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Construction of second and third generation of 2DA6-CAR constructs and their expression in NK-92 cells to control the Paracoccidioides brasiliensis infection

Grant number: 22/13035-6
Support Opportunities:Scholarships in Brazil - Master
Effective date (Start): March 01, 2023
Effective date (End): June 30, 2024
Field of knowledge:Biological Sciences - Immunology - Applied Immunology
Principal Investigator:Thiago Aparecido da Silva
Grantee:Edanielle Silva de Moura
Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:18/18538-0 - Bioengineered of T- and NK-cells by CAR against invasive fungal infections, AP.JP


Paracoccidioidomycosis (PMC), caused mainly by the fungus Paracoccidioides brasiliensis, is a systemic granulomatous mycosis of higher prevalence in South America with high correlation with individuals having rural activities. PMC caused about 51% of deaths caused by systemic mycoses in Brazil between the years 1996 and 2006, according to data from the Ministry of Health. The infection occurs by inhaling conidia present in the soil, which reach the lung tissue of the host, and the clinical manifestations are divided into the acute/subacute form with patients having high fever, generalized or intra-abdominal lymphadenomegaly, and skin lesions; in the chronic form there is a high involvement of lung tissue, skin lesions on the oral and nasal mucosa. The conventional treatment, based on azolic derivatives and amphotericin B, is used for a period of up to 24 months; however, there is a constant search for immunotherapeutic approaches to reduce the adverse effects caused by the antifungal drugs available in the clinic. In this sense, vaccination strategies and adoptive transfer of immune cells have been investigated in the treatment of PCM and with a promising effect in immunotherapy against PCM. With this, our research group has advanced the bioengineering of T and NK cells to express chimeric antigenic receptors (CAR) that redirect the modified cells to recognize the target fungus. CAR receptors by redirecting cells to interact with the fungal forms under study mediate cellular activation, through the signal transduction domain, with the release of cytotoxic components that act to control fungal growth. Recently, our group constructed a CAR, named 2DA6-CAR, with specificity for mannan containing ±-1,6-mannose backbone, and our preliminary data demonstrate the ability of 2DA6-CAR to recognize P. brasiliensis. With this, the current proposal search to evaluate the effect of different variants of 2DA6-CAR, by constructing second and third generation CAR, with modification in the intracellular activation domain via the co-stimulatory molecules CD28, CD137 and ICOS. The different 2DA6-CAR constructs will be expressed by Jurkat cells to evaluate the following specific objectives: (i) to evaluate the expression of 2DA6-CAR on the cell surface by flow cytometry; (ii) to determine the molecular weight of the different 2DA6-CAR constructs by western blot; (iii) to analyze the capacity of the different 2DA6-CAR constructs expressed to induce cell activation, by measuring IL-2 levels, in the presence of the yeast form of P. brasiliensis. Next, the 2DA6-CAR construct with the highest functionality will be expressed in NK-92 cells and the following objectives will be considered: (i) activation of NK-92 cells expressing 2DA6-CAR in co-culture with P. brasiliensis, by dosing INF-³ by ELISA and measuring cytotoxic granules by flow cytometry; (ii) evaluating the fungicidal and fungistatic effect of NK-92 cells expressing 2DA6-CAR on P. brasiliensis by quantifying fungal burden.

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