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.

Conflict of interest statement

Disclosures:

C.L.S. has patents and financial interests in NK receptor–based CAR therapies. C.L.S. is a scientific founder for Celdara Medical, a consultant, and receives research support from Celdara Medical. These conflicts are managed under the policies of Dartmouth College. M.-L.S. has an immediate family member with financial interests in NK receptor–based CAR therapies. J.M.M., J.R., M.W.F., T.W., and A.S. are employed by Celdara Medical, which has a material financial interest in NK receptor–based CAR intellectual property assigned to the Trustees of Dartmouth College. DEG, SS and FLE are employees of Celyad S.A, which is the clinical trial sponsor. G.D. is currently an employee of Novartis, which has material financial interests in other CAR T cell therapies. S.B., S.N. received salary support through an SBIR grant awarded to Celdara Medical. The other authors have no financial conflicts of interest.

Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Figures

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

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