Background Triple-negative breast cancer (TNBC) is an aggressive cancer with low survival rates, still seeking for efficient targeted therapies. Genetic modification of human T cells to express chimeric antigen receptors (CAR-T cells) redirects T cells activity towards pre-established tumor-associated antigens (TAAs).¹ Despite highly successful against hematological malignancies, CAR-T cells efficacy against solid tumors has been limited, owing to lack of accessible TAAs and tumor microenvironment (TME)-mediated immunosuppression.² In this context, nucleolin, a membrane-nucleus shuttling phosphoprotein, has been demonstrated to be overexpressed on both TNBC cancer and endothelial cells from angiogenic blood vessels,³ emerging as an accessible target. In this respect, this work focuses on the first steps of anti-nucleolin CAR-T cells manufacturing, aiming at optimizing lentiviral-based CAR-transduction conditions.

Materials and Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor buffy-coats by density gradient centrifugation using Ficoll-Paque PLUS and activated with a polymeric nanomatrix agonist for CD3 and CD28 T cell receptors, in IL-2-containing culture medium. Activated PBMCs were transduced with produced CAR-encoding lentivirus - transient transfection of 293ET cells with different ratios of CAR-transgene, packaging, rev and envelope plasmids - either in flat- or round-bottom 96-well plates, in the presence or absence of the activation reagent. Transduction efficacy in T cells was assessed by both tdTomato fluorescence (reporter gene) and CAR surface expression through flow cytometry.

Results The transgene: packaging:rev:envelope plasmid ratio enabling the highest transduction rate - 50% of both tdTomato and surface CAR expression - was chosen for further CAR-transductions.The presence of T-cell-activating nanomatrix in transduction-media has demonstrated to influence CAR-transduction efficacy. Moreover, differences in tdTomato fluorescence levels between T cells transduced in flat- or round-bottom microplates were also observed.

Conclusions Overall, the presentwork provides insight on relevant experimental parameters that impact anti-nucleolin CAR-transduction in human T cells.

This work was funded by the FCT R&D Project EXPL/MED-FAR/1512/2021 and by CIBB contract programmes UIDB/04539/2020, UIDP/04539/2020 and LA/P/0058/2020.

References

1. Maher, J, Brentjens, R. J, Gunset, G, Rivière, I, Sadelain, M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCR ζ/CD28 receptor. Nat. Biotechnol 2002; 20:71–75

2. Newick, K.; Moon, E.; Albelda, S. M., Mol. Chimeric antigen receptor T-cell therapy for solid tumors. Ther. - Oncolytics 2016; 3:1–6.

3. Gregório, A., Meeting the needs of breast cancer: a nucleolin’s perspective, PhD Thesis, Univ. Coimbra, Coimbra, Port. 2015

Disclosure Information T.R. Abreu: None. A. Godinho-Santos: None. J.N. Moreira: None. J. Gonçalves: None.