Proceedings of Oncobiology Symposiums
Proceedings of XIV Oncobiology Symposium

INTRODUCTION AND OBJECTIVES: Adoptive T cell transfer therapy with chimeric antigen receptors (CAR) redirects the action of lymphocytes to a specific antigen, such as CD19 or HER2, present on the surface of target cells (CHICAYBAM et al, 2020). Several factors can impact the function of CAR-T cells, such as exposure to cytokines and inhibitory molecules in the tumor microenvironment. The aim of this work is to generate T lymphocytes with different CAR receptors and to evaluate the modulation of these cells when promoting the expression of membrane-bound IL-15 fusion protein (mIL15). This cytokine is related to increased proliferation of T lymphocytes. MATERIAL AND METHODS: Different plasmids were generated carrying the anti-CD19 CAR isolated or accompanied by mIL-15. In parallel, the anti-HER2 CAR clones: 4D5 and FRP5 were tested. All constructions were cloned in the sleeping beauty system and electroporated into peripheral blood mononuclear cells (PBMC) from healthy adults donors. Finally, the cells were activated, expanded in culture and CAR expression was confirmed by flow cytometry. RESULTS AND CONCLUSION: After 8 days of expansion, the expression of the receptors was quite heterogeneous when evaluating cells from different donors. Two of them showed a substantial reduction in the expression of CARs 4D5 and FRP5 throughout the expansion, while a third one reached 11.2% and 14.5% of expression respectively on day +8. In the case of CAR anti-CD19 cells, there was better efficiency in gene expression, reaching 30% and 40% in the different donors used; however, contrary to expectations, the addition of mIL-15 expression has been shown to decrease the percentage of transgenic cells to 10% and 5% in the same donors. In general, the cells showed low expression of the receptors, with the exception of CAR 19BBZ alone, which supports expression compatible with the pattern observed in our previous studies. Several factors may be related to this variation, such as the size of the plasmids, in the case of construction with mIL-15, or the amino acid sequence itself in the case of HER2 CARs. In order to optimize these aspects, we will test alternatives in the design of the molecules and in the carrier vector of the transgene.