Development of GPC3-Specific Chimeric Antigen Receptor-Engineered Natural Killer Cells for the Treatment of Hepatocellular Carcinoma - PubMed (original) (raw)
Development of GPC3-Specific Chimeric Antigen Receptor-Engineered Natural Killer Cells for the Treatment of Hepatocellular Carcinoma
Min Yu et al. Mol Ther. 2018.
Abstract
Chimeric antigen receptor (CAR)-modified natural killer (NK) cells represent a promising immunotherapeutic modality for cancer treatment. However, their potential utilities have not been explored in hepatocellular carcinoma (HCC). Glypian-3 (GPC3) is a rational immunotherapeutic target for HCC. In this study, we developed GPC3-specific NK cells and explored their potential in the treatment of HCC. The NK-92/9.28.z cell line was established by engineering NK-92, a highly cytotoxic NK cell line with second-generation GPC3-specific CAR. Exposure of GPC3+ HCC cells to this engineered cell line resulted in significant in vitro cytotoxicity and cytokine production. In addition, soluble GPC3 and TGF-β did not significantly inhibit the cytotoxicity of NK-92/9.28.z cells in vitro, and no significant difference in anti-tumor activities was observed in hypoxic (1%) conditions. Potent anti-tumor activities of NK-92/9.28.z cells were observed in multiple HCC xenografts with both high and low GPC3 expression, but not in those without GPC3 expression. Obvious infiltration of NK-92/9.28.z cells, decreased tumor proliferation, and increased tumor apoptosis were observed in the GPC3+ HCC xenografts. Similarly, efficient retargeting on primary NK cells was achieved. These results justified clinical translation of this GPC3-specific, NK cell-based therapeutic as a novel treatment option for patients with GPC3+ HCC.
Keywords: chimeric antigen receptor; glypican-3; hepatocellular carcinoma; natural killer cell.
Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.
Figures
Figure 1
Generation of NK-92/9.28.z Cells Harboring the Second Generation of GPC3-Specific CAR by Lentiviral Vector Transduction (A) Schematic representation of GPC3-specific CAR and the lentiviral vector. The construct consisted of a CD8α signal peptide (SP), a humanized GPC3-specific single-chain variable fragment (scFv) (hu9F2), a CD8α signal hinge region, and CD28 transmembrane region (TM), followed by the intracellular domains of co-stimulatory CD28 and the intracellular domain of CD3ζ. (B) Flow cytometric analysis of CAR expression on the surface of parental NK-92, mock, and NK-92/9.28.z cells with goat anti-human biotin-conjugated anti-Fab antibody followed by PE-conjugated streptomycin. Gating was based on the same cells stained with isotype-matched antibody. Data shown are representatives of experiments with similar results. (C) Western blotting analysis of CAR expression in parental NK-92 and NK-92/9.28.z cells. A CD3ζ-specific antibody was used to detect endogenous and chimeric CD3ζ. Data shown are representatives of at least three experiments with similar results.
Figure 2
Enhanced Cytotoxicities of NK-92/9.28.z Cells against GPC3+, but Not GPC3−, HCC Cell Lines (A) Cytotoxic activity of NK-92/9.28.z, mock, or parental NK-92 cells against GPC3+ (including GPC3+ variants SK-HEP-1/GPC3 and SMMC-7721/GPC3) and negative targets. The effector cells were co-cultured for 6 hr with target cells (1 × 104) at an E:T ratio of 3:1, 1:5:1, and 0.75:1 in a total volume of 100 μL. (B) Cytotoxicity of NK-92/9.28.z, mock, or parental NK-92 cells against Huh-7 at a low E:T ratio while extending the co-culture time to 18 hr. (C) Cytotoxicity of NK-92/9.28.z cells or parental NK-92 cells against Huh-7 under normoxia (20%) or hypoxia (1%). (D) Cytotoxicity of NK-92/9.28.z cells or their counterpart CAR-T cells against Huh-7 in the presence of 5 to 20 ng/mL of human TGF-β1 at an E:T ratio of 3:1 for 24 hr. (E) cytotoxicity of NK-92/9.28.z cells against Huh-7 in the presence of mammalian cell-expressed, soluble GPC3N, GPC3ΔGPI, and BSA at an E:T ratio of 3:1 for 6 hr. NS, not significant. Cytotoxicity was determined by LDH release assays. Data reflect the mean ± SEM of three separate experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with parental NK-92 cells at the same E:T ratios or concentrations.
Figure 3
Phenotypic Characterization and IFN-γ Production of NK-92/9.28.z Cells (A) Flow cytometric analyses of phenotypic characterization of NK-92/9.28.z cells. NK-92/9.28.z or parental NK-92 cells were either cultured with or without Huh-7 or K562 cells for 6 hr, and surface expression of NKp30, NKp44, Nkp46, NKG2D, and CD107a was assessed using mean fluorescence intensity (MFI). Gating was based on the same cells stained with isotype-matched antibody. Data presented are the quantitative data. (B) Representative flow cytometric dot plots illustrating CD107a expression on NK-92/9.28.z or parental NK-92 cells after 6 hr of co-culture with medium, Huh-7, SK-HEP-1, SK-HEP-1/GPC3, and phorbol-12-myristate-13-acetate or ionomycin (PMA/IONO). The same cells stained with isotype-matched controls were used for gating. (C) Depicted data for histograms shown in (B). (D) IFN-γ production by NK-92/9.28.z, mock, or parental NK-92 cells when co-cultured with the indicated HCC cell lines at an E:T ratio of 1.5:1 for 24 hr. (E) Correlation between the IFN-γ production from NK-92/9.28.z cells and the MFI of GPC3 expression on the HCC cell lines. Data presented are the mean ± SD of three separate experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with parental NK-92 cells.
Figure 4
Target-Dependent Growth-Suppressive Effects of NK-92/9.28.z Cells on GPC3-Transfected SK-HEP-1 Tumor Xenografts (A) Growth curves of SK-HEP-1/GPC3 and SK-HEP-1 xenografts treated with NK-92/9.28.z or parental NK-92 cells or PBS (n = 6). Red arrows, days on which the cyclophosphamide pretreatments were delivered; black arrows, days on which the indicated treatments were administered; treatment was repeated every 5–6 days for 4 weeks. (B) Tumor weight of the individual mice from each treatment group the day the experiment was terminated. (C) Accumulation of NK-92/9.28.z cells in SK-HEP-1/GPC3 xenografts. NK-92/9.28.z or parental NK-92 cells were labeled with CFSE and intravenously injected into mice bearing SK-HEP-1/GPC3. After 36 hr, tumors were excised and analyzed for the presence of CFSE-labeled cells. Representative flow cytometric data from one animal of each group are shown (n = 3). (D) Representative tumor sections stained with CD56, Ki67, and cleaved caspase-3 are shown. The specimens were harvested from SK-HEP-1/GPC3 xenografts sacrificed after the study was terminated. Nuclei are stained with hematoxylin. Magnification, 200×. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with mice treated with parental NK-92 cells.
Figure 5
Therapeutic Effectiveness of NK-92/9.28.z Cells against HCC Xenografts with High or Low Endogenous GPC3 Expression (A) Growth curve of Huh-7 subcutaneous xenografts treated with the indicated NK-92 cells or PBS (n = 6). Treatment was repeated every 4 days. (B) Body weights of the individual mice from each treatment group at the endpoint. (C) Growth curve of PLC/PRF/5 xenografts treated with the indicated NK-92 cells or PBS (n = 6). Treatment was repeated every 4–5 days for 4 weeks. (D) Tumor weights of the individual mice from each treatment group at the endpoint. (E) NK-92/9.28.z cells were irradiated with 10 Gy, and viabilities were determined by counting viable cells at different time points using trypan blue exclusion. (F) Tumor-killing activities of irradiated or non-irradiated NK-92/9.28.z cells against PLC/PRF/5 cells varied with time at an E:T ratio of 3:1. (A–D) Red arrows, days on which the cyclophosphamide pretreatments were administered; black arrows, days on which the indicated treatments were delivered. Data are presented as the mean ± SD. NS, not significant. ***p < 0.001, compared with mice treated with parental NK-92 cells. (E and F) Data reflect the mean ± SEM of three triplicates. ***p < 0.001, compared with non-irradiated NK-92/9.28.z cells at the same time or E:T ratios.
Figure 6
Efficient Redirecting of GPC3-Specific CAR in Primary Human NK Cells (A) Representative flow cytometry dot plots illustrate expression of GPC3-specific CAR in untransduced and transduced NK cells. (B) Intracellular IFN-γ in response to SK-HEP-1 and SK-HEP-1/GPC3 was detected with anti-human IFN-γ-PE antibody by flow cytometry. The same cells stained with isotype-matched controls were used for gating. Data shown are representatives of experiments with similar results. (C) Cytotoxicity of untransduced and CAR-expressing NK cells against Huh-7 and HepG2 cells determined by LDH release assays. (D) Cytotoxicity of untransduced and CAR-expressing NK cells against non-transformed PBMCs determined by LDH release assay. Data reflect the mean ± SEM of three triplicates. ***p < 0.001, compared with untransduced NK cells at the same E:T ratios.
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