pCDCAR1 GPC3 h(CD3ζ) (CAR-LC046)

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.1 Flow analysis of GPC3-specific CARs expression on human T cells transduced with lentiviral particles.

GPC3-specific CARs expression on human T cells transduced with lentiviral particles were analyzed using flow cytometry by detection of GFP autofluorescence.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.2 Antigen-specific reproliferation analysis of αGPC3-28BBZ CAR-T.

In vitro expansion of T cells with stimulation of αCD3/αCD28-coated magnetic beads on day 0 and transduction with the indicated CARs on day 1. Cultured 2 weeks. On day 18, T cells were restimulated by aK562-64/86 with coexpression of the extracellular domain of CD64 and CD86, in the presence of OKT3 (100 ng/mL) and recombinant human IL2 (300 U/mL). D, on day 18, the general or antigen-specific reproliferation of αGPC3-28BBZ ransduced T cells were quantified.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.3 IL2 produced by GPC3-targeted CAR T cells cocultured with various HCC cell lines.

IL2 were secreted by the indicated genetically modified T cells cocultured with various HCC cell lines for 24 hours.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.4 IFNγ produced by GPC3-targeted CAR T cells cocultured with various HCC cell lines.

IFNγ were secreted by the indicated genetically modified T cells cocultured with various HCC cell lines for 24 hours.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.5 Correlation between the IFNγ secretion from GPC3-targeted T cells and the MFI of GPC3 expression on the five HCC cell lines.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.6 In vitro cytotoxic activities of GPC3-targeted CAR T cells with hepg2 cell.

Primary human T cells transduced with the indicated lentiviral vectors were coincubated with the five HCC cell lines at the varying effector: target ratios for 18 hours, respectively.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.7 In vitro cytotoxic activities of GPC3-targeted CAR T cells with huh-7 cell.

Primary human T cells transduced with the indicated lentiviral vectors were coincubated with the five HCC cell lines at the varying effector: target ratios for 18 hours, respectively.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.8 In vitro cytotoxic activities of GPC3-targeted CAR T cells with hep3B CELL.

Primary human T cells transduced with the indicated lentiviral vectors were coincubated with the five HCC cell lines at the varying effector: target ratios for 18 hours, respectively.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.9 In vitro cytotoxic activities of GPC3-targeted CAR T cells with plc/prf/5 CELL.

Primary human T cells transduced with the indicated lentiviral vectors were coincubated with the five HCC cell lines at the varying effector: target ratios for 18 hours, respectively.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.10 In vitro cytotoxic activities of GPC3-targeted CAR T cells with SK-HEP-1 CELL.

Primary human T cells transduced with the indicated lentiviral vectors were coincubated with the five HCC cell lines at the varying effector: target ratios for 18 hours, respectively.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.11 Cytotoxicity of αGPC3-28BBZ CAR T cells on Huh-7 cells in the absence or presence of mammalian cell-expressed GPC3ΔGPI and GPC3N.

E.coli-expressed GST-fused GC33-binding peptide or BSA at the effector:target ratio of 3:1 for 18 hours.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.12 In vivo antitumor activities of αGPC3-28BBZ CAR T cells on established subcutaneous Huh-7 HCC tumor xenografts.

Growth curve of Huh-7 xenografts treated with the indicated T cells or saline.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.13 In vivo antitumor activities of αGPC3-28BBZ CAR T cells on established subcutaneous PLC/PRF/5 HCC tumor xenografts.

Growth curve of PLC/PRF/5 xenografts treated with the indicated T cells or saline.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.14 In vivo antitumor activities of αGPC3-28BBZ CAR T cells on established subcutaneous Huh-7 HCC tumor xenografts.

The quantities of circulating human CD4+ and CD8+ T cells from mice bearing Huh-7 xenografts treated with the indicated genetically modified T cells.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.15 In vivo antitumor activities of αGPC3-28BBZ CAR T cells on established subcutaneous PLC/PRF/5 HCC tumor xenografts.

The quantities of circulating human CD4+ and CD8+ T cells from mice bearing PLC/PRF/5 treated with the indicated genetically modified T cells.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.16 αGPC3-28BBZ CAR T cells potently suppressed the established orthotopic Huh-7 xenografts in vivo.

The growth of tumors treated with αGPC3-28BBZ CAR T cells was potently suppressed when compared with the control groups.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.17 αGPC3-28BBZ CAR T cells potently suppressed the established orthotopic Huh-7 xenografts in vivo.

The quantitative analysis of human CD4+ and CD8+ T cells by TruCount tubes.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.18 αGPC3-28BBZ CAR T cells potently suppressed the established orthotopic Huh-7 xenografts in vivo.

The quantitative analysis of GFP-positive peripheral blood cells.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD3ζ; GC33 scfv-CD28-41BB-CD3ζ

Fig.19 αGPC3-28BBZ CAR T cells potently suppressed the established orthotopic Huh-7 xenografts in vivo.

The overall survival of mice treated with the indicated T cells or saline.

Gao, H., Li, K., Tu, H., Pan, X., Jiang, H., Shi, B., ... & Li, Z. (2014). Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular CarcinomaGPC3-Targeted CAR T Cells for HCC Treatment. Clinical Cancer Research, 20(24), 6418-6428.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.20 Cytotoxicity and cytokine production of different CARTs.

The fold of increase over mock-T cells were presented. Each data was from the combined media of triplicate wells. Two Luminex testing repeats were conducted.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.21 Comparison of the cytotoxicity of CARs with and without mCherry tag.

Triplicate wells were used at each time point. The experiment was repeated 3 times with similar observation.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.22 GC33 CARTs do not kill normal primary human cells.

Primary human cells were co-cultured with CARTs for 24hrs and the media were used for LDH assay. HepG2 cells were used as positive control.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.23 In vivo antitumor activities of αGPC3 CAR T cells.

Bioluminescent imaging (BLI) of the IP tumors. All images at different time points and from different groups were adjusted to the same scale.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.24 Tumor BLI kinetics of αGPC3 CAR T cells antitumor activities in vivo.

Tumor BLI kinetics of each individual mouse, 5 mice in each treated group.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.25 Detection of hCD45 T cells and CAR+ T cells in the blood.

Statistical analysis was done with SAS System GLM procedure.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.26 The change of hCD45+T cells in the blood from DI 2 to DI 6 after ACT.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.27 Tumor BLI kinetics after CART treatment.

Each line represented an individual tumor.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.28 Representative IHC staining of tumor infiltraing CD8T cells at indicated time points after ACT.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.29 Summary of the tumor infiltrating CART numbers of 5 mice from 3 time points (DI 3, D18 and D2l).

The number was normalized by dividing the total CART number in the tumor by the corresponding tumor mass BLI. The experiment was repeated twice and similar data was observed.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.30 The gating strategy for enumerating the tumor infiltrating CARTs.

The viable cells from the single tumor cell suspension were counted by Trypan blue. The CART number in the tumor was calculated by the total viable cells in the tumor x% of hCD45 x %of CAR+.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.31 The exhaustion and apoptosis of tumor-infiltrating CARTs at D21 after ACT.

The tumor infiltrating CARTs from the single cell suspensions were stained and gated.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.32 Summary of the exhaustion and apoptosis of tumor infiltrating CARTs.

The % of PD1 + and LAG+ of CARTs, and the MFI of Annexin V on CARTs were summarized from 3 mice.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.33 The apoptosis staining of CAR- and CAR+T cells after HepG2 stimulation.

The numbers in the figure indicate the MFI of Anne×in-VThe dynamic changes of apoptosis was also summarized.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.34 The %and MFI of exhaustion markers, and the %of PD1 and LAG3 double positive cells among the CARTs after HepG2 stimulation were presented.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.35 The %of PD1 and LAG3 double positive cells among the CARTs after HepG2 stimulation were presented.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-CD28-CD3ζ; GC33 scfv-41BB-CD3ζ

Fig.36 The % of central memory (Tern) and naive-like cells (Tnaive) cells in the CAR+ cells after HepG2 co-culture were shown.

Each data point represents mean+/-SD of triplicate wells. The experiments were repeated 3 times with similar observation.

Caraballo Galva, L. D., Jiang, X., Hussein, M. S., Zhang, H., Mao, R., Brody, P., ... & He, Y. (2022). Novel low‐avidity glypican‐3 specific CARTs resist exhaustion and mediate durable antitumor effects against HCC. Hepatology, 76(2), 330-344.

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.37 GPC-3.CAR/sIL-15 Vδ1 T cells exert robust in vitro antitumor activity against GPC-3-expressing tumor cell lines

GPC-3.CAR Vδ1 were cocultured with RFluc-expressing GPC-3+ HepG2 and PLC/PRF/5 (PLC) liver cancer cell lines (top panels) or GPC-3- SKMEL5 and HCT116 melanoma and colon cancer cell lines (bottom panels) across listed E:T ratios for ~18-hour.

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.38 Cytotoxic potential of GPC-3.CAR/sIL-15 Vδ1 T cells against HepG2 and PLC cells at a fixed 1:1 ratio was assessed in the presence of soluble GPC-3 (0.3μg/mL-20 μg/mL) in an 18 hours cytotoxicity assay.

Per cent inhibition was calculated relative to the T cell alone control.

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.39 Cytokine production by GPC-3.CAR/sIL-15 and GPC-3.CAR Vδ1 T cell effectors after a 24hours co-culture with HepG2 cells at 2:1 E:T ratio.

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.40 Cytokine production by GPC-3.CAR/sIL-15 and GPC-3.CAR Vδ1 T cell effectors after a 24hours co-culture with PLC cells at 2:1 E:T ratio.

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.41 Cytotoxic potential of GPC-3.CAR and GPC-3.CAR/sIL-15 Vδ1 T cells against PLC cells in a long-term cytotoxicity assay (2.5:1 E:T ratio).

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.42 Cytotoxic potential of GPC-3 CAR Vδ1 T cells against PLC spheroids.

CAR and GPC-3.CAR/sIL-15 Vδ1 T cells against PLC spheroids in a long-term cytotoxicity assay (2:1 E:T ratio).

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.43 Proliferation monitoring analysis of GPC-3 CAR Vδ1 T cells in cytotoxicity assay.

On days 1, 3, 5, and 7 from the long-term cytotoxicity assay in fig.42, replicate wells were sacrificed for Vδ1 cell quantification (top) and proliferation monitoring (bottom) by flow cytometry.

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.44 Proliferative potential of GPC-3.CAR and GPC-3.CAR/sIL-15 Vδ1 T cells in tumor, bone marrow, spleen, blood, and lungs for ex vivo analysis.

(A) Schematic for ex vivo analysis of Vδ1 T cells in HepG2 tumor-bearing NSG mice. (B) Proliferative potential of GPC-3.

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.45 Differentiation potential of GPC-3.CAR and GPC-3.CAR/sIL-15 Vδ1 T cells in the spleen and tumor for ex vivo analysis.

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.46 In vivo efficacy of a single dose of car Vδ1 T cells in HepG2 tumor-bearing NSG mice.

(D) Study schematic. (E) Tumor growth kinetics (left) and tumor volumes on day 38 (right).

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.47 In vivo efficacy of a single dose of GPC-3.CAR and GPC-3.CAR/sIL-15 Vδ1 T cells in combination with interleukin-2 in HepG2 tumor-bearing NSG mice.

(F) Study schematic. (G) Tumor growth kinetics (left) and tumor volumes on day 35 (right).

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).

CAR Construction : GC33 scfv-41BB-CD3ζ-IL15

Fig.48 GPC-3.CAR/sIL-15 Vδ1 T cells do not induce toxicity or GvHD as determined by histopathological analysis.

On day 45 post-treatment, mice treated with GPC-3.CAR/sIL-15 Vδ1 T cells and from remaining control mice were euthanized and spleens, lungs, livers and skin were collected from each animal for histopathology analysis and for assessing infiltration of human CD3+ cells by immunohistochemistry.

Makkouk, A., Yang, X. C., Barca, T., Lucas, A., Turkoz, M., Wong, J. T., ... & Herrman, M. (2021). Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. Journal for immunotherapy of cancer, 9(12).