Cryptococcosis is a disease caused by fungi of the genus Cryptococcus, a WHO priority pathogen, and nearly one million cases diagnosed annually. Patients affected by lymphoproliferative neoplasms are more susceptible to Cryptococcus spp. infection, and novel therapeutic approaches are encouraged to address invasive fungal infection (IFI) in lymphoproliferative disorders. The polysaccharide GXM (glucuronoxylomannan) composes Cryptococcus spp. capsule that is targeted by CAR (Chimeric Antigen Receptor) technology previously reported as GXMR-CAR. This chimeric receptor expressed by T cells mitigated the progression of cryptococcosis, however, the improvement of signaling transduction triggered by GXMR-CAR is in progress by our group. The success of CAR application to treat hematological neoplasms opened a research field related to CAR beyond cancer, and the association of CAR cell therapy redirecting immune cells to target cancer cells and Cryptococcus spp., side-by-side, is the main goal of the current proposal.

Then, this work is focused on the modification of human T cells to co-express GXMR-CAR and CD19-CAR for the controlling of cryptococcosis and neoplastic B cell expansion.

(i) Generation of GXMR-CAR variants and CD19-CAR using Gibson assembly and overlap PCR approaches; (ii) Production of lentiviral particles through transfection of HEK-293T cells; (iii) Isolation of human T cells from PBMCs (Peripheral Blood Mononuclear Cells) using Ficoll density gradient centrifugation, followed by stimulation with anti-CD3/CD28 beads before the transduction of T cells using the spinoculation method; (iv) Evaluation of cytotoxic activity by luciferase assay using the Raji-Luc cell line, a neoplastic B cell line expressing luciferase; (v) Quantification of pro-inflammatory mediator secretion by ELISA (OptEIA Kit; BD Biosciences); (vi) evaluation of the fungicidal effect by CFU assay.

Thus, we generated GXMR-CAR variants containing CD28, CD137, or iCOS as costimulatory domains, and CD19-CAR. So far, preliminary data demonstrated the cytotoxic activity of CD19-CAR modified T cells against Raji-Luc cell line, after incubation for 24 hours RLU determination. CD19-CAR T cells in the presence of Raji cell line showed a significant increase in the production of IFN-γ or IL-2, compared to the unmodified T cells. The expression of cellular exhaustion markers was also investigated, and both PD-1 and TIM-3 had increased expression in CD19-CAR T cells. In addition, T cells modified with GXMR-CAR variants were co-cultivated with C. gattii or C. neoformans yeasts for 24-hours. Cryptococcus burden was determined through the CFU assay, and GXMR-CD137-CAR T cells promoted a significant reduction in the C. neoformans burden, compared to unmodified T cells.

Notably, additional experiments are required to validate these findings. The next steps, GXMR-CAR variants and CD19-CAR will be co-expressed by human T cells verifying the effect on in vitro control of C. neoformans and C. gattii infection, as well as the ability to reduce the expansion of neoplastic B cells. Finally, modified T cells co-expressing GXMR-CAR and CD19-CAR will be used in the therapy of NSG mice previously infected with Cryptococcus spp. or infused with neoplastic B cell line.

CAPES - PROEX; CNPQ (process 167848/2023-2); FAPESP (process number: 2024/00300-9).