Development of chimeric antigen receptors for redirecting T and NK cells to control invasive candidiasis
Invasive candidiasis, primarily caused by Candida albicans, is a severe infection with significant clinical implications. The World Health Organization has classified Candida infections as a critical priority for new therapeutic strategies due to the increasing prevalence of antifungal resistance an...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2025 |
| País: | Brasil |
| Institución: | Universidade de São Paulo (USP) |
| Repositorio: | Biblioteca Digital de Teses e Dissertações da USP |
| Idioma: | inglés |
| OAI Identifier: | oai:teses.usp.br:tde-04042025-100316 |
| Acceso en línea: | https://www.teses.usp.br/teses/disponiveis/17/17136/tde-04042025-100316/ |
| Access Level: | acceso abierto |
| Palabra clave: | Candidíase invasiva Cell therapy Célula NK Célula T Chimeric antigen receptor Invasive candidiasis NK cells Receptor de antígeno quimérico T cells Terapia celular |
| Sumario: | Invasive candidiasis, primarily caused by Candida albicans, is a severe infection with significant clinical implications. The World Health Organization has classified Candida infections as a critical priority for new therapeutic strategies due to the increasing prevalence of antifungal resistance and limited therapeutic options. The Candida cell wall is mainly composed of mannans, chitin, B-1,3- and B-1,6- glucans, which are pathogen-associated molecular pattern (PAMP) ligands recognized by host immune receptors. Natural killer (NK) cells play an essential role in early immune responses, releasing cytotoxic granules and cytokines that promote the differentiation of T helper (Th)1, Th17 cells, and cytotoxic T cells. However, Candida species can evade these immune responses through morphological switching between yeast cells, pseudohyphae, and hyphae forms - an important virulence factor that leads to a more challenging treatment. To address these limitations, chimeric antigen receptors (CARs), synthetic receptors designed to redirect immune cells against specific targets, represent a promising therapeutic approach for invasive fungal infections. This study aimed to bioengineer T and NK cell lines (Jurkat and NK-92) to express Candida-specific CARs, targeting Candida species both in vitro and in vivo. Four secondgeneration CARs specific to C. albicans were developed, each based on distinct single-chain variable fragments (scFvs) derived from monoclonal antibodies against C. albicans: scFv3-CAR, scFv5-CAR, scFv12-CAR, and scFvk3-1-CAR. These CAR constructs were subcloned into lentiviral vectors, and lentiviral particles were produced in HEK-293T cells by transfection. Jurkat cells were modified with each CAR separately, and GFP+ cell populations were enriched using fluorescence-activated cell sorting (FACS). Among these, scFvk3-1-CAR demonstrated the most robust activation, mediating the production of high IL-2 levels against both yeast and hyphal forms of C. albicans, as well as other species such as C. tropicalis, C. glabrata, and clinical isolates of C. auris. In contrast, scFv3-CAR and scFv12-CAR did not show notable activation, while scFv5-CAR mediated only modest activation. Given its superior performance, scFvk3-1-CAR was selected for further investigation. Several activation mechanisms in Jurkat cells were evaluated, including the CAR signaling pathway and the expression of exhaustion and activation markers on the cell surface, detected by flow cytometry. NK-92 cells were then modified with scFvk3-1-CAR to assess its potential to enhance antifungal activity. scFvk3-1-CAR-NK-92 cells exhibited increased IFN-γ production and CD107a expression, leading to significant damage to C. albicans in vitro. In a preclinical model of invasive candidiasis, NOD scid gamma (NSG) mice infected with C. albicans and treated with scFvk3-1-CAR-NK-92 cells showed a reduction in fungal burden in the kidneys compared to the untreated animals. Finally, scFv k3-1 was expressed as a soluble protein in a bacterial system, purified, and analyzed using a glycan microarray. The analysis confirmed that scFv k3-1 binds specifically to C. albicans mannan and not to any of the other 672 glycan probes tested, highlighting its highly specific binding capability. This study presented a proof-ofconcept for a CAR that effectively targets Candida species and enhances the antifungal activity of NK cells, representing a promising approach for future invasive candidiasis therapies. |
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