Original ResearchFull Report: Basic and Translational—LiverPersistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice
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Section snippets
Generation of Glypican 3–Targeted Chimeric Antigen Receptor T Cells
The antigen recognition region from the HN3 (pMH288), hYP7 (pMH289), and the anti-CD19 antibody FMC63 (pMH376) was subcloned into the second-generation (2G) CAR construct, which contains expressing cassettes encoding the CD8α hinge and transmembrane region, a 4-1BB costimulatory domain, the intracellular CD3ζ, the self-cleaving T2A sequence, and the truncated human epidermal growth factor receptor for cell tracking and ablation. The truncated human epidermal growth factor receptor lacks the
Chimeric Antigen Receptor (Humanized YP7) T Cells Are More Potent Than Chimeric Antigen Receptor (HN3) T Cells
To evaluate the effect of GPC3 epitopes in CAR T-cell killing, we compared the HN323 and hYP724,25 antibodies that recognizes the N-lobe and C-lobe of GPC3, respectively (Figure 1A). The HN313 and YP7 antibodies are highly tumor-specific for their binding on tumor cells and tissues (Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, Supplementary Figure 4). To produce CAR T cells for testing in HCC cell and animal models (Figure 1B), the antigen recognition region of the
Discussion
In the present study, we used the antibodies hYP7 and HN3 specifically for a membrane-proximal C-lobe epitope and a membrane-distal N-lobe epitope of GPC3 to make CAR T cells and analyzed their antitumor activities. The CAR (hYP7) T cells targeting the C-lobe of GPC3 close to the cell membrane showed superior antitumor activity by producing CAR T cells that not only induce perforin/granzyme-mediated apoptosis, but also inhibit Wnt/β-catenin signaling in tumor cells. We also used ddPCR and
Acknowledgments
The authors thank Dr Steven Rosenberg (National Cancer Institute [NCI]) for valuable advice in the early stage of this study and for reading the manuscript. We thank the CCR LGI Flow Cytometry Core Facility in Building 37 for assistance of cell sorting. We also thank National Institutes of Health (NIH) Fellows Editorial Board, NIH Library Editing Service, Jessica Hong (NCI) and Bryan Fleming (NCI) for editorial assistance. The NCI holds patent rights to anti-GPC3 antibodies including YP7 and
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Conflicts of interest The authors disclose no conflicts.
Funding This work was supported by the Intramural Research Program of NIH, NCI (Z01 BC010891 and ZIA BC010891) to MH. DL was a recipient of a predoctoral fellowship from the China Scholarship Council and supported by the Center for Cancer Research at the NCI and the NIH Graduate Partnerships Program in Bethesda, Maryland. HF was a recipient of a visiting fellowship from the China Scholarship Council. AK is a predoctoral fellow supported by the Mayo Clinic in Rochester, Minnesota (Clinical and Translational Science award UL1 TR000135), the Center for Cancer Research at the NCI and the NIH Graduate Partnership Program. The Leidos Biomedical Research, Inc in Frederick, Maryland was in part supported with federal funds from the NCI, NIH, under contract no. HHSN261200800001E.
Author names in bold designate shared co-first authorship.
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Authors share co-first authorship.