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

Cytotherapy. 2018 Jul;20(7):952-963. doi: 10.1016/j.jcyt.2018.05.001. Epub 2018 Jun 29.


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 CAR T cells for treatment of acute myeloid leukemia and multiple myeloma ( 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.

Methods: 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.

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

Conclusions: 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.

Keywords: CAR T; Good manufacturing practice cell therapy; NKG2D; acute myeloid leukemia; chimeric antigen receptor; gamma retrovirus; multiple myeloma.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cell Proliferation
  • Clinical Trials as Topic
  • Cytokines / metabolism
  • Humans
  • Immunotherapy, Adoptive*
  • Ligands
  • NK Cell Lectin-Like Receptor Subfamily K / metabolism*
  • Phenotype
  • Receptors, Antigen, T-Cell / immunology
  • Receptors, Chimeric Antigen / metabolism*
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology*
  • Transplantation, Autologous


  • Cytokines
  • Ligands
  • NK Cell Lectin-Like Receptor Subfamily K
  • Receptors, Antigen, T-Cell
  • Receptors, Chimeric Antigen

Associated data