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P06.02 Anti-nucleolin CAR (chimeric antigen receptor)-T cell targeting triple-negative breast cancer – critical parameters in CAR-T cells generation
  1. TR Abreu1,2,3,
  2. A Godinho-Santos3,
  3. JN Moreira1,2 and
  4. J Gonçalves3
  1. 1CNC – Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine, Coimbra, Portugal
  2. 2Univ Coimbra – University of Coimbra, CIBB, Faculty of Pharmacy, Pólo das Ciências da Saúde, Coimbra, Portugal
  3. 3iMed.ULisboa – Research Institute for Medicines, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal


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.


  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.

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