Redirecting T Cells to Glypican-3 with 4-1BB Zeta Chimeric Antigen Receptors Results in Th1 Polarization and Potent Antitumor Activity - PubMed Skip to main page content
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. 2017 May;28(5):437-448.
doi: 10.1089/hum.2016.025. Epub 2016 Aug 16.

Redirecting T Cells to Glypican-3 with 4-1BB Zeta Chimeric Antigen Receptors Results in Th1 Polarization and Potent Antitumor Activity

Wenpeng Li  1   2   3 , Linjie Guo  1   2   3 , Purva Rathi  4 , Ekaterina Marinova  1   2   3 , Xiuhua Gao  1   2   3 , Meng-Feng Wu  4 , Hao Liu  4 , Gianpietro Dotti  5 , Stephen Gottschalk  1   2   3   6 , Leonid S Metelitsa  1   2   3   6 , Andras Heczey  1   2   3   4
Affiliations

Redirecting T Cells to Glypican-3 with 4-1BB Zeta Chimeric Antigen Receptors Results in Th1 Polarization and Potent Antitumor Activity

Wenpeng Li et al. Hum Gene Ther. 2017 May.

Abstract

T cells engineered to express CD19-specific chimeric antigen receptors (CARs) have shown breakthrough clinical successes in patients with B-cell lymphoid malignancies. However, similar therapeutic efficacy of CAR T cells in solid tumors is yet to be achieved. In this study we systematically evaluated a series of CAR constructs targeting glypican-3 (GPC3), which is selectively expressed on several solid tumors. We compared GPC3-specific CARs that encoded CD3ζ (Gz) alone or with costimulatory domains derived from CD28 (G28z), 4-1BB (GBBz), or CD28 and 4-1BB (G28BBz). All GPC3-CARs rendered T cells highly cytotoxic to GPC3-positive hepatocellular carcinoma, hepatoblastoma, and malignant rhabdoid tumor cell lines in vitro. GBBz induced the preferential production of Th1 cytokines (interferon γ/granulocyte macrophage colony-stimulating factor) while G28z preferentially induced Th2 cytokines (interleukin-4/interleukin-10). Inclusion of 4-1BB in G28BBz could only partially ameliorate the Th2-polarizing effect of CD28. 4-1BB induced superior expansion of CAR T cells in vitro and in vivo. T cells expressing GPC3-CARs incorporating CD28, 4-1BB, or both induced sustained tumor regressions in two xenogeneic tumor models. Thus, GBBz CAR endows T cells with superior proliferative potential, potent antitumor activity, and a Th1-biased cytokine profile, justifying further clinical development of GBBz CAR for immunotherapy of GPC3-positive solid tumors.

Keywords: T-cell therapy; chimeric antigen receptor; glypican-3; hepatoblastoma; hepatocellular carcinoma; immunotherapy; rhabdoid tumor.

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Conflict of interest statement

The Center for Cell and Gene Therapy has a research collaboration with Cell Medica and Bluebird Bio. S.G., L.M., G.D., W.L., and A.H. have patent applications in the field of T-cell and gene-modified T-cell therapy for cancer.

Figures

<b>Figure 1.</b>
Figure 1.
Generation of glypican-3 chimeric antigen receptor (GPC3-CAR) T cells. The GPC3-specific single chain variable fragment (scFv) derived from GC33 monoclonal antibodies was cloned in frame into retroviral vectors encoding the immunoglobin 1 (IgG1) short hinge, the CD28 transmembrane domain and a CD3ζ signaling domain with or without the costimulatory endodomains derived from CD28, 4-1BB, or their combination. (a) Schematic map of CAR constructs. (b, c) GPC3-CAR cell surface expression determined by flow cytometry for 1 representative donor and summary data for 10 independent donors (mean and standard deviation [SD]: Gz, 79.2 ± 11.13; G28z, 69.2 ± 12.97; GBBz, 73.2 ± 14.9; G28BBz, 65.8 ± 11.9). Parental T cells are shown as controls. No difference was detected between the expression levels of GPC3 CARs (analysis of variance [ANOVA]).
<b>Figure 2.</b>
Figure 2.
GPC3-CAR T cells recognize and kill GPC3-positive tumor cells. Tumor cell lysis was measured with standard 4 h chromium 51 (Cr) release assay at indicated effector to target ratios against GPC3-positive solid tumor cell lines HepG2, Huh-7, Hep3B, and G401. GPC3-negative A549 cell line served as negative control, and A549, genetically modified to express GPC3 (A549.GPC3), as positive control. (a–f) Combined results of sux independent experiments. Nontransduced T cells and T cells expressing CAR-GD2 were used as controls. GPC3-CAR T cells recognized and killed GPC3-positive cell lines (GPC3-CAR T cells vs. controls: p < 0.001 for all GPC3-positive targets) but did not lyse GPC3-negative A549.
<b>Figure 3.</b>
Figure 3.
Differential cytokine production of GPC3-CAR T cells dependent on costimulatory endodomains. GPC3-positive HepG2, Huh-7, Hep3B, and G401 cells were cocultured with GPC3-CAR T cells for 24 h at 1:1 ratio and indicated cytokine levels in tissue culture supernatant were measured using Luminex. To correct for donor to donor variability of absolute cytokine concentrations, the data were normalized as a ratio relative to the maximum in each experiment, with the maximal cytokine level in each experiment being 1 (formula: sample value / maximum value of the experiment for the tested cytokine; 3–5 independent donors for all GPC3 CAR constructs). Mean and SD are shown. *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA with Bonferoni post-test analysis. ns, not significant (p ≥ 0.05).
<b>Figure 4.</b>
Figure 4.
4-1BB improves survival of GPC3-CAR T cells after repeated stimulation by GPC3-positive target in vitro. GPC3-positive Huh-7 was cocultured at 1:1 ratio with GPC3-CAR T cells and cells were replated every 3 days with fresh tumor cells without the addition of cytokines. (a) Carboxyfluorescein succinimidyl ester dilution of proliferating GPC3 CAR T cells on day 3. (b) Staining of GPC3-CAR T cells with 7-amino-actynomycin on day 7. (c) Combined results from four experiments measuring the absolute number of CAR-positive T cells at indicated time points. For nontransduced T cells, absolute T cell number is shown. Day 7: G28z mean 3.2, SD 0.021; GBBz mean 6.0, SD 0.25; G28z vs. GBBz p < 0.001. Day 10: G28z mean 4.9, SD 0.19; GBBz mean 7.2, SD 0.54; G28z vs. GBBz p < 0.001). (d) Area under the curve (AUC) of cell number of GPC3 CAR T cells and controls are shown (mean and SD): Gz vs. GBBz p < 0.001; Gz vs. G28BBz p < 0.001; G28z vs. GBBz p = 0.002; G28z vs. G28BBz p = 0.001.
<b>Figure 5.</b>
Figure 5.
4-1BB improves the expansion of GPC3-CAR T cells induced by GPC3-positive target in vivo. NOD/SCID/IL2γnull (NSG) mice were injected with 2 × 106 GPC3-positive Huh-7 tumor cells intraperitoneally (IP) followed by injection of 5 × 107 green fluorescent protein/firefly luciferase fusion gene (eGFP.Ffluc)–expressing GPC3-CAR T cells intravenously (IV) on day 21. Parental T cells and CAR-CD19 T cells coexpressing eGFP.Ffluc served as controls. (a) Serial bioluminescence images of mice at indicated time points. (b) Mean photon count with SDs of mice groups shown at indicated time points. (c) Bioluminescence on day 6 of indicated treatment groups. Bioluminescence from each mouse (n = 5 per group) with SD from the mean is shown (Control, 4.01 × 105 ± 90.41 × 104; CD19-CAR, 4.81 × 105 ± 1.11 × 105; Gz, 8.63 × 105 ± 1.86 × 105; G28z, 3.86 × 106 ± 8.41 × 105; GBBz, 1.16 × 107 ± 2.5 × 106; and G28BBz, 1.5 × 107 ± 2.56 × 106). *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA with Bonferoni post-test analysis.
<b>Figure 6.</b>
Figure 6.
GPC3-CAR T cells eliminate HCC and MRT xenografts in vivo. (a) NSG mice (n = 5 per group) were injected with 2 × 106 GPC3-positive Huh-7.Ffluc tumor cells IP followed by injection of 1 × 107 GPC3-CAR T cells and controls (parental T cells and CAR-GD2 T cells) IV on day 14. Serial tumor bioluminescence imaging of mice at indicated time points. (b) Tumor bioluminescence as mean photon count with standard deviations of mice groups. Control groups vs. CAR T cell groups p < 0.001 in weeks 4 and 5. (c) Kaplan–Meier survival curve of tumor-bearing mice after treatment with GPC3-CAR T cells. Control groups vs. GPC3-CAR T cell groups p < 0.001 by Gehan-Breslow-Wilcoxon test. (d) NSG mice (n = 4–5 per group) were injected with 5 × 106 GPC3-positive G401.Ffluc tumor cells IP followed by IV injection of GPC3-CAR T cells and controls (parental T cells and CAR-GD2 T) cells on day 21. Serial tumor bioluminescence imaging of mice at indicated time points. (e) Mean photon count with standard deviations of mice groups shown at indicated time points. G28z, GBBz, and G28BBz vs. Gz p = 0.0083 (AUC). (f) Survival of tumor-bearing mice after GPC3 CAR T cell treatment. Control groups vs. GPC3-CAR T cell groups p < 0.001 by Gehan-Breslow-Wilcoxon test.
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