Non-MHC-Dependent Redirected T Cells Against Tumor Cells

  • Hilde Almåsbak
  • Marianne Lundby
  • Anne-Marie Rasmussen
Part of the Methods in Molecular Biology book series (MIMB, volume 629)


Adoptive transfer of T cells with restricted tumor specificity provides a promising approach to immunotherapy of cancers. However, the isolation of autologous cytotoxic T cells that recognize tumor-associated antigens is time consuming and fails in many instances. Alternatively, gene modification with tumor antigen-specific T-cell receptors (TCR) or chimeric antigen receptors (CARs) can be used to redirect the specificity of large numbers of immune cells toward the malignant cells. Chimeric antigen receptors are composed of the single-chain variable fragment (scFv) of a tumor-recognizing antibody cloned in frame with human T-cell signaling domains (e.g., CD3ζ, CD28, OX40, 4-1BB), thus combining the specificity of antibodies with the effector functions of cytotoxic T cells. Upon antigen binding, the intracellular signaling domains of the CAR initiate cellular activation mechanisms including cytokine secretion and cytolysis of the antigen-positive target cell.

In this chapter, we provide detailed protocols for large-scale ex vivo expansion of T cells and manufacturing of medium-scale batches of CAR-expressing T cells for translational research by mRNA electroporation. An anti-CD19 chimeric receptor for the targeting of leukemias and lymphomas was used as a model system. We are currently scaling up the protocols to adapt them to cGMP production of a large number of redirected T cells for clinical applications

Key words

mRNA electroporation chimeric antigen receptor gene therapy T lymphocytes 



This work was supported in part by CHILDHOPE (EU contract #037381) and the authors would like to thank Dr. Martin Pulè at UCL Cancer Institute, University College London, UK, for providing the vector for the chimeric antigen receptor. The authors would also thank Dr. Stein Sæebøe-Larssen at our laboratory for providing the pCIpA102 expression vector and Dr. Rainer Loew at EUFETS, Idar-Oberstein, Germany, for the IRES-EGFP construct.


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Hilde Almåsbak
    • 1
  • Marianne Lundby
    • 2
  • Anne-Marie Rasmussen
    • 1
  1. 1.Department of ImmunologyRadiumhospitalet-Rikshospitalet, University HospitalOsloNorway
  2. 2.Department of Cellular TherapyRadiumhospitalet-Rikshospitalet, University HospitalOsloNorway

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