Dasatinib enhances potency of a novel CD123 CAR against AML by improving cell fitness Free

Background: CD123, the interleukin-3 receptor alpha chain (IL-3Ra), is overexpressed in hematologic malignancies including acute myeloid leukemia (AML), at both the level of leukemic stem cells (LSCs) and leukemic blasts. This makes CD123 an attractive therapeutic target. However, fratricide has been identified as a confounding issue impacting CD123 CAR-T production and efficacy of the therapeutic cell product. CD123 expression is typically negligible on naïve T cells and is upregulated, at low levels, following T cell activation. A potential approach for improved production yields and CAR activity is to modulate tyrosine kinase activation during production. Dasatinib, a small molecule Src-and Abl-family tyrosine kinase inhibitor, prevents T cell activation upon CD3, CD28, TCR or CAR stimulation. This effect is achieved through inhibition of proximal T cell receptor-associated kinases. Transient rest, induced by intermittent application of dasatinib during CAR T cell manufacturing, was reported to slow down terminal differentiation, abrogate tonic signaling, promote formation of T cell memory, prevent fratricide and reverse exhaustion of CAR T cells. Here, we developed a second-generation anti-CD123 CAR and investigated the effect of dasatinib on T cell fitness and potency of CAR-T against AML.

Method: A novel fully human scFv anti-CD123 CAR featuring a 4-1BB (CD137) co-stimulatory domain and a CD3ζ activation domain was generated. Primary human T cells from healthy donors were enriched and transduced with lentiviral vector encoding the CD123 CAR. Transduced T cells were manufactured +/- dasatinib, and CAR expression was detected by flow cytometry. CAR123 or untransduced (UTD) control T cells were co-cultured with target (CD123⁺ MOLM-14 ), or control (CD123^-293T) cells for 18h. Target/control cell numbers and co-culture IL-2, IFN-γ, and TNF-a cytokine levels were evaluated by luciferase assay and ELISA, respectively. CAR T cell surface expression of phenotype markers were measured by flow cytometry. Fratricide was quantified by co-culture of CD123 CAR T cells with labeled CD123+ or CD123- (untransduced control) cells. CAR123 T cell phenotype and potency was evaluated in vivo using a MOLM-14 acute myeloid leukemia NSG xenograft model. In addition, myelotoxicity was investigated in colony-forming unit assays.

Results: Lentiviral T cell transduction resulted in robust CD123 CAR. However, increased CD123 expression was observed on activated T cells during CAR T manufacturing raising concerns of potential fratricide. In fratricide CAR-T co-culture assays, a significant reduction was noted in the CD123⁺ target cell fraction. Importantly, dasatinib treatment during manufacture of CAR-T cells mitigated the reduction of the CD123+ T cell population. Surprisingly, dasatinib engineered CD123 CAR T cells demonstrated improve cytotoxicity of MOL14 targets in vitro. During manufacture, dasatinib treated CAR123 T cells had reduced fratricide (90% to 50%), reduced exhaustion (20-30% to 5-10%) and increased central memory phenotype (60% to 95% ) compared to controls. Dastainib engineered CAR T cells were evaluated in in vitro cytotoxicity assays and demonstrated maintenance of cytotoxic function and a 3-5 fold increase inpro-inflammatory cytokines. In AML xenograft mouse models, the rate of tumor clearance during the first 7 days was increased with dasatinib engineered CAR T cells. Robust cytokine production and improved expansion/persistence was observed in dasatinib-engineered CAR123 T cells in vivo. To evaluate potential toxicity, colony-forming unit (CFU) assays were performed on healthy donor CD34+ HSPCs. CAR123 T cells, engineered +/- dasatinib were co-cultured with CD34+ cells overnight. No significant difference in total counts or colony proportions was observed when compared to controls. CAR123-associated myelotoxicity was not observed in vitro.

Conclusion: In summary dasatinib engineered fully human CAR123 T cells demonstrated potent cytotoxicity against AML in vitro and in vivo. Dastinib treatment also improved the exhaustion and cytokine phenotype of CAR123. Additionally, dasatinib engineered CAR123 maintained the desired safety characteristics. The current data supports the potential for dasatinib to improve CAR123 during manufacturing and result in an improved clinical product for treatment of AML.