1. Volume 26, Issue 2, Pages 366-378 (February 2018)
Development of GPC3-Specific Chimeric Antigen Receptor-Engineered Natural Killer Cells for the Treatment of Hepatocellular Carcinoma 
Min Yu, Hong Luo, Mingliang Fan, Xiuqi Wu, Bizhi Shi, Shengmeng Di, Ying Liu, Zeyan Pan, Hua Jiang, Zonghai Li 
Molecular Therapy 
Volume 26, Issue 2, Pages (February 2018)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. 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.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. 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.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. 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.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. 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.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. 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.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. 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.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions