Abstract
Abstract 1177
The adoptive transfer of antigen-specific T cells can be a powerful tool for immunotherapy of malignant diseases or infectious complications after allogeneic stem cell transplantation (Riddell et al., 1992; Heslop et al. 2010). Human adenovirus (AdV), Epstein-Barr virus (EBV) or cytomegalovirus (HCMV) infections are frequent and often life-threatening complications post allogeneic stem cell transplantation. To reduce the time required to isolate antigen-specific T cells for adoptive transfer we have developed a method to isolate IFN-gamma-secreting CD4+ as well as CD8+ T cells after antigen-specific restimulation, the Cytokine Capture System IFN-gamma (CCS). Several preclinical studies demonstrate the efficient enrichment of functional CD4+ and CD8+ T cells specific for HCMV (Rauser et al., 2004), EBV (Hammer et al., 2007) or AdV (Feuchtinger et al. 2008) using the CCS. First clinical data of adoptively transferred HCMV-, EBV- or AdV-specific T cells into patients post allogeneic stem cell transplantation are very encouraging (Feuchtinger et al., 2010; Moosmann et al., 2010; Feuchtinger et al., 2006) with low T cell doses infused varying from 1–97×10e3 cells/kg. We have now developed a cell processing device for the automation of the CCS procedure. Antigen-specific stimulation, labeling with the CCS reagents, washing steps, cytokine capture, magnetic enrichment and potentially expansion of the isolated cells are performed fully automated in a closed system. At the beginning of the procedure all components including the cellular starting product, antigen(s), reagents, buffer, and media are connected to a sterile single-use closed system processing set in the device. Due to usage of sterile filters and sterile docking the whole process runs under sterile conditions. The cellular end product can be obtained in the medium/buffer of choice, with the desired cell concentration and volume. The cellular starting product can be leukapheresis or bone marrow and the yield of antigen-specific T cells depends on the frequency of IFN-gamma producing cells. When starting with 1×10e9 cells 1–20×10e5 HCMV-specific T cells could be isolated. Cell processing is possible overnight and the isolated cells might be used directly after enrichment or after a phase of in vitro expansion. Using this cell processing device, IFN-gamma secreting HCMV-specific T cells were enriched to the same purity (>80% IFN-gamma secreting CD4+ and CD8+ T cells) as with the semi-automated procedure. Cell loss during the procedure is markedly reduced, leading to an increased yield of IFN-gamma positive cells. An improved viability was observed resulting in better expansion rates. In conclusion, the automation in a closed system enables the fast and robust generation of antigen-specific T cells for adoptive therapy and will reduce clean room requirements.
Disclosures:
Fahrendorff: Miltenyi Biotec GmbH: Employment. von Oppen:Miltenyi Biotec GmbH: Employment. Rauser:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Biehl:Miltenyi Biotec GmbH: Employment. Miltenyi:Miltenyi Biotec GmbH: Membership on an entity's Board of Directors or advisory committees.
Source Cell Preparation for the Manufacture of DUOC‐01 Using a Closed System Cell‐Processing Device