NKG2D-CAR-T(IBGM)

Despite the successful application of chimeric antigen receptor (CAR)‐T cell therapy in hematological malignancies, the treatment efficacy in solid tumors remains unsatisfactory, largely due to the highly immunosuppressive tumor microenvironment and low density of specific tumor antigens. Natural killer group 2 member D (NKG2D) CAR‐T cells have shown promising treatment effects on several cancers such as lymphoma and multiple myeloma. However, the application and efficacy of NKG2D‐CAR‐T cells in gastric cancer (GC) still needs further exploration. This study identified a novel combination immunotherapy strategy with Dickkopf‐1 (DKK1) inhibition and NKG2D‐CAR‐T cells, exerting synergistic and superior antitumor effect in GC. We show that the baseline expression of NKG2D ligands (NKG2DLs) is at low levels in GC tissues from The Cancer Genome Atlas and multiple GC cell lines including NCI‐N87, MGC803, HGC27, MKN45, SGC7901, NUGC4, and AGS. In addition, DKK1 inhibition by WAY‐262611 reverses the suppressive tumor immune microenvironment (TIME) and upregulates NKG2DL expression levels in both GC cell lines and GC tissues from a xenograft NCG mouse model. DKK1 inhibition in GC cells markedly improves the immune‐activating and tumor‐killing ability of NKG2D‐CAR‐T cells as shown by cytotoxicity assays in vitro. Moreover, the combination therapy of NKG2D‐CAR‐T and WAY‐262611 triggers superior antitumor effects in vivo in a xenograft NCG mouse model. In sum, our study reveals the role of DKK1 in remodeling GC TIME and regulating the expression levels of NKG2DLs in GC. We also provide a promising treatment strategy of combining DKK1 inhibition with NKG2D‐CAR‐T cell therapy, which could bring new breakthroughs for GC immunotherapy.

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 CAR T 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 CD3ζ 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.

CAR T 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.

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-γ and tumor necrosis factor-α were selectively up-regulated.

The process resulted in production of required cell doses for the first-in-human phase I NKG2D CAR T clinical trial and provides a robust, flexible base for further optimization of NKG2D CAR T-cell manufacturing.