Rapid Generation of TCR and CD8αβ Transgenic Virus Specific T Cells for Immunotherapy of Leukemia

Abstract

Background: Virus-specific T cells (VSTs) are an attractive cell therapy platform for the delivery of tumor-targeted transgenic receptors. However, manufacturing with conventional methods may require several weeks and intensive handling. Here we evaluated the feasibility and timelines when combining IFN-γ cytokine capture (CC) with retroviral transduction for the generation of T cell receptor (TCR) and CD8αβ (TCR8) transgenic VSTs to simultaneously target several viral and tumor antigens in a single product. Methods: Healthy donor peripheral blood mononuclear cells were stimulated with cytomegalovirus (CMV) and Epstein-Barr-Virus (EBV) peptide mixtures derived from immunogenic viral proteins, followed by CC bead selection. After 3 days in culture, cells were transduced with a retroviral vector encoding four genes (a survivin-specific αβTCR and CD8αβ). TCR8-transgenic or control VSTs were expanded and characterized for their phenotype, specificity and anti-viral and anti-tumor functions. Results: CC selected cells were efficiently transduced with TCR8. Average fold expansion was 269-fold in 10 days, and cells contained a high proportion of CD8+ T central memory cells. TCR8+ VSTs simultaneously expressed native anti-viral and transgenic anti-survivin TCRs on their cell surface. Both control and TCR8+ VSTs produced cytokines to and killed viral targets, while tumor targets were only recognized and killed by TCR8+ VSTs. Conclusions: IFN-γ cytokine capture selects and activates CMV and EBV-specific memory precursor CD8+ T cells that can be efficiently gene-modified by retroviral transduction and rapidly ex vivo expanded. Our multi-specific T cells are polyfunctional and recognize and kill viral and leukemic targets expressing the cognate antigens.