OPTIMIZATION OF MANNAN-SPECIFIC CHIMERIC ANTIGEN RECEPTOR (M-CAR) TO ENHANCE T CELL ACTIVATION AGAINST FUNGAL SPECIES

Invasive fungal infections (IFI) remain severe and underappreciated causes of disease, and associated with high morbidity and mortality worldwide. Candida albicans impacts strongly the incidence of IFI, and non-albicans Candida species have emerged as a growing global public health concern. Paracoccidioides brasiliensis is the main responsible for an endemic IFI identified in the Latin America. Additionally, Rhizopus oryzae is the most common cause of mucormycosis, a severe and often life-threatening IFI. The host immune response against IFI relies on the effector T cells. However, T cell activity is impaired in immunocompromised individuals, increasing susceptibility to IFI. Chimeric antigen receptor (CAR) technology offers a promising strategy to redirect T cells to specifically recognize and attack fungal pathogens at the site of infection. Our group developed a novel CAR, called M-CAR, capable to induce T cell activation after recognition of Candida spp., P. brasiliensis and R. oryzae . The antigen recognition domain of M-CAR is compound of a single-chain variable fragment (scFv) with specificity to α-1,6 mannose backbone of fungal mannan, a carbohydrate presents on the fungi cell wall. M-CAR has a FLAG-tag on the N-terminus, the CD8 molecule as hinge/transmembrane and CD3ζ as signaling domain. As costimulatory domain, the original M-CAR construct contained CD137 molecule. However, in this study, we evaluated alternative costimulatory molecules in both second- and third-generation M-CARs to screen costimulatory domains able to enhance T cell activation against the target. We generated M-CAR variants incorporating either CD28 or iCOS as coestimulatory domain, as second generation. Additionally, we developed two variants of third-generation M-CARs combining costimulatory domains along with a CD3ζ, as follows: iCOS and CD137; iCOS and CD28. The M-CAR constructs were generated using the Gibson Assembly method. Lentiviral particles containing the coding sequence of the different constructs of M-CAR were made in HEK 293T cells after transfection. These lentiviral particles were used to transduce Jurkat cell line and primary human T cells, which was isolated from peripheral blood mononuclear cells (PBMC) using Ficoll. The modified cells were co-cultured with soluble mannan obtained from S. cerevisiae , different species and morphologies of Candida, P. brasiliensis yeasts or R. oryzae spores. All different M-CAR variants were efficiently expressed on the surface of Jurkat cells. After 24 hours of co-culture with the target fungi Jurkat cells expressing M-CAR variants exhibited increased expression of CD69, an early marker of T cell activation. This upregulation was observed in response to mannan, as well as heat-killed C. albicans and C. tropicalis hyphae, C. parapsilosis pseudohyphae, C. glabrata yeast, P. brasiliensis yeast and R. oryzae spores. Cells expressing M-CAR containing as costimulatory domains iCOS, CD28, iCOS-CD28 and iCOS-CD137 enhanced the CD69 expression, whereas the strongest activation cell came from M-CAR containing CD137 alone as costimulatory domain. Primary T cells were effectively transduced with second-generation M-CAR constructs, and preliminary data suggest that these receptors enhance IFN-γ production in response to C. albicans . These data open the way to evaluate the fungicidal activity of human T cells expressing the M-CAR variants in response to fungal species reported in the current work. FAPESP 2024/00060-8