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

Hum Gene Ther. 2017 May;28(5):437-448. doi: 10.1089/hum.2016.025. Epub 2016 Aug 16.


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.

Publication types

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

MeSH terms

  • Animals
  • Antigens, CD19 / genetics
  • Antigens, CD19 / therapeutic use
  • CD28 Antigens / genetics*
  • CD28 Antigens / therapeutic use
  • Cell Polarity / immunology
  • Glypicans / genetics*
  • Glypicans / therapeutic use
  • Humans
  • Immunotherapy
  • Lymphoma, B-Cell / genetics
  • Lymphoma, B-Cell / immunology
  • Lymphoma, B-Cell / therapy*
  • Mice
  • Receptors, Antigen, T-Cell / genetics*
  • Receptors, Antigen, T-Cell / immunology
  • Receptors, Antigen, T-Cell / therapeutic use
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • T-Lymphocytes, Cytotoxic / immunology
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / genetics*
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / immunology
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / therapeutic use
  • Xenograft Model Antitumor Assays


  • Antigens, CD19
  • CD28 Antigens
  • Glypicans
  • Receptors, Antigen, T-Cell
  • Tumor Necrosis Factor Receptor Superfamily, Member 9