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Impact of scFv on Functionality and Safety of Third-Generation CD123 CAR T Cells.

Maxime FredonMargaux PoussardSabeha BiichleFrancis BonnefoyCharles-Frédéric MantionEvan SeffarFlorian RenosiElodie Bôle-RichardRomain BoidotSandrine ChevrierFrançois AnnaMaria LoustauJulien CaumartinMathieu Gonçalves-VenturelliEric RobinetPhilippe SaasErick DeconinckEtienne DaguindauXavier RousselYann GodetOlivier AdotéviFanny Angelot-DelettreJeanne GalaineFrancine Garnache-Ottou
Published in: Cancer immunology research (2024)
Chimeric antigen receptor (CAR) T cells express an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface tumor-associated antigen. scFv selection should involve safety profiling with evaluation of the efficacy/toxicity balance, especially when the target antigen also is expressed on healthy cells. Here, to assess differences in terms of efficacy and on-target/off-tumor effects, we generated five different CARs targeting CD123 by substituting only the scFv. In in vitro models, T cells engineered to express three of these five CD123 CARs were effectively cytotoxic on leukemic cells without increasing lysis of monocytes or endothelial cells. Using the IncuCyte system, we confirmed the low cytotoxicity of CD123 CAR T cells on endothelial cells. Hematotoxicity evaluation using progenitor culture and CD34 cell lysis showed that two of the five CD123 CAR T cells were less cytotoxic on hematopoietic stem cells. Using a humanized mouse model, we confirmed that CD123- cells were not eliminated by the CD123 CAR T cells. Two CD123 CAR T cells reduced tumor infiltration and increased the overall survival of mice in three in vivo models of blastic plasmacytoid dendritic cell neoplasm. In an aggressive version of this model, bulk RNA sequencing analysis showed that these CD123 CAR T cells upregulated genes associated with cytotoxicity and activation/exhaustion a few days after the injection. Together, these results emphasize the importance of screening different scFvs for the development of CAR constructs to support selection of cells with the optimal risk-benefit ratio for clinical development.
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