Electroporation of Dicer-Substrate siRNA Duplexes Targeting Endogenous TCR Enhance Tumor Killing Activity of Wilms' Tumor 1 (WT1)-Specific TCR-Redirected Cytotoxic T Cells

In adoptive cellular immunotherapy, T cells can be genetically engineered to express a novel T-cell receptor (TCR) that recognizes a tumor-associated antigen. However, mispairing between transgene and endogenous TCR chains may result in a reduction of transgene TCR expression and potentially harmful off-target reactivities. Here, we sought to develop a novel clinically safe strategy to promote transgene expression of a Wilms' tumor 1 (WT1)-specific TCR by Dicer-substrate small interfering RNA (DsiRNA)-mediated silencing of the endogenous TCR, using a double electroporation protocol.

First, we isolated and cloned an HLA-A*0201-restricted WT1 peptide-specific TCR derived from a leukemia patient who demonstrated clinical benefit after receiving a WT1-targeted DC vaccine. Next, we produced a codon-optimized TCR sequence from the wild-type TCR construct and both TCR mRNAs were generated by in vitro transcription. TCR expression levels were validated by electroporation of TCR-deficient Jurkat J76.7 cells stably transduced with CD8 and an NFAT-driven GFP reporter gene. TCR functionality was confirmed by high expression levels of GFP (70% GFP⁺cells) upon TCR signaling after co-culture with WT1 peptide-pulsed T2 cells.

In order to suppress the translation of endogenous TCR mRNA in CD8⁺ T cells, DsiRNA duplexes were designed to specifically target the constant regions of wild-type TCR α- and β-chains, but not the codon-optimized TCR. We further developed a double electroporation protocol combining DsiRNA and TCR mRNA transfection in which DsiRNA electroporation was performed 24 hours prior to TCR mRNA electroporation. Our results show more than 2-fold increase in WT1-specific TCR expression by HLA-A2/WT1 tetramer staining after DsiRNA treatment as compared to TCR mRNA electroporation only. This specific TCR expression was maintained at least 5 days after TCR mRNA electroporation in resting peripheral blood CD8⁺ lymphocytes from healthy donors. The enhanced TCR expression in DsiRNA-transfected CD8⁺T cells was also correlated with an increase of epitope recognition as shown by interferon (IFN)-γ ELISpot.

To determine the killing capacity of DsiRNA/TCR mRNA-transfected CD8⁺ T cells against epitope-bearing target cells, we performed a flow cytometry-based cytotoxicity assay using WT1 peptide-pulsed T2 cells. Specific cytotoxicity, which was already present in WT1 TCR-transfected cells, was significantly enhanced in TCR mRNA-electroporated T cells following suppression of the endogenous TCR expression by DsiRNA treatment. Accordingly, DsiRNA-treated TCR mRNA transfected CD8⁺T cells presented higher levels of CD137 and CD69 activation markers and secretion of cytokines (IFN-γ and tumor necrosis factor-α), granzyme B, and perforin upon TCR triggering as compared to the non-DsiRNA treated T cells.

In summary, we show a marked enhancement of transgene WT1-specific TCR expression upon silencing of the endogenous TCR using DsiRNA electroporation prior to TCR mRNA electroporation. Importantly, this enhancement in TCR expression was correlated with a significant increase in WT1-specific CD8⁺ T-cell killing activity, expression of CD69 and CD137 activation markers and cytokine secretion after recognition of WT1 peptide-bearing target cells. These results pave the way for developing a clinically safer strategy for T cell-based adoptive immunotherapy of patients with WT1-expressing malignancies.