Manufacturing development and clinical production of NKG2D chimeric antigen receptor-expressing T cells for autologous adoptive cell therapy

Background aims. Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CART cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3 xi cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins. Methods. CART cells were infused fresh after a 9-day process wherein OKT3-activated T cells were genetically modified with replication-defective gamma-retroviral vector and expanded ex vivo for 5 days with recombinant human interleukin-2. Results. Despite sizable interpatient variation in originally collected cells, release criteria, including T-cell expansion and purity (median 98%),T-cell transduction (median 66% CD8(+)T cells), and functional activity against NKG2D ligand-positive cells, were met for 100% of healthy donors and patients enrolled and collected. There was minimal carryover of non-T cells, particularly malignant cells; both effector memory and central memory cells were generated, and inflammatory cytokines such as granulocyte colony-stimulating factor, RANTES, interferon-gamma and tumor necrosis factor-alpha were selectively up-regulated. Conclusions. The process resulted in production of required cell doses for the first-in-human phase I NKG2D CART clinical trial and provides a robust, flexible base for further optimization of NKG2D CAR .cell manufacturing.