Dados do Trabalho

Título

CAR T-lymphocyte anti-carbonic anhydrase IX releasing programmed cell death ligand 1 antibody in the treatment of glioblastoma

Introdução

Glioblastoma (GBM) is the most prevalent of all brain tumors. Hypoxia is a frequent feature of GBM that leads to the overexpression of several proteins, including carbonic anhydrase IX (CAIX), expressed in GBM cases. Solid tumors have a microenvironment that promotes T cell exhaustion, which favors tumor development. Considering GBM, the programmed cell death ligand-1 (PD-L1), a molecule related to exhaustion induction, is overexpressed in GBM cases. A new therapeutic strategy has been highlighted in the fight against cancer: T cells containing chimeric antigen receptors (CAR T cells) designed against a tumor antigen.

Objetivo

This project aims to evaluate the in vitro efficacy of anti-CAIX CAR T cells, releasing anti-PD-L1 antibodies in the tumor microenvironment, in combating different GBM cell lines in vitro, and in an orthotopic model to be established in NSG mice.

Métodos

In this project, we will use glioblastoma cell lines U251, A172, and U87. The expression levels of CAIX and PD-L1 will be evaluated by flow cytometry. The lentiviruses will be produced by transfection of five plasmids, including a plasmid coding for CAR, into 293T cells and will be transduced into T lymphocytes. Secreted IgG will be detected by ELISA. The cytotoxic effects of CAR T cells will be analyzed by viability using the lactate dehydrogenase assay. CAR T cells will be injected into the in vivo model 7, 14, and 21 days after tumor implantation, and the effects of the CAR T cells will be analyzed by immunohistochemistry after three months.

Resultados

Results indicate that only U251 cell lines express CAIX and PD-L1 significantly, A172 doesn’t present it, even using CoCl2 treatment. All anti-CAIX CAR T cells were able to induce U251 (CAIX+/PD-L1+) cytotoxicity, and the anti-CAIX CAR T cells secreting anti-PD-L1 are slightly more cytotoxic for these cells than the anti-CAIX CAR T cells only and secrete more IL-2 and IFNγ, demonstrating the CAIX dependent activation of these CAR T cells.

Conclusões

From this study, we were able to conclude that molecular constructs anti-CAIX with a CD28 co-stimulatory domain have a better response when it comes to interventional therapy against glioblastoma multiforme. The construct with 4-1BB releasing anti-PD-L1 antibodies, despite lower IFN-γ release, could be used as an interesting option, potentially capable of reducing the exhaustion of LT infiltrating the tumor. In vivo tests are being planned to continue this research.

Palavras-chave

CAR T cell, glioblastoma, solid tumors