Exploiting Cytokine Secretion to Rapidly Produce Multivirus-Specific T Cells for Adoptive Immunotherapy

Viral infections remain a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT), and conventional small-molecule therapeutics often have modest benefit, high cost and adverse effects. Adoptive transfer of donor-derived, virus-specific T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) proved feasible and safe after HSCT, and reconstituted immunity to all 3 viruses. Current protocols to generate multivirus-specific T cells are lengthy, taking up to 12 weeks. Since viral infections often occur <30 days after HSCT, more rapid production is required. The IFN-γ capture (Miltenyi) and tetramer methods can select antigenspecific T cells directly from peripheral blood and have been used clinically to prevent infections with AdV and CMV respectively. Tetramer selection is limited to certain HLA class I molecules with known epitope specificities. By contrast, the IFN-γ capture assay can be used to select multi-epitope and multi-antigen, CD4+ and CD8+ T cells from any donor, irrespective of HLA type. We now describe a modified rapid selection method for production and characterization of CD4+ and CD8+ T cells specific for CMV, EBV and AdV in a single infusate using the IFN-γ capture assay. We could manufacture a specific product from small volumes of peripheral blood over a 48 hour time period. These cells are suited for infusion into immunocompromised patients. Activated PBMCs were transduced either with Ad5f35pp65 vector or Ad5f35LMP2 vector and then combined. After overnight culture, IFN-γ-secreting antigen-specific T cells were captured. The number of selected cells ranged from 0.06 ×10⁶ to 0.36 ×10⁶ cells (mean: 0.14 ± 0.06 ×10⁶; n=5) per 20 × 10⁶ PBMCs.Importantly, we demonstrate that the selected cells lack alloreactivity, indicating that the infused cells should be safe in vivo. By expanding a small fraction of the selected product we could characterize the phenotype, specificity and function of the infused product, to allow subsequent comparison with clinical outcome. All products were able to secrete IFN-γ in response to each antigen and kill CMV, EBV and AdV antigen-expressing targets. These results show that we can successfully generate trivirus-specific T cells for infusion within 3 days without sacrificing the product characterization required to correlate product with clinical outcome.