Gilteritinib Augments Preclinical FLT3 and CD19 CAR T Cell Immunotherapy in High-Risk Pediatric Leukemias

Background: Despite impressive remission induction rates achieved with CD19 CAR T cells (CD19CART) in pediatric patients with relapsed/refractory B-ALL, long-term CD19-positive and CD19-negative relapse risk remains approximately 50%. We recently established potent preclinical efficacy of FLT3-directed CAR T cells (FLT3CART) against FLT3-mutant AML and KMT2A-rearranged (KMT2A-R) ALL, two high-risk pediatric leukemias characterized by activated intracellular FLT3 kinase signaling and/or high FLT3 cell surface expression (Niswander Haematologica 2023). We tested a dual FLT3 targeting strategy combining FLT3CART immunotherapy and second generation FLT3i kinase inhibitor gilteritinib which demonstrated enhanced in vivo leukemia clearance in preclinical models (Niswander ASH 2021). We initially hypothesized that the superior activity of dual therapy was attributable to combined direct anti-leukemia activity of each distinct therapeutic modality, but observed in pilot studies that co-treatment with FLT3i gilteritinib also directly increased the number of FLT3CART in vivo. In the current studies, we sought to determine the mechanism(s) of this combined effect and to test the applicability of gilteritinib co-therapy to other clinically-in-use CAR T cell immunotherapies for high-risk pediatric leukemias.

Methods and results: Co-treatment of human FLT3-ITD AML or KMT2A-R ALL cell line and patient-derived xenograft mouse models with FLT3CART and sub-curative doses of gilteritinib in vivo significantly improved leukemia clearance and animal survival with surprising concomitant dose-dependent increases in detectable peripheral blood FLT3CART. As FLT3 is not reported to be expressed in human T cells, we first hypothesized that increased FLT3CART in vivo was mediated indirectly by the anti-leukemia effects of gilteritinib co-treatment. However, naïve NSG mice treated with FLT3CART and gilteritinib (in the absence of human leukemia) demonstrated a significant increase in circulating peripheral blood FLT3CART, suggesting an antigen-independent mechanism of T cell augmentation. Surprisingly, in vitro gilteritinib exposure of FLT3CART alone or co-cultured with leukemia target resulted in a 10-20% increase in FLT3CART number with no detected differences in CD4:CD8 ratio or CD8+ memory/effector T cell subsets. FLT3CART co-cultured with gilteritinib also exhibited diminished surface activation marker expression upon target leukemia exposure. Similarly, CD8+ FLT3CART analyzed on the Isoplexis single-cell multiplexed cytokine platform with gilteritinib treatment displayed a decreased polyfunctionality strength index (PSI), the product of the percentage of polyfunctional cells and the signal intensity of secreted cytokines. Gene set enrichment analysis (GSEA) of FLT3CART exposed to gilteritinib confirmed lack of FLT3 gene expression and revealed upregulation of pathways related to mitochondrial oxidative phosphorylation, consistent with a less activated/more quiescent metabolic phenotype. In vitro exposure of FLT3CART to other second generation FLT3i crenolanib and quizartinib interestingly did not increase T cell numbers, suggesting a gilteritinib-specific phenomenon that may be mediated in part by inhibition of other T cell kinase(s) not targeted or less targeted by crenolanib and quizartinib. Finally, we also saw that the gilteritinib-mediated improvement in FLT3CART efficacy against FLT3-ITD AML and KMT2A-R ALL extended to other antigen-targeting CAR T cells. Indeed, co-treatment of AML or ALL xenograft mice with gilteritinib and CD33CART (Qin JITC 2021) or CD19CART, respectively, resulted in enhanced and sustained anti-leukemia activity compared to monotherapies, as well as notably increased CD33CART or CD19CART in murine peripheral blood, underscoring a potential broad applicability of gilteritinib-based dual therapy to improve CAR T cell immunotherapies already in the clinic.

Conclusions: Our data suggest that gilteritinib co-treatment directly enhances desired anti-leukemia activity of FLT3CART immunotherapy against FLT3-activated AML and KMT2A-R ALL. More broadly, these studies also highlight the potential for gilteritinib co-therapy to augment the efficacy of other clinically-in-use CAR T cell immunotherapies and present a promising combinatorial translational strategy for prospective clinical investigation.