Gastroenterology

Gastroenterology

Volume 158, Issue 8, June 2020, Pages 2250-2265.e20
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice

https://doi.org/10.1053/j.gastro.2020.02.011Get rights and content

Background and Aims

Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties.

Methods

We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice.

Results

Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active β-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice.

Conclusions

In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

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

References (45)

  • D.L. Porter et al.

    Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia

    N Engl J Med

    (2011)
  • J. Filmus et al.

    Identification of a new membrane-bound heparan sulphate proteoglycan

    Biochem J

    (1995)
  • H.C. Hsu et al.

    Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: biological significance and temporospatial distribution

    Cancer Res

    (1997)
  • D. Baumhoer et al.

    Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples

    Am J Clin Pathol

    (2008)
  • M. Montalbano et al.

    Biology and function of glypican-3 as a candidate for early cancerous transformation of hepatocytes in hepatocellular carcinoma (review)

    Oncol Rep

    (2017)
  • M.I. Capurro et al.

    Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling

    Cancer Res

    (2005)
  • W. Gao et al.

    Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy

    Hepatology

    (2014)
  • N. Li et al.

    A frizzled-like cysteine rich domain in glypican-3 mediates Wnt binding and regulates hepatocellular carcinoma tumor growth in mice

    Hepatology

    (2019)
  • W. Gao et al.

    Immunotoxin targeting glypican-3 regresses liver cancer via dual inhibition of Wnt signalling and protein synthesis

    Nat Commun

    (2015)
  • B.D. Fleming et al.

    The engineered anti-GPC3 immunotoxin, HN3-ABD-T20, produces regression in mouse liver cancer xenografts via prolonged serum retention

    Hepatology

    (2019 Sep 2014)
  • Y. Fu et al.

    Glypican-3-specific antibody drug conjugates targeting hepatocellular carcinoma

    Hepatology

    (2019)
  • H. Gao et al.

    Development of T cells redirected to glypican-3 for the treatment of hepatocellular carcinoma

    Clin Cancer Res

    (2014)
  • Cited by (96)

    View all citing articles on Scopus

    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.

    Authors share co-first authorship.

    View full text