CHIMERIC ANTIGEN RECEPTOR (CAR) T CELL THERAPY FOR ASPERGILLOSIS.

Abstract

Background & Aim

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 CAR T cells, are direly needed. Here, we propose a novel approach to generate Aspergillus fumigatus (AF)-CAR T cells using the scFv domain of AF-269-5 mAb and a lentiviral vector system.

Methods, Results & Conclusion

Targeting domain of the AF-CAR construct was designed from the CDR sequence of AF-269-5 monoclonal antibody. The targeting domain was fused to the hinge and transmembrane domains of human CD8α and the cytoplasmic signaling domains of CD137/4-1BB and CD3ζ and subcloned in LV vector dCAS9-VP64-GFP (Addgene, Water Town, USA).

These cells successfully targeted mature hyphal filaments of representative clinical and reference AF isolates and elicited 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 cytokine stimulation (IL-2, IL-7+IL-15) regimens significantly suppressed mycelial growth of AF-293 after 18 h 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 on days 0 and 2 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 to mice receiving non-targeting control T cells. This newly developed construct has several major advantages in its design and mode of action compared to published AF-reactive CAR T-cell products: (i) Unlike Dectin-1 CAR T cells that have weak affinity to mature mycelium, our present product was designed to efficiently target mature AF hyphae. (ii) Our AF-CAR T cells could enable generation of clinical-grade AF-CAR T cells for large scale production in a time frame of 3-7 days.

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. These results underscore the promise of anti-Aspergillus immunotherapy and provide a novel approach for efficient production of AF-CAR T cells for future clinical translation after further in-depth evaluation.