A novel automated process generates virus-specific T lymphocytes for immunotherapy that maintain their in vivo phenotype and functional competence

      Adoptive transfer of virus-specific T cells is an option to treat severe infections after allogenic stem cell transplantation. To generalize this approach, a cGMP-compliant process for enrichment of virus-specific CD4+ and CD8+ T cells is required. Here, we use a newly established automated manufacturing process for rapid ex vivo isolation of multi-virus- or CMV pp65 peptide-specific T cells. During this process, first 109 white blood cells from healthy donors were stimulated with either a combination of peptide pools (CMV pp65, EBV EBNA-1, BZLF1, and LMP2, and ADV hexon) or with a pp65 peptide for 4 hours. Subsequently, virus-specific T cells were magnetically enriched according to IFN-γ secretion. To examine, if cell processing influence the phenotype, virus-specific T cells were analyzed before and up to 4 days after the selection process. Isolated T cell products consist of 86.6% IFN-γ+ CD8+ multi-virus-specific T cells and 75.5% IFN-γ+ pp65 peptide-specific CD8+ T cells. However, after a resting phase in vitro, IFN-γ production decreased drastically. In addition, we detected an upregulation of CD69 and CD137 in T cell products directly and 24 hours after selection, respectively. The transient nature of activation could again be confirmed, as both, CD69 and CD137 were downregulated later on. To test functionality we re-incubated 3-days rested cells with antigens. Induction of IFN-γ in up to 100% of T cells was observed demonstrating cells maintained their in vivo imprinted physiological role. We also monitored CD45RA, CD28, and CCR7 expression on pp65-peptide specific CD8+ T cells before and directly after the enrichment. The enrichment process did not induce phenotypic changes. Thus, the newly developed automated manufacturing process provides a product, where the original phenotypic and functional characteristics of virus-specific T cells are conserved. Hence this cellular product is expected to fight viral infections effectively upon adoptive transfer.
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