Effect on the cytotoxic activity of human T cells modified with distincts CAR specific to carbohydrates of Cryptococcus neoformans surface

• Grant number: 23/06496-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: October 01, 2023
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Immunology - Applied Immunology
• Principal Investigator: Thiago Aparecido da Silva
• Grantee: Michele Procópio Machado
• Host Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Invasive fungal infections are a serious public health problem that affects more than one billion people worldwide. A species of particular concern is Cryptococcus neoformans, which is responsible for most cases of fungal meningitis in immunocompromised patients. The annual death rate due to cryptococcosis is alarming, reaching around 625,000 deaths. C. neoformans has mechanisms that make it highly pathogenic, allowing it to evade the host's immune response. The presence of a polysaccharide capsule, composed of GXM (glucuroxylomannan) and GXMGAL (galactoxylomannan), associated with a cell wall containing chitin, are components involved in the mechanism of evasion of C. neoformans. These carbohydrates mask the surface of the fungus, impairing the recognition of molecular patterns by innate immune cells. As a result, the host immune response is compromised and, consequently, the development of a fungus-specific adaptive response is inefficient. Currently, the drugs of choice in the treatment of cryptococcosis cause adverse effects and an increase in the antifungal resistance of Cryptococcus spp. has advanced. Given this, immunotherapies are considered a promising approach in the treatment of cryptococcosis. One therapeutic strategy involves the use of T cells modified with a chimeric antigenic receptor (CAR) specific for the GXM of Cryptococcus spp, called GXMR-CAR. Previous studies have shown that this redirection of T cells expressing GXMR-CAR was capable of triggering a cytotoxic effect on the fungus at the site of infection in vivo. Therefore, the objective of this study is to expand the construction of new CARs with specificity for GXMGal (monoclonal antibody anti-GXMGal clone D8; D8-GXMGALR-CAR) or chitin (monoclonal antibody anti-chitin clone C55 or DD11; C55-CHITINR-CAR and DD11-CHITINR-CAR) from C. neoformans.In addition, a comparison between the cytotoxic activity triggered by modified T cells with the different specific CARs for C. neoformans will be performed to list the construct with the greatest impact on the control of cryptococcosis. In possession of this result, a combined therapeutic approach between modified T cells and the association with antifungal drugs such as the echinocandins class is foreseen. This proposal aims to increase the susceptibility of C. neoformans to the antifungal agent in order to result in a reduction in dosage and, therefore, mild adverse effects. In the present proposal, the coding sequences of the variable fragments of the monoclonal antibodies D8, C55 or DD11 will be inserted into a lentiviral vector containing the other domains composing a second-generation CAR. Lentiviral particles will be produced in HEK-293T cells and Jurkat and PBMC cells will be transduced to evaluate the specificity of CAR against C. neoformans, and also the ability of CAR to induce cytotoxic activity against the fungus. Furthermore, the proposal will investigate the redirection of T-CAR cells in controlling the establishment and progression of C. neoformans infection in NSG mice. Therefore, the research proposal has great potential to generate new CARs with high specificity for C. neoformans, which expands the possibility of characterizing CARs with high redirection capacity and capable of mediating a greater cytotoxic effect on C. neoformans.