Rapid Manufacture and Isolation of Highly Viable Interferon-γ Expressing Virus-Specific T Cells: Validation Results for the Virus-Specific Cytotoxic T-Lymphocyte Consortium for Refractory EBV, CMV, and Adenovirus Infections

Abstract

Background Children and young adults who develop treatment-refractory infection with EBV, CMV, and/or adenovirus (AdV) after allogeneic hematopoietic stem cell transplant (allo-HSCT) have a dismal prognosis (≥90% mortality). Adoptive transfer of ex-vivo expanded virus-specific cytotoxic T lymphocytes (CTLs) is a treatment option for these patients, but the cell manufacturing process is labor-intensive and typically takes several weeks of culturing to complete. More recently, a culture-free method has been developed to directly isolate virus-specific T cells from peripheral blood for adoptive transfer. The process is fully automated using the CliniMACS Prodigy device (Miltenyi Biotec), a cGMP-compliant, temperature-controlled, closed system. Importantly, virus-specific T cells ready for infusion can be isolated with Methods Peripheral blood mononuclear cells from volunteer donors were stimulated with PepTivators (CMV, EBV, and/or AdV), followed by isolation of the virus-specific T cells using the interferon-γ (IFNγ) cytokine capture system (CCS) on the CliniMACS Prodigy device (n=11). Expression of lineage and T-cell subset markers, IFNγ, and cell viability were determined by flow cytometry. Results The viability of recovered T cells was remarkably and consistently high (92.9% ± 7.4%, Table 1). The number of recovered viable T cells was 5.03 × 106 ± 5.09 × 106. The purity of IFNγ+ cells was 69.0% ± 23.8% for CD4 T cells and 82.9% ± 15.3% for CD8 T cells. At a dose of 2.5 × 104 T cells/kg, the mean T cell collection for a 70 kg recipient would suffice for 3 separate infusions. Conclusion The results presented here show that manufacture of virus-specific CTLs by the CCS method reproducibly produced sufficient quantities of viable T cells for patient treatment. These results thus support the feasibility of the multicenter VIRCTLC trial. The trial is currently enrolling patients.