHC class I chain-related protein A (MICA) and B (MICB) family, and the UL16-binding proteins (ULBPs) family, also known as retinoic acid early transcript 1 (RAET1) proteins, which comprises six members (ULBP1 – 6). NKG2DL biogenesis is stimulated in cells under stress conditions as viral infection, cellular senescence and tumorigenesis. However, NKG2DL expression on healthy and unstressed cells has also been reported as dendritic cells, activated T cells, normal myelomonocytic cells and epithelial cells of gastric mucosa. NKG2DL are expressed on various tumor types including different pediatric solid and hematological malignancies, providing suitable targets for cancer therapy. Ligand engagement of human NKG2D triggers the phosphorylation of the signaling adaptor protein DNAX-activating protein of 10kDa (DAP10) and the recruitment of either phosphatidylinositol-3-kinase (PI3K) or a Grb2-Vav1 complex, both of which are required for full activation. NKG2D activation on NK cells results in cytokine secretion (including IFN-γ, GM-CSF and MIP-1b) and exocytosis of cytotoxic granules. NKG2D expression can be modulated by different regulatory mechanisms. For instance, IL-4 and TGF-β decrease NKG2D surface expression and, thereby, NKG2D-mediated cytotoxic function. However, several cytokines as IL-2, IL-7 and IL-15 induce NKG2D expression. The activating receptor NKG2D is particularly relevant for cancer immunosurveillance. For instance, interaction between NKG2DL and NKG2D receptor is essential for NK-cell elimination of osteosarcoma tumor-initiating cells. However, tumor cells may develop immune scape strategies like ligand release (NKG2DLs) mediated by distinct matrix metalloproteinases, which reduces NKG2DL expression and may cause NKG2D receptor downregulation, thus limiting their clinical efficacy. In addition, chronic exposure of NKG2D receptor with membrane-bound ligand reduces NKG2D-mediated cytotoxic activity upon a trogocytosis process which produce induced-self recognition named fratricide. The use of NKG2D-CAR on memory (45RA-) T cells may overcome these limitations. In our experience, we have observed preclinical evidence of the efficacy of a NKG2D-CAR expressed on memory (45RA-) T cell based therapy as treatment for different pediatric cancers. In addition, we have developed a protocol to expand clinical grade NKG2D-CAR on memory (45RA-) T cells in a fully automated closed system, CliniMACS Prodigy. Finally, we report the safety of NKG2D-CAR on memory (45RA-) T cells infusions in two pediatric patients suffering from refractory acute leukemia.

CD45RA+ cells were depleted from non-mobilized apheresis by using CD45RA microbeads and CliniMACS device. NKG2D-CAR T cells were obtained by lentiviral (NKG2D-41BB-CD3z) transduction of CD45RA-T cells (MOI=2). The expression of NKG2DL was analyzed in primary samples of osteosarcoma (22 patients), as well as in the peripheral blood or bone marrow samples from 97 leukemia patients (AML=13, B-ALL=52 and T-ALL=19), at different status of the disease (Diagnosis, Remission, Relapse/refractory). Three osteosarcoma and ten leukemia cell lines were also analyzed for NKG2DL expression by flow cytometry (FCM). Cytotoxicity of NKG2D-CAR on memory (45RA-) T cells against pediatric cancer cells was evaluated in vitro by performing 4-hours Europium-TDA assays. For the in vivo orthotopic model, 531MII YFP-luc human osteosarcoma cells were used as targets in NOD-scid IL2Rgnull mice. The effect of sNKG2DL on NKG2D-CAR T cells was explored by culturing with different concentrations of sNKG2DL for 7 days. Cell proliferation and CAR downregulation were measured by FCM. The production of IFN-γ and TNF-α was measured in the supernatants by ELISA. The effect on cytotoxicity was evaluated in a 2 hours-degranulation assay by co-culturing sNKG2DL pretreated NKG2D-CAR T cells against K562 cell line. An automated manufacturing protocol to obtain clinical-grade NKG2D-CAR memory (45RA-) T cells were developed using CliniMACS Prodigy instrument. NKG2D-CAR memory (45RA-) T cells were infused into two patients. Patient #1 suffered from r/r biphenotypic ALL. She received two cycles of NKG2D CAR memory T cells infusions. In the first cycle, a total of 3×107 cells/kg were infused into three doses with no conditioning. In the second cycle, a total of 4×107 cells/kg were administered into two doses after lymphodepleting conditioning with Cy/Flu and low dose bortezomib. Patient#2 suffered from r/r B-ALL. She received a single dose of 1×107 cells/kg after lymphodepleting conditioning and low dose bortezomib.

NKG2DL were expressed in pediatric primary tumor cells and cell lines. We observed that NKG2DL expression changed with disease status. In osteosarcoma samples, metastatic tumors showed significantly higher expression of MICA, MICB and ULBP1. In leukemia samples, we found a trend of NKG2DL to decrease at diagnosis and relapse/refractory compared to remission. NKG2D-CAR on memory (45RA-) T cells were cytotoxic against 3 osteosarcoma cell lines and 8/10 leukemia cell lines with specific lysis over 50%. Myeloid and T-ALL cell lines were more susceptible to NKG2D-CAR T cells (specific lysis ranging from 50-78%) compared to B-ALL cell lines (19-52%). NKG2D-CAR memory (45RA-) T cells had considerable antitumor activity in a mouse model of human osteosarcoma, whereas untransduced T cells were ineffective. Physiological concentrations of sNKG2DL increased NKG2D-CAR expression. However, supra-physiological levels of sNKG2DL decreased NKG2D-CAR expression up to 5 times and increased cell proliferation up to 4 times. The effects of sNKG2DL were dose-dependent and attenuated by IL-2. NKG2D-CAR memory (45RA-) T cells manufactured at CliniMACS Prodigy expanded up to 2076±697 million with 77,8±20% NKG2D-CAR expression and 76±10% viability. Harvested CAR T cells showed 90±14% of specific lysis against Jurkat cells and 31±16% against 531MII osteosarcoma cell line. Vector copy number was≤5 in all validations except for one. CGH and karyotype showed no genetic alterations. Free viral particles were undetectable in the supernatants. No overexpression of myc/tert was found except for one validation. Endotoxins were≤0.25EU/ml. NKG2D CAR memory T cells infusions were well tolerated by patients, although a therapeutic effect was only observed in Patient#2.

NKG2D-CAR expression on memory (CD45RA-) T cells are cytotoxic against pediatric tumor cells in vitro and in vivo, and thus could be a novel therapeutic approach for children suffering from cancer. Supraphysiological levels of sNKG2DL may downregulate NKG2D-CAR expression, that is softened by IL-2. The changes observed in NKG2DL surface expression at the different stages of the disease could be related to ligands shedding and immune escape. Automated manufacturing of clinical-grade NKG2D-CAR on memory (CD45RA-) T cells using CliniMACS Prodigy is feasible and reproducible. NKG2D CAR memory (CD45RA-) T cells infusions are essentially safe and therapeutic effect mainly relies on lymphodepleting conditioning, NKG2DL expression on tumor cells and sNKG2DL.