Optimization of the manufacture of CAR-T cells with TCR deleted via CRISPR/Cas9 to obtain a less differentiated allogeneic cell product with greater cytotoxic potential

• Grant number: 23/15546-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): February 01, 2024
• Effective date (End): December 31, 2024
• Field of knowledge: Biological Sciences - Immunology - Applied Immunology
• Principal Investigator: Lucas Eduardo Botelho de Souza
• Grantee: Karolina Santos Esteves
• Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil

Abstract

Since the approval of the first cell therapy product for the treatment of neoplasms with CAR-T lymphocytes in 2017, impressive clinical results have been achieved. However, even though this type of immunotherapy is increasingly becoming a form of cancer treatment, especially in hematological tumors, there are several limitations to its use. One of the main restrictions for expanding the clinical use of this therapy is its autologous nature, depending on a long and complex manufacturing process, which increases its costs and is not always possible depending on the patient's clinical condition. Therefore, the generation of allogeneic CAR-T cells appears as a promising alternative to reduce the time and costs of this cellular product. Nevertheless, another challenge is the low efficacy of modified lymphocytes in solid neoplasms, making necessary the development of processes that increase the persistence and infiltration of CAR-T cells in the tumor niche. Therefore, the objective of this research project is to optimize the manufacture of allogeneic CAR-T lymphocytes, in order to obtain a less differentiated cellular product with greater cytotoxic potential. To achieve this, allogeneic anti-GD2 CAR-T lymphocytes will be generated through knockout of the TRAC gene using the CRISPR/Cas9 editing system. These cells will be cultivated in a glucose-depleted medium supplemented with inosine and interleukins 7 and 15, and will be subsequently evaluated for their antitumor action in vitro in co-culture with human glioblastoma cells. It is expected that the results obtained in this project will allow the development of a platform for the production of ready-to-use allogeneic CAR-T cells with greater persistence and antineoplastic action, with a focus on increasing the therapeutic efficiency of CAR-T cells.