CAR T cells targeting <i>Aspergillus fumigatus</i> are effective at treating invasive pulmonary aspergillosis in preclinical models.

<i>Aspergillus fumigatus</i> is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to <i>A. fumigatus</i> are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered <i>A. fumigatus</i>-specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of <i>A. fumigatus</i> hyphae. T cells expressing the Af-CAR recognized <i>A. fumigatus</i> strains and clinical isolates and exerted a direct antifungal effect against <i>A. fumigatus</i> hyphae. In particular, CD8⁺ Af-CAR T cells released perforin and granzyme B and damaged <i>A. fumigatus</i> hyphae. CD8⁺ and CD4⁺ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8⁺ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8⁺ Af-CAR T cells conferred greater antifungal efficacy compared to CD4⁺ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA.