ALBERT

Description: Invasive fungal diseases (IFDs) caused by the filamentous fungal pathogens of the genera Aspergillus, fusarium, zygomycetes, scedosporium and the yeast candida are the most important causes of fungus related deaths in immunosuppressed hematology patients1. Clinical efficacy of A. fumigatus specific T cells has been demonstrated in haploidentical hematopoietic stem cell transplant (HSCT) recipients2. We recently published a method to culture A. fumigatus specific T cells from normal allogeneic donors using procedures compliant with the code of Good Manufacturing Practice3. The safety and efficacy of cells generated using our method is currently being tested in a Phase 1 study in HSCT recipients. Here, we investigated the possibility of manufacturing a single T cell product with activity against a broad target range of fungal pathogens for clinical adoptive immunotherapy. We made water-soluble lysates from germinated spores of fungal pathogens, A. flavus, A. terreus, C. albicans, C. krusei, F. oxysporum, F. solani, R. oryzae and S. prolificans and used these to pulse monocyte derived dendritic cells (MoDC) from PBMC of 4 normal donors. Pulsed MoDC were washed, irradiated and used to stimulate autologous PBMC on Days 0 and 7. Cells were expanded with the addition of IL-2, IL-7 and IL-15 from Days 7 to 21. Cell numbers, phenotype and fungus-specificity were assessed on Day 21. Cross-reactivity of cultured T cells was tested against lysates of other fungi to identify a combination of antigens likely to induce broad anti-fungal T cell reactivity. Products were generated from PBMC using either no selection or TNFα capture on Day 7 of culture to select antigen-specific cells. Three products were generated from G-CSF mobilized peripheral blood stem cell products (PBSC) without selection. Antifungal activity was mapped to specific HLA molecules using blocking antibodies to HLA-DR, -DP and –DQ. In cultures from normal donor PBMC, expansion occurred with lysates from all fungi (range 2.3-109.6 fold) generating 85-97% T cells of which 〉80% were CD4+ T cells. The percentage of fungus-specific (TNFα+) CD4+ cells were A flavus 6.8±5.5%, A terreus 13.2±12.8%, C albicans 10.5±8.5%, C krusei 6.4±6.8%, F oxysporum 6.4±3.8%, F solani 5.2±7.8%, R oryzae 7.6±6.4 and S prolificans 4.4±3.8% (n=4). T cells also produced IFNγ and IL-2. T cells from cultures generated with Aspergillus, Fusarium and Scedosporium cross-reacted with one another and with lysates from most other fungi. We selected a combination of A. terreus, C. krusei and R. oryzae to generate multifungus T cell products from PBMC and PBSC. All but one multifungus culture generated 〉89% T cells. The majority of T cells had terminally differentiated effector and effector memory phenotypes. Regulatory T cells were