Clinical grade generation of human anti-adenoviruscytotoxic T-cells for adoptive immunotherapy

Adenovirus (ADV) infections represent one of the major cause of morbidity and mortality following Hematopoietic stem cell transplantation. The incidence of ADV infections ranges between 5 to 30%, with paediatric recipients showing the highest rate of infection. The mortality rate is very high in those patients: 60 to 73%, despite new antiviral treatment strategies. Actually, it has been demonstrated that a sufficient host T-cell response is essential to clear the virus. We describe here a complete clinical grade generation of Human anti-Adenovirus cytotoxic T cells in order to propose an adoptive immunotherapy to the recipient. Healthy donor mononuclear cells (PBMC), known for their good cellular immunity against ADV, are stimulated during 6 hours with the Peptivator-ADV5 (Miltenyi Biotec) which is a synthetic peptide pool covering the ADV5 Hexon protein. Gamma Interferon (IFN[Florin]x) secreting cells are isolated on the CliniMACS device using the Cytokine Capture System (Miltenyi Biotec) (Feuchtinger et al, 2007). The results of 4 immunomagnetic selections are presented in the table below: Isolated T lymphocytes (CTL) are cultured with IL2 and autologous feeder cells (irradiated cells from the negative fraction) in order to perform the functional quality controls. We first controlled the ability of the amplified CTL to secrete IFN[Florin]x when restimulated with the Peptivator ADV. A cytotocity of 40% against autologous dendritic cells (DC) as target cells (10/1) loaded with ADV5 or ADV 2 lysates could be observed when a cytotoxicity of 6% was reported with non loaded DC. Finally, we could observe a low allogeneic reaction with CTL against non HLA identical healthy donor PBMC, decreased of more than one log compared to the autologous PBMC. Good Manufacturing Practice-grade generation of ADV-specific T cells for adoptive immunotherapy could be achieved with a synthetic antigen. This technology presents the advantages to be fast, without any in vitro amplification before infusion, and to allow a good reactivity to propose immunotherapy in case of anti-viral treatment failure.