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Aspergillus fumigatus-specific CAR T cells are effective at treating invasive pulmonary aspergillosis

SEIF M. ¹, KAKOSCHKE T. ², EBEL F. ², BELLET M. ³, TRINKS N. ⁷, ROMANI L. ³, TAPPE B. ¹, ESPIE D. ⁶, DONNADIEU E. ⁶, SAUER M. ⁷, HÜNNIGER K. ⁵, KURZAI O. ^4,5, EINSELE H. ¹, HUDECEK M. ¹, LÖFFLER J. ¹

¹ Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany; ² Institut für Infektionsmedizin und Zoonosen, Medizinische Fakultät, LMU, München, Germany; ³ Dipartimento di Medicina e Chirurgia, Università degli Studi di Perugia, Perugia, Italy; ⁴ Institut für Hygiene und Mikrobiologie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany; ⁵ Fungal Septomics Research Group, Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie – Hans-Knöll-Institut (HKI), Jena, Germany; ⁶ Université de Paris, Institut Cochin, INSERM, CNRS, Paris, France; ⁷ Biozentrum und RVZ - Center for Integrative and Translational Bioimaging, Julius-Maximilians-Universität Würzburg, Würzburg, Germany

Invasive pulmonary aspergillosis (IPA), mainly caused by Aspergillus fumigatus (A. fumigatus), is a leading cause of mortality in immunocompromised patients. Aspergillus-specific T cells are essential for fungal clearance. Such T cells are infrequent in the endogenous repertoire and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus-specific chimeric antigen receptor (Af-CAR) T cells and explored their potential in IPA therapy.

We expressed the Af-CAR in T cells using non-viral sleeping beauty transposon system. We verified the CAR expression and specificity by flow cytometry, microscopy, calcium flux, and IFN-? secretion. In co-cultures with A. fumigatus hyphae, we analyzed the antifungal activity of Af-CAR T cells alone or together with innate immune cells, we measured cytokine secretion, ROS production, and hyphal damage. Finally, we analyzed Af-CAR T cells therapeutic potential in vivo.

T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and showed no off-targets in the human lung or cross-reaction with other fungi. CD8⁺ Af-CAR T cells released perforin and granzyme B and exerted a direct anti-fungal effect against A. fumigatus hyphae. Furthermore, CD4⁺ and CD8⁺ Af-CAR T cells produced mainly Th1 cytokines that activated macrophages and neutrophils to potentiate the anti-fungal effect. In an in vivo model of IPA in immunodeficient mice, CD8⁺ Af-CAR T showed a higher potential to reduce fungal burden than CD4⁺ T cells. They controlled the local inflammation in the lung by recruiting mainly mononuclear cells and reducing the damage induced to the lung. Adoptive transfer of CD8⁺ Af-CAR T cells in a neutropenic IPA mouse model improved overall survival.

Taken 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.