A novel lentiviral vector-based approach to generate chimeric antigen receptor T cells targeting Aspergillus fumigatus

Invasive aspergillosis (IA) is a common and deadly mold infection in immunocompromised patients. As morbidity and mortality of IA are primarily driven by poor immune defense, adjunct immunotherapies, such as chimeric antigen receptor (CAR) T cells, are direly needed. Here, we propose a novel approach to generate Aspergillus fumigatus (AF)-CAR T cells using the single-chain variable fragment domain of monoclonal antibody AF-269-5 and a lentiviral vector system. These cells successfully targeted mature hyphal filaments of representative clinical and reference AF isolates and elicited a potent release of cytotoxic effectors and type 1 T cell cytokines. Furthermore, AF-CAR T cells generated from peripheral blood mononuclear cells of four healthy human donors and expanded with either of three cytokine stimulation regimens (IL-2, IL-2 + IL-21, or IL-7 + IL-15) significantly suppressed mycelial growth of AF-293 after 18 hours of co-culture and synergized with the immunomodulatory antifungal agent caspofungin to control hyphal growth for 36 hours. Moreover, cyclophosphamide-immunosuppressed NSG mice with invasive pulmonary aspergillosis that received two doses of 5 million AF-CAR T cells (6 and 48 hours after AF infection) showed significantly reduced morbidity on day 4 post-infection ( P < 0.001) and significantly improved 7-day survival ( P = 0.049) compared with mice receiving non-targeting control T cells, even without concomitant antifungal chemotherapy. In conclusion, we developed a novel lentiviral strategy to obtain AF-CAR T cells with high targeting efficacy, yielding significant anti-AF activity in vitro and short-term protection in vivo . Our approach could serve as an important steppingstone for future clinical translation of antifungal CAR T-cell therapy after further refinement and thorough preclinical evaluation.