Development of a platform for in vivo tracking of CAR-T cells in an experimental model of glioblastoma.

• Grant number: 25/06059-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2025
• End date: August 31, 2026
• Field of knowledge: Biological Sciences - Immunology - Applied Immunology
• Principal Investigator: Lucas Eduardo Botelho de Souza
• Grantee: Maria Helena Zech Batista
• 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

Improving the therapeutic efficacy of CAR-T cells depends on the development of tools that allow real-time, highly sensitive monitoring of their infiltration and persistence. Bioluminescence-based imaging using the reporter enzyme firefly luciferase (FLuc) is the most widely used method for this purpose in nonclinical studies of CAR-T cells. However, the maximum wavelength of light emitted by FLuc is 562 nm, causing absorption by hemoglobin and melanin, resulting in signal attenuation and loss of sensitivity in vivo. Therefore, the objective of this project is to establish a platform for highly sensitive monitoring of the biodistribution kinetics of CAR-T cells using the luciferase enzyme AkaLuc and its substrate Aka-HCl. AkaLuc and AkaHCl were engineered to emit high levels of bioluminescence at the near-infrared wavelength (677 nm), leading to greater penetration into biological tissues and a signal intensity up to 1,000 times higher than that of conventional luciferases. Thus, we will generate CAR-T cells co-expressing an anti-GD2 CAR and the AkaLuc reporter enzyme (CAR-T/GD2-AkaLuc). We will then determine the detection limit of CAR-T/GD2-AkaLuc cells by bioluminescence quantification. Finally, we will evaluate the in vitro persistence and cytotoxicity of CAR-T/GD2-AkaLuc cells against GD2-positive and -negative tumor cells. We hope that the development of this platform will facilitate the understanding of CAR-T cell kinetics in vivo and accelerate the development of strategies to enhance their infiltration, persistence, and antitumor activity against solid neoplasms. (AU)