426. Multi-Pathogen-Specific T Cells: A Single T-Cell Product Simultaneously Targeting Multiple Pathogens for Adoptive Immunotherapy

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Viral infections, most commonly by cytomegalovirus (CMV), Epstein-Barr virus (EBV), polyoma virus type I(BK), andfungal infections, mainly by Aspergillus Fumigatus (Asp), are among the deadliest complications for patients undergoing allogeneic hematopoieticstem cell transplantation. Treatment with antiviral and antifungal pharmacological agents, which are today's standard therapy, is often ineffective or toxic whereas it can lead to the outgrowth of drug-resistant strains. Adoptive immunotherapy with the use of antigen-specific T-cells to restore antigen-specific immunity post-transplant, offers an attractive alternative approach to conventional drugs. We here, aimed to generate at large scale, multipathogen-specific T cells (mp-STs) that simultaneously target CMV, EBV, BK and Asp, from healthy donors, with a rapid, simplified, low-cost and minimally laborious protocol. A total of 1,5×107mononuclear cells, derived from 15-20ml blood of eight CMV and EBV seropositive donors, were pulsed with viral pepmixes(CMV: IE1, pp65;EBV: EBNA1, LMP2, BZLF1;BK: Large T, VP1) combined with either Asp lysate or Asp pepmixes (Crf1, Gel1 and SHMT) and cultured in the presence of IL-4/IL-7 for 10 days in G-rex bioreactors. The cells were characterized immunophenotypically by flow cytometry and their specificity/functionality was assessed by IFN-γ Elispot assay. Cells stimulated with either Asp lysate (n=4) or Asp pepmixes (n=4), had a similarexpansion, reaching a mean of 170±36×106and 206±46×106 cells, respectively. All cell lines were polyclonal, comprised predominantly of CD4+ cells (70±5% and 72±3% respectively) and CD8+ cells (19±4% and 18±2% respectively) and expressed central (CD45RA-/CD62L+: 36±6%, and 35±2% respectively) and effector memory markers (CD45RA-/CD62L-: 53±5%, and 54±3% respectively). Importantly, allmp-STs lines (8/8) were specific against all targeted pathogens [mean±SEM spot forming cells (SFC)/2×105 input cells, with lysate:CMV:211±41; EBV:1137±165; BK:551±109; Asp: 143±8; with peptides: CMV: 251±111; EBV: 1031±238; BK: 616±119 SFC; Asp: 463±132). Given that all donors were CMV and EBV seropositive and BK or Asp prior donor exposure wasnot tested, our data suggest that BK or Aspspecificity can be obtained practically from all healthy individuals, due to the particularly high exposure of the general population to these pathogens. Notably, the combination of Asp targeted-proteins (Crf1, Gel1 and SHMT) induced stronger Th1 responses compared to Asp lysate (p=0,049). Overall, we established arapid and simple, optimizedprotocol of generating clinically relevant numbers of mp-STs from a small amount of donor blood. Should mp-STs move to the clinic and prove safe and effective, they will be an ideal treatment for patients suffering from life-threatening, post-transplant infections.