Development of Clinical Scale Cytomegalovirus-Specific T Cells for Adoptive Immunotherapy Using pp65.

Cytomegalovirus (CMV) represents a major cause of morbidity in patients undergoing allogeneic stem cell transplantation (SCT). Although antiviral prophylaxis has contributed to the reduction of early CMV disease, recovery of CMV-specific cytotoxic lymphocytes is still necessary for the long-term control of CMV reactivation after SCT. It has been demonstrated that CMV-specific CD4⁺ and CD8⁺ cytotoxic T lymphocytes from the donor are important in resolving CMV infection and protecting recipients from developing CMV disease post SCT. Recently CMV recombinant protein pp65, an immunodominant target for CD4⁺ and CD8⁺ T cell responses, has been developed for the stimulation and isolation of donor CMV-specific T cells. The aim of this study was to investigate the potential of pp65 to generate CMV-specific CD8⁺ and CD4⁺ T cells from seropositive subjects for use as adoptive immunotherapy after SCT. Peripheral blood mononuclear cells (5x10⁸ cells) were isolated from single unit CMV-positive buffy-coats and were stimulated with pp65 for 16 hours. CMV-specific T cells that produced IFNγ were captured using the IFNγ-secretion assay and were enriched using the CliniMACS system. The phenotype of the cells, both pre and post enrichment was evaluated by flow cytometry using monoclonal antibodies against CD3, CD4 and IFNγ. Enriched IFNγ producing T cells were cultured with irradiated feeder cells in the presence of IL-2 for 18 days. Alloreactivity experiments were performed by mixing 10⁵ enriched T cells at day 0 and day 18 of culture with 10⁵ third party cells for seven days. Proliferation was assessed by the ³H-thymidine incorporation assay. Experiments to date have shown that pp65 is capable of stimulating both CD4⁺ and CD8⁺ T cells to secrete IFNγ. Flow cytometric analyses revealed that prior to enrichment, IFNγ⁺ cells represented 0.13% of the CD3⁺/CD4⁺ cell population and 2.1% of the CD3⁺/CD4⁻ cell population. Whereas, following magnetic enrichment and an 18-day expansion phase, the proportion of IFNγ⁺ cells increased significantly (554-fold and 6-fold) to 72% and 13.3% in the CD3⁺/CD4⁺ and CD3⁺/CD4⁻ subfractions, respectively. The alloreactivity of the expanded positive cells had decreased by an average 95.5% (SD=5.7, n=3) compared to prior to enrichment and cell expansion. The proliferation of the expanded cells was still high (Stimulation Index >3), but considerably reduced compared to the cells at day 0. The cytotoxicity of these cells will be assessed to determine their suitability for immunotherapeutic use post-SCT. Large scale CMV-specific T cell capture experiments, performed under GMP conditions, are ongoing.