Accomplices in Cure: Blinatumomab + Dasatinib Reduces TCR Signaling and Effector Function Stronger Than Ponatinib and Ameliorates T-Cell Exhaustion
Authors: Philipp Nora , Marcinek Anetta , Hänel Gerulf , Nixdorf Daniel , Buecklein Veit L. , Subklewe Marion 
Published: December 2023

The tyrosine kinase inhibitors (TKIs) dasatinib (dasa) and ponatinib (pona) are being tested in clinical trials in philadelphia chromosome-positive (Ph +) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients in combination with the CD19xCD3 bispecific blinatumomab (blina). Pona but not dasa is able to bind the BCR-ABL T315I mutation, which frequently occurs in BCP-ALL patients. Therefore, clinical studies are moving away from dasa and favor pona. Nevertheless, both TKIs in combination with blina have led to striking remission rates in Ph + BCP-ALL (complete response dasa: 98%, Foá 2020; pona: 92%, Jabbour 2023). Interestingly, increased lymphocyte frequencies were reported in patients under blina + dasa therapy (Puzzolo 2021), indicating that the effect of dasa is not limited to toxicity towards ALL cells conferred by BCR-ABL inhibition. Indeed, dasa modulates T-cell function by interfering with TCR signaling (Mestermann 2019). Also, we have shown that resting of T cells by high-dose dasa treatment ameliorates bispecific-induced T-cell exhaustion (Philipp 2022). In contrast, less is known about the effect of pona on T-cell function. Based on these data, we hypothesize that TKIs fine-tune TCR signaling, which ameliorates T-cell exhaustion during continuous blina infusion. Here, we systematically compared the influence of dasa and pona on blina-mediated TCR signaling and T-cell function.

Inhibition of T-cell activation and function by dasa or pona at 0.3 - 100 nM was tested in vitro over 3 days: (1) CD69 expression, (2) blina-mediated killing as specific lysis of CD19 + tumor cells (3) T-cell expansion analyzed by dye dilution or CD2+ fold change (FC), (4) cytokine secretion measured by cytokine catch or cytometric bead array.

Inhibition of TCR signaling was evaluated using a Jurkat-NFAT T-cell line stimulated for 6h with blina and CD19 + tumor cells. Phosphoflow of pAKT and pZAP70 was performed on CD3/CD28 activated primary human T cells. Toxicity of the TKIs against BCR-ABL +/- cell lines was evaluated after 3 days.

T-cell exhaustion upon continuous blina + dasa stimulation was assessed in an in vitro long-term culture system for continuous bispecific exposure (Philipp 2022). Functional assays as described in (1)-(4) were performed every 7 days with isolated T cells from long-term cultures.

Both TKIs inhibited T-cell function in a dose-dependent manner. Surprisingly, dasa was more potent than pona at low-intermediate doses in reducing T-cell function, shown by CD69 expression (mean % at TKI = 3 nM: basal = 70, dasa = 25, pona = 64, p&lt;0.0001), cytotoxic function (mean % specific lysis at TKI = 12.5 nM: basal = 55, dasa = 0, pona = 54, p&lt;0.0001), T-cell proliferation (mean % proliferated at TKI = 1.5 nM: basal = 62, dasa = 25, pona = 51, p&lt;0.0001), as well as IFN secretion (normalized mean fluorescence intensity (MFI) at TKI = 12.5 nM: basal = 655, dasa = 0, pona = 924, p&lt;0.0001). We also observed that NFAT engagement was significantly lower in dasa-treated Jurkat-NFAT cells (relative luminescence units at TKI 12.5 nM: basal = 1620, dasa = 392, pona = 996, p&lt;0.001). Similarly, phosphoflow analyses revealed lower levels of pAKT and pZAP70 upon CD3/CD28 + dasa versus pona treatment (MFI of pAKT at TKI = 25 nM: basal = 2347, dasa = 1642, pona = 2045). Importantly, both TKIs induced cell death of BCR-ABL + but not BCR-ABL - cell lines at concentrations &gt;3 nM.

Interestingly, 14 days continuously stimulated T cells with blina + dasa (12.5 nM) did not lead to co-expression of inhibitory checkpoints (mean % of PD-1 +Tim-3 +LAG-3 + T cells on day 14: blina = 31, +dasa = 0.6, p&lt;0.01). Furthermore, when isolated from long-term cultures and tested in functional assays, blina + dasa treated T cells maintained high cytokine secretion (mean IL-2 in pg/ml on day 14: blina = 730, blina + dasa = 5638, p&lt;0.01), granzyme B expression (mean MFI ratio of CD8 + on day 14: blina = 96, blina + dasa = 335, p&lt;0.01) and cytotoxic activity (mean % specific lysis on day 14: blina = 61, blina + dasa = 80).

Together, these findings indicate that while both TKIs confer comparable toxicity to BCR-ABL + leukemia cells, dasa dampens TCR signaling and T-cell effector function more potently than pona. Strikingly, dasa can ameliorate T-cell exhaustion upon continuous blina exposure. Our data suggest that low-dose TKIs can fine-tune T-cell activity and might play a considerable role for the clinical success of combinatorial bispecific and TKI therapy in Ph + BCP-ALL.