CHIMERIC ANTIGEN RECEPTOR T CELLS AGAINST BREAST CANCER CELLS BY TARGETING FOLATE RECEPTOR ALPHA PROTEIN
Breast cancer (BCA) is the most common malignancy in women worldwide. Although treatments have greatly advanced, the patients with BCA still suffer from side effects, metastasis and relapse, suggesting the need of new therapies. Adoptive chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable efficiency against several malignancies. Folate receptor-alpha (FRα) is a promising target protein for CAR T cells due to its high expression on various types of BCA cells but rare on normal cells. Therefore, in this study, we aimed to construct CAR T cells targeting FRα and evaluate their cytotoxicity against BCA cells.
FRα expression in Thai BCA tissues and breast cell lines (MCF10A, MCF-7 and MDA-MB-231) were studied by immunohistochemistry and flow cytometry, respectively. FRα-specific single-chain variable fragment (scFv) gene was cloned into lentiviral vector bearing CAR generation 4 (CAR4) cassette genes (CD28, 4-1BB, CD27 and CD3ζ). Lentiviruses containing this construct were used to transduce into human primary lymphocytes. Surface FRα-CAR4 protein expression was monitored by flow cytometry. Cytotoxicity against FRα+ MDA-MB-231 cell line was then evaluated by using luciferase assay.
Different levels of FRα expression in Thai BCA tissues were observed. Surface FRα was present in BCA cell lines (10% in MCF-7 and 30% in MDA-MB-231) but not in breast normal-like cell line (1% in MCF10A). The FRα-CAR4 construct was effectively transferred to primary T cells by lentiviral system with transduction efficiency up to 48%. The FRα-CAR4 T cells, containing both CD4+ and CD8+ cells, conferred superior cytotoxicity against MDA-MB-231 cells over CD19-CAR and untransduced T cells.
We have successfully generated human FRα-CAR4 T cells by lentiviral system. The FRα-CAR4 T cells held specific anti-FR+ BCA cells. This study thus provided fundamental knowledge for generation of FRα-CAR4 T cells, which can potentially be developed as a treatment for FRα+ BCA patients.