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 singlechain variable fragment domain of monoclonal antibody AF2695 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, AFCAR T cells generated from peripheral blood mononuclear cells of four healthy human donors and expanded with either of three cytokine stimulation regimens (IL2, IL2 + IL21, or IL7 + IL15) significantly suppressed mycelial growth of AF293 after 18 hours of coculture and synergized with the immunomodulatory antifungal agent caspofungin to control hyphal growth for 36 hours. Moreover, cyclophosphamideimmunosuppressed NSG mice with invasive pulmonary aspergillosis that received two doses of 5 million AFCAR T cells (6 and 48 hours after AF infection) showed significantly reduced morbidity on day 4 postinfection (P < 0.001) and significantly improved 7day survival (P = 0.049) compared with mice receiving nontargeting control T cells, even without concomitant antifungal chemotherapy. In conclusion, we developed a novel lentiviral strategy to obtain AFCAR T cells with high targeting efficacy, yielding significant antiAF activity in vitro and shortterm protection in vivo. Our approach could serve as an important steppingstone for future clinical translation of antifungal CAR Tcell therapy after further refinement and thorough preclinical evaluation. IMPORTANCE Invasive aspergillosis (IA) remains a formidable cause of morbidity and mortality in patients with hematologic malignancies and those undergoing hematopoietic stem cell transplantation. Despite the introduction of several new Aspergillusactive antifungals over the last 30 years, the persisting high mortality of IA in the setting of continuous and profound immunosuppression is a painful reminder of the major unmet need of effective antifungal immune enhancement therapies. The success of chimeric antigen receptor (CAR) Tcell therapy in cancer medicine has inspired researchers to translate this approach to opportunistic infections, including IA. Aiming to refine antiAspergillus CAR Tcell therapy and improve its feasibility for future clinical translation, we herein developed and validated a novel antibodybased CAR construct and lentiviral transduction method to accelerate the production of CAR T cells with high targeting efficacy against Aspergillus fumigatus. Our unique approach could provide a promising platform for future clinical translation of CAR Tcellbased antifungal immunotherapy.