Characterization of T cell responses to EBV-associated gastric carcinoma using a single TCR cloning platform

Abstract

Adoptive T-cell therapy has emerged as a powerful treatment for many cancers. T-cell receptor (TCR)-engineered T-cells are genetically modified to express full-length tumour-specific TCRs and have demonstrated encouraging results in the treatment of numerous solid cancers. Here, I outline the development and validation of a highly accessible novel single TCR cloning platform. Using optimized TCR-specific PCR and cloning primers, and Gibson Assembly techniques, the streamlined platform allows efficient cloning of any TCR without expansion in culture. The platform clones TCR genes into a novel universal retroviral vector backbone for stable expression in target cells for crucial functional characterization, epitope mapping and MHC-restriction analysis. The capability of the platform was demonstrated via identification and cloning of dominant TCRs specific to ZEBRA, a highly immunogenic lytic stage EBV antigen. These TCRs comprised up to 21% of sequences in two healthy donors and were successfully expressed in a TCR-deficient T-cell line and in primary T-cells. Transduced T-cells upregulated the early activation marker CD69 and secreted TNF-α and IFN-γ upon stimulation, demonstrating preserved functional ability of the cloned TCRs. Characterization of a previously undiscovered TCR from one healthy donor revealed its curious ability to recognise antigen presented on two different autologous HLA alleles, HLA-A*02:01 and HLA-B*35:01. Such cross-reactivity to HLA molecules within the same donor has not been described in literature and may hold promise for T-cell therapy for patients carrying either, or both, HLA types. Moreover, functional studies suggested that ZEBRA-specific TCR-transduced primary T-cells could recognise and mediate killing of EBV-associated gastric carcinoma cells after virus latency reversal with HDAC inhibitors. The data suggests that combination therapy comprising latency reversal agents to reactivate the lytic stage of EBV infection, followed by adoptive T-cell therapy with TCR-engineered T-cells specific to highly immunogenic lytic stage antigens, may be a promising novel approach to the treatment of EBV-associated malignancies.