Improving persistence of anti-CD19 CAR T cells via overexpression of the IL-21 receptor

Toskov, Vasil (2023): Improving persistence of anti-CD19 CAR T cells via overexpression of the IL-21 receptor. Dissertation, LMU München: Medizinische Fakultät

Abstract

Adoptive transfer of anti-CD19 chimeric antigen receptor (CAR) T cells has revolutionized the therapy of relapsed/refractory B-cell precursor ALL. High initial remission rates are nevertheless hampered by a lack of long-term persistence due to T cell exhaustion. Sustained proliferation and persistence of less differentiated T cell populations can counteract the effects of exhausted immune cells and confer a superior anti-tumor response to immunotherapy. Of particular interest in this regard is the interaction between interleukin-21, a common gamma chain receptor cytokine, and its receptor complex, IL-21R, which consists of the specific alpha chain and the common gamma chain (γC). The ligand/receptor interplay leads to improved proliferation and the generation of long-lived memory CD8+ T cells. This in vitro study characterizes the role of IL-21R expression in the setting of CAR T cells with the aim of improving long-term persistence of T cell-based immunotherapy. First, primary T cells with altered expression of the IL-21R alpha chain were examined. A CRISPR/Cas9 genomic knock-out of the receptor had no detrimental effects on T cell functionality, whereas retroviral overexpression of the IL-21R led to slower expansion, a less differentiated phenotype and reduced secretion of pro-inflammatory cytokines in CD8+ cells, even after stimulation with IL-21. Co-expression of the common γC played no additional role in the IL-21/IL-21R interaction in primary T cells. To examine this effect in a CAR setting, first- and second-generation bicistronic CAR constructs overexpressing the IL-21R alpha chain were compared to conventional CAR T cells. Specific anti-CD19 functionality was observed in all CAR constructs, with direct cytotoxicity and proliferation comparable after stimulation with CD19+ target cells. Interestingly, INF-γ secretion was consistently lower in IL-21R overexpressing CAR T cells, while addition of IL-21 partially reversed this trend. Further experiments focused on the effect of IL-21/IL-21R interaction in a second-generation CAR construct with a 4-1BB co-stimulatory domain. Not surprisingly, CAR T cells with a CRISPR/Cas9 genomic knock-out of IL-21R performed similarly to conventional CAR T cells. In contrast, IL-21R overexpressing CAR T cells demonstrated signs of improved persistence, as they retained a high proliferative capacity despite the rapid increase in effector functions after addition of IL-21 to the co-culture. No signs of early T cell dysfunction were detected, since no marked rise in expression of the co-inhibitory markers TIM-3 and PD-1 was observed. Moreover, the interplay between IL-21 and its receptor in the setting of IL-21R overexpressing CAR T cells led to the increased secretion of pro-inflammatory cytokines as well as IL-10. In a final experiment, IL-21 was overexpressed in CD4+ and IL-21R in CD8+ CAR T cells with the aim of mimicking the physiologic interaction between the two T cell populations. This novel approach led to superior functionality when compared to standard-of-care CAR T cells: the highest IFN-y Secretion was observed when IL-21 was overexpressed in CD4+ CAR T cells while IL21R was expressed in CD8+ CAR T cells. These promising results underscore the in vitro benefits to T cell functionality after interaction between IL-21 and its receptor, which may improve the long-term persistence of CAR T cell-based immunotherapy. Combining IL-21 producing CD4+ with IL-21R overexpressing CD8+ CAR T cells can help further examine the in vivo persistence of IL-21 receptor overexpressing CAR T cells.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 11. Oktober 2023
• 1. Bericht­erstatter:in: Feuchtinger, Tobias