Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model

Gene Ther. 2015 May;22(5):391-403. doi: 10.1038/gt.2015.4. Epub 2015 Feb 5.


Cancer therapy with T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical responses in recent trials, but also severe side effects. Whereas most protocols use permanently reprogrammed T cells, we have developed a platform for transient CAR expression by mRNA electroporation. This approach may be useful for safe clinical testing of novel receptors, or when a temporary treatment period is desirable. Herein, we investigated therapy with transiently redirected T cells in vitro and in a xenograft mouse model. We constructed a series of CD19-specific CARs with different spacers and co-stimulatory domains (CD28, OX40 or CD28-OX40). The CAR constructs all conferred T cells with potent CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti-leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the CH2-domain eradicated leukemia in vivo, without notable toxicity. Follow-up studies demonstrated that the CH2CH3-spacer bound soluble mouse Fcγ-receptor I and mediated off-target T-cell activation towards murine macrophages. Our findings highlight the importance of non-signalling CAR elements and of in vivo studies. Finally, the results show that transiently redirected T cells control leukemia in mice and support the rationale for developing an mRNA-CAR platform.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, CD19 / genetics
  • Antigens, CD19 / immunology
  • CD28 Antigens / genetics
  • CD28 Antigens / immunology
  • Cell Line, Tumor
  • Cells, Cultured
  • Genetic Therapy
  • HEK293 Cells
  • Humans
  • Immunotherapy, Adoptive
  • Leukemia / therapy*
  • Macrophage Activation
  • Macrophages / immunology
  • Mice
  • Mice, Inbred NOD
  • Receptors, Antigen, T-Cell / genetics*
  • Receptors, Antigen, T-Cell / immunology
  • Receptors, IgG / genetics*
  • Receptors, IgG / immunology
  • Receptors, OX40 / genetics
  • Receptors, OX40 / immunology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / transplantation
  • Xenograft Model Antitumor Assays


  • Antigens, CD19
  • CD28 Antigens
  • Receptors, Antigen, T-Cell
  • Receptors, IgG
  • Receptors, OX40
  • Recombinant Proteins