Dataset Information

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

ABSTRACT: Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR ? and ? expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.

SUBMITTER: Campillo-Davo D

PROVIDER: S-EPMC6234959 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Campillo-Davo Diana D Fujiki Fumihiro F Van den Bergh Johan M J JMJ De Reu Hans H Smits Evelien L J M ELJM Goossens Herman H Sugiyama Haruo H Lion Eva E Berneman Zwi N ZN Van Tendeloo Viggo V

Frontiers in immunology 20181107

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8<sup>+</sup> T ce ...[more]

PMID: 30464762

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Project description:BACKGROUND:High-affinity tumor antigen-specific T-cell receptor (TCR) gene is required to engineer potent T cells for therapeutic treatment of cancer patients. However, discovery of suitable therapeutic TCR genes is hampered by the fact that naturally occurring tumor antigen-specific TCRs are generally of low-affinity, and artificial modification of TCRs can mediate cross-reactivity to other antigens expressed in normal tissues. Here, we discovered a naturally occurring T-cell clone which expressed high-affinity HLA-A*02:01 (A*02)-restricted TCR against NY-ESO-1 from a patient who had NY-ESO-1-expressing ovarian tumor. METHODS:A*02-restricted NY-ESO-1-specific T-cell clones were established from peripheral blood of patients who had NY-ESO-1-expressing ovarian tumors. TCR ? and ? chain genes were retrovirally transduced into polyclonally activated T cells. Phenotype and function of the parental and TCR-transduced T cells were analyzed by flow cytometry, ELISA and cytotoxicity assay. In vivo therapeutic efficacy was investigated in a xenograft model using NOD/SCID/IL-2R?-deficient (NSG) mice. RESULTS:A rare population of NY-ESO-1-specific T cells, which we named 19305DP, expressed cell surface CD4, CD8?, and CD8? but not CD56 and recognized A*02+NY-ESO-1+ cancer cell lines in a CD4- and CD8-independent manner. 19305DP showed a gene expression profile that is consistent with a mixed profile of CD4+ and CD8+ single-positive T cells. Both CD4+ and CD8+ T cells that were retrovirally transduced with 19305DP-derived TCR gene (19305DP-TCR) showed strong reactivity against A*02+NY-ESO-1+ cancer cells, whereas TCR genes from the conventional A*02-restricted NY-ESO-1-specific CD8+ single-positive T-cell clones functioned only in CD8+ T cells. Both 19305DP-TCR gene-engineered CD4+ and CD8+ T cells eliminated A*02+NY-ESO-1+ tumor xenografts in NSG mice. Finally, based on reactivity against a series of alanine-substituted peptides and a panel of normal human tissue-derived primary cells, 19305DP-TCR was predicted to have no cross-reactivity against any human non-NY-ESO-1 proteins. CONCLUSION:Together, our results indicate that the naturally occurring 19305DP-TCR derived from CD4+CD8+ double-positive ?? T cells, is a promising therapeutic TCR gene for effective and safe adoptive T-cell therapy in A*02+ patients with NY-ESO-1-expressing tumor.

Dataset Information

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Publications

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

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