Multi-cistronic vector encoding optimized safety switch for adoptive therapy with T-cell receptor-modified T cells.

T-cell receptor (TCR) gene transfer is an attractive strategy to equip T cells with defined antigen-specific TCRs using short-term in vitro procedures to target both hematological malignancies and solid tumors. TCR gene transfer poses different safety issues that might warrant the inclusion of a suicide gene. High affinity TCRs may result in on-target toxicity, and off-target reactivity directed against healthy tissue can be observed due to mixed TCR dimers. Inclusion of a suicide gene as a safety switch may abrogate these unwanted toxicities. Human CD20 has been proposed as a nonimmunogenic suicide gene targeted by widely used clinical-grade anti-CD20 antibodies that can additionally function as a selection marker. However, transduction of T cells with a multi-cistronic vector encoding both TCR and CD20 resulted in poor coexpression. In this study, we demonstrated that codon optimization of TCR and CD20 resulted in profound coexpression of both the preferentially expressed antigen in melanoma (PRAME)-TCR and CD20, allowing selective as well as efficient elimination of these engineered T cells in vitro. These results demonstrate the great potential of codon optimized CD20 to be broadly used in clinical trials as a safety switch.

New tools to monitor the impact of viral infection on the alloreactive T-cell repertoire.

Accumulating evidence suggests that alloreactive memory T-cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross-reactivity of virus-specific memory T-cells is not available. We therefore aimed to develop a novel system for the detection of cross-reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein-Barr Virus (EBV) EBNA3A specific CD8 memory T-cell clones (HLA-B*0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon-gamma Elispot as readout. Generation of the T-cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex-specific tetramer. Monoclonality of the T-cell clones was confirmed by T-cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A-specific T-cell clones against SAL-expressing HLA-B*4402. Further screening against the entire panel of SALs also showed additional cross-reactivity against SAL-expressing HLA-B*5501. Functionality of the cross-reactive T-cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross-reactivity of viral-specific CD8 memory T-cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.