Improving EBV-specific T cell therapy: lineage tracing and EBV stem cell memory T cells

Viral infections remain an important cause of morbidity and mortality, especially after transplantation, and treatment options are often limited. Adoptive therapies with virus-specific T cells (VST) have shown to be promising in restoring virus-specific immunity and thereby preventing and treating viral infections over the past 25 years. Donor-derived Epstein Barr virus (EBV)-specific cytotoxic T-cell lines (CTLs) have demonstrated prolonged overall survival in patients with EBV-driven post-transplant lymphoproliferative disease (PTLD), lymphomas and diseases, but approximately 30% of patients show no response indicating a need for further improvements (the overview of the clinical trials with EBV-CTLs is summarized in Appendix I).
Naturally, after antigen stimulation and activation, T cells undergo a series of cell proliferation and differentiation stages, from naïve to stem cell memory (TSCM), central memory (TCM), transitional memory (TTM) effector memory (TEM), and terminally differentiated, short-lived effector T cells (TEMRA). During differentiation, the effector function of T cells gradually increases, while their self-renewal capacity correspondingly declines. As one of the major challenges of adoptively transferred T cells is their poor ability to persist in vivo upon the infusion., early differentiated i.e., stem cell memory T cells (TSCM) became relevant for the adoptive therapies due to their high proliferation, engraftment, and persistence potential in different human diseases and have shown promising results in ACT against cancer. Protocols that are used for EBV-specific ACT to date mainly exploit late-differentiated TEM or at best TCM. Data on EBV-specific TSCM in adoptive therapy is very limited and no protocol for clinical application is available.
In chapter II, I present a clinically-scalable protocol for TSCM-enriched expansion of Epstein-Barr virus (EBV)-specific T cells. In collaboration with Prof. Dr. Christian Münz and the group of Dr. Obinna Chijioke from the University of Zurich we compared its anti-tumor efficiency with conventionally expanded, EBV-transformed lymphoblastoid cell line-stimulated T cells in the in vivo model of post-transplant lymphoproliferative disorder (PTLD). Rapidly expanded TSCM-enriched EBV-specific T cells efficiently controlled the PTLD, showed a better tumor infiltration rate, robust in vivo proliferation and persistence potential with functional CD4+ and CD8+ cells and a broader reconstitution of EBV specificity. The method and these data together should help to establish the next generation of unmodified antigen-specific cell therapies beyond EBV diseases.
Another way to improve the cell therapies is to study their dynamic after infusion, thus being able to characterize the most promising clones.
Determining the fate of T cells following patient infusion hinges on the ability to track them in vivo. While this is possible by genetic labeling of parent cells, the applicability of this approach has been limited by the non-specificity of the edited T cells. Recently, it was shown that CAR T cells can persist in patients as many as 10 years after infusion. While CAR T cells are readily identifiable, non-engineered therapeutic T cells are difficult to distinguish from naïve T cells. In chapter II, I describe a novel method for CRISPR-targeted genome integration of a barcoded gene into Epstein-Barr virus-antigen-stimulated T cells that we devised in collaboration with the group of Prof. Sai T. Reddy from ETH Zurich. We demonstrated its use for exclusively identifying expanded virus-specific cell lineages. Our method facilitated the enrichment of antigen-specific T cells, which then mediated improved cytotoxicity against EBV-transformed target cells. Single-cell and deep sequencing for lineage tracing revealed the expansion profile of specific T cell clones and their corresponding gene expression signature. This method has the potential to enhance the traceability and the monitoring capabilities during immunotherapeutic T cell regimens.