CAR (chimeric antigen receptor)-T cell therapy, which involves genetically modifying T cells with synthetic receptors that target specific tumor antigens, has shown remarkable success in hematologic malignancies. However, its application in solid tumors remains challenging due to limited tumor infiltration and strong immunosuppression. Nucleolin, a membrane-nucleus shuttling protein, has been demonstrated to be overexpressed on the surface of cancer and endothelial cells from tumor blood vessels, across different solid tumors. Accordingly, anti-nucleolin nanobodies, previously developed by our group, showed strong binding to nucleolin-positive cell lines, suggesting nucleolin as a potential CAR-T cell target. Here, we describe the initial steps in generating anti-nucleolin CAR-T cells and evaluate their in vitro activity against solid tumor-derived cell lines of diverse histological origin.

Peripheral blood mononuclear cells were isolated from healthy donors, activated with CD3/CD28 agonists, and transduced with CAR-encoding lentivirus. Transduction efficiency and stability were evaluated by both reporter gene and CAR surface expression, at multiple timepoints. To assess CAR-T cells anti-tumor activity, specific lyse of different nucleolin expressing cancer cell lines was analyzed by real-time impedance-based co-culture assays, at different E(effector):T(target) ratios. Untransduced and mock-transduced T cells were used as controls. Proliferation in the presence or absence of target cells and immunophenotyping studies by spectral flow cytometry were also performed.

CAR expression ranged between 40% and 60% and remained stable for at least six days in culture. Anti-nucleolin CAR-T cells displayed significantly higher cytotoxicity than respective controls, and higher effector cell ratios led to increased target cell death. This effect was more pronounced in tumor cells with superior surface nucleolin expression. Phenotypic analysis revealed a predominance of central memory (CCR7+CD45RO+) CAR-T cells, and differences on exhaustion markers expression, namely PD-1 and LAG-3, were observed over 10 days in culture, suggesting dynamic changes in activation and exhaustion profiles.

In summary, this work demonstrates the successful generation and in vitro validation of anti-nucleolin CAR-T cells, against different cancer cell lines, supporting nucleolin as a potential new target in CAR-based immunotherapy for solid tumors. This work was funded by: Fellowship 2020.04685.BD (FCT). Projects: EXPL/MED-FAR/1512/2021 (FCT); CIBB (FCT UIDB/04539/2020, UIDP/04539/2020 and LA/P/0058/2020), Fundación La Caixa BREAST-BRAIN-N-BBB; CInTech (PRR-30) C644865576-00000005; 2022.07746.PTDC.