Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice

Gastroenterology. 2020 Jun;158(8):2250-2265.e20. doi: 10.1053/j.gastro.2020.02.011. Epub 2020 Feb 12.

Abstract

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.

Keywords: Hepatic; Immunotherapy; Lymphocyte; Tumor-Specific T Cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Aged, 80 and over
  • Animals
  • Apoptosis
  • Carcinoma, Hepatocellular / immunology
  • Carcinoma, Hepatocellular / metabolism
  • Carcinoma, Hepatocellular / pathology
  • Carcinoma, Hepatocellular / therapy*
  • Cell Proliferation
  • Female
  • Gene Expression Regulation, Neoplastic
  • Glypicans / genetics
  • Glypicans / immunology
  • Glypicans / metabolism*
  • Granzymes / metabolism
  • Hep G2 Cells
  • Humans
  • Immunotherapy, Adoptive*
  • Liver Neoplasms / immunology
  • Liver Neoplasms / metabolism
  • Liver Neoplasms / pathology
  • Liver Neoplasms / therapy*
  • Male
  • Mice, Inbred NOD
  • Mice, SCID
  • Middle Aged
  • Perforin / metabolism
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / immunology
  • Receptors, Chimeric Antigen / metabolism*
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • T-Lymphocytes / transplantation*
  • Tumor Burden
  • Tumor Microenvironment
  • Wnt Signaling Pathway
  • Xenograft Model Antitumor Assays

Substances

  • GPC3 protein, human
  • Glypicans
  • Receptors, Chimeric Antigen
  • Perforin
  • Granzymes