Transiently redirected T cells for adoptive transfer

Cytotherapy. 2011 May;13(5):629-40. doi: 10.3109/14653249.2010.542461. Epub 2010 Dec 21.


Background aims: T cells can be redirected to reject cancer by retroviral transduction with a chimeric antigen receptor (CAR) or by administration of a bispecific T cell engager (BiTE). We demonstrate that transfection of T cells with messenger (m) RNA coding for CAR is an alternative strategy.

Methods: We describe the pre-clinical evaluation of a method based on transient modification of expanded T cells with a CD19 CAR directed against B-cell malignancies. CAR mRNA was generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation was used to load the mRNA into the cytoplasm of T cells with no risk of insertional mutagenesis.

Results: After transfection >80% of T cells were viable, with 94% CAR expression. Transfected T cells were cytolytic to CD19(+) targets and produced interferon (IFN)-γ in response. Killing of CD19(+) target cells was demonstrated even at day 8 with undetectable CAR expression. Increasing the concentration of mRNA resulted in higher surface CAR expression, better killing and more IFN-γ release but at the expense of increased activation-induced cell death. Finally, we demonstrated that a second transgene could be introduced by co-electroporation of CXCR4 or CCR7 with CAR to also modify chemotactic responses.

Conclusions: We advocate the transient redirection approach as well suited to meet safety aspects for early phase studies, prior to trials using stably transduced cells once CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in pre-clinical studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adoptive Transfer / methods*
  • Antigens, CD19 / analysis
  • Cell Line, Tumor
  • Cells, Cultured
  • Cytotoxicity, Immunologic
  • Humans
  • Leukemia / therapy*
  • Lymphoma / therapy*
  • Receptors, Antigen / genetics*
  • Receptors, CCR7 / genetics*
  • Receptors, CXCR4 / genetics*
  • Retroviridae
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / transplantation*
  • Transduction, Genetic


  • Antigens, CD19
  • CCR7 protein, human
  • CXCR4 protein, human
  • Receptors, Antigen
  • Receptors, CCR7
  • Receptors, CXCR4