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

Abstract

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 (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.

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.

Figure 1

Manufacturing process with healthy donors (HD) and donors with malignancies (DM) – pre-clinical results. (A) Manufacturing workflow for production of Mock-treated and NKG2D CAR-transduced T cells. (B) Fold-expansion of NKG2D CAR T cells from Day −4 to 0. Each dot represents a donor. Bars indicate the mean. (C) Viability of NKG2D CAR-transduced T-cell product measured at Day 0 (harvest) by Trypan Blue exclusion. Error bars represent standard deviation (SD). HD RG n=20 runs from 17 donors; HD GMP n=3; DM GMP n=6. IL-2: Interleukin-2; Ab: Antibody; RG: Research-grade manufacturing; GMP: Clinical-grade manufacturing.

Figure 2

NKG2D CAR-transduced T-cell product phenotype and in vitro functional activity – pre-clinical results. Surface expression analysis by flow cytometry of (A) CD3 (open bars), CD4 (black bars) and CD8 (striped bars) in product samples manufactured from HDs (n=17) and DMs (n=6); (B) Transduction efficiency in the final product from a representative healthy donor based on surface vector-driven NKG2D CAR expression on CD8+ T cells. Any expression above 5% Mock background (dashed vertical line) was considered due to vector expression. (C) IFN-γ production by ELISA of Mock-treated (open bars) and NKG2D CAR-transduced T cells (black bars) in response to cells with (RPMI8866) and without (P815) NKG2D ligand expression, incubated with either anti-NKG2D blocking Abs or control IgG Abs. NKG2D CAR-transduced T cells alone were plated as controls. Data shown is from one representative donor. Error bars represent SD of triplicates. ** p<0.01; *** p<0.001; n.s indicates not significant, p> 0.05.

Figure 3

Cytokine secretion by Multiplex assay of Mock-treated and NKG2D CAR-transduced T cells alone or after coculture with ligand-expressing tumor cells RPMI8866. Human Mock-treated T cells (solid bars) or NKG2D CARs-transduced T cells (striped bars) derived from 3 healthy donors were co-cultured with the NKG2D ligand-expressing RPMI8866 cell line. Tumor cells alone are shown in white bars. Culture supernatants were analyzed for the levels of cytokines (A) GM-CSF, (C) TNF-α, (D) IL-5, (E) IL-10, and (F) IL-13, and the chemokine (B) CCL5 (RANTES). Error bars represent SD of triplicate wells within one run. * p<0.05; ** p<0.01; *** p<0.001; n.s indicates not significant, p≥0.05.

Figure 4

Transduction efficiency, phenotype and functional activity of AML and MM-derived NKG2D CAR-transduced T-cell products in clinical trial. (A) Transduction efficiency in products derived from AML (n=7) and MM patients (n=5). (B) Median distribution of differentiation stages by CD45RO and CCR7 expression on Mock-treated and NKG2D CAR-transduced CD4+ (left panel) and CD8+ (right panel) T cells. Naïve: CD45RO− CCR7+; Central Memory (CM): CD45RO+ CCR7+; Effector Memory (EM): CD45RO+ CCR7−; Terminal Effector Memory RA (Temra): CD45RO− CCR7−; (C) IFN-γ production by ELISA of NKG2D CAR-transduced T cells in response to ligand-positive cells (RPMI 8866, Panc-1 and K562 cells) from a representative AML (top panel) and MM patient (bottom panel); P815 cells (ligand negative); or NKG2D CAR-transduced T cells alone. NKG2D CAR-transduced T cells were incubated with blocking Abs (open bars) or control IgG Abs (closed bars). Error bars represent SD of triplicate wells within a single assay. (D) Stability of 5 NKG2D CAR-transduced products based on lymphocyte viability by 7-AAD exclusion (left panel) and vector-specific NKG2D CAR expression on CD8+ T cells (right panel). * p<0.05; ** p<0.01; *** p<0.001; n.s indicates not significant, p≥ 0.05.