Abstract:
Background: Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is an aggressive malignant hemopathy characterized by constant overexpression of the CD123 antigen on 100% of blasts identifying CD123 as an antigenic target for the development of adoptive cell therapy. Chimeric Antigen Receptor-T (CAR-T) are composed of an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface antigen expressed on tumor cells. In view of the low expression of CD123 on hematopoietic stem cells, monocytes and endothelial cells, it is necessary to evaluate and select the CD123 CAR-T allowing the best cytotoxicity differential between leukemic and healthy cells.
Methods: We developed five third-generation CD123 CAR-T by substituting scFv, in order to assess changes in efficacy and on-target/off-tumor side effects. Using various in vitro and in vivo BPDCN models, we evaluated the functionality on BPDCN and the on-target/off-tumor effect on endothelial cell line, monocytes and hematopoietic stem cell (HSC).
Results: Using Incucyte, we confirmed the low cytotoxicity on endothelial cells. We showed an increase in CD123 expression on endothelial cells, dependent on the activation of CAR-T through cytokine secretion. Evaluated by CFU-GM culture, we show that two CAR-T are less cytotoxic against hematopoietic stem cells and these two CAR-T significantly reduce tumor infiltration and increase overall survival of mice in three in vivo BPDCN models. Finally, we demonstrate in bulk RNA-sequencing that the most effective CAR-T in an aggressive BPDCN model, upregulates genes associated with cytotoxicity and activation/exhaustion at day 34, with fewer regulatory signatures
Conclusions: Together, these results emphasize the importance of evaluating different scFv for the development of CAR-T, to select the best one, with high cytotoxicity potential and the best safety profile for clinical development.
Journal Link: Research Square Other Link: Download PDF Other Link: Google Scholar
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