T-Cell Receptor Signaling and Dysfunction during Blinatumomab Therapy in Pediatric Acute Lymphoblastic Leukemia

Introduction: While blinatumomab induces minimal residual disease (MRD) negativity in most patients with MRD+ B-cell acute lymphoblastic leukemia (B-ALL), the response is transient in a substantial proportion of patients. Moreover, the biology underlying the loss of response is not well understood; one small study reported T-cell exhaustion with continued blinatumomab exposure in adults with relapsed/refractory B-ALL. The effects of blinatumomab on the different components of the T-cell receptor (TCR) signaling cascade remain undefined. Notably, blinatumomab is frequently administered as a consolidation therapy during complete remission (CR) in pediatric and adult B-ALL regimens. Differences in T-cell function between high and low leukemia burden states and the age-related T-cell dysfunction in adults underscore the need for T-cell studies during blinatumomab therapy for pediatric patients in CR. However, the challenges in conducting T-cell biology studies on low volume blood samples in children have resulted in a paucity of pediatric data. The goal of this study was to assess changes in TCR signaling, T-cell memory subsets, and T-cell function during a 28-day blinatumomab cycle in pediatric CD19+ leukemia with low burden disease. Methods: We conducted a single center prospective study of patients aged 0-25 years with CD19+ acute leukemia who received blinatumomab. Blood samples were obtained pre-blinatumomab (baseline) and on ~days 14 (mid) and 28 (end) during a 28-day blinatumomab cycle; 23-color spectral flow cytometry was used to evaluate T-cell naïve/memory subsets, activation markers, and exhaustion profiles in these blood samples. T-cells were also cocultured with B-ALL cells (REH cell line) with and without blinatumomab (0.5 ng/mL, biosimilar reagent) at a 1:3 effector to target ratio (E:T) for 72 hours. Flow cytometry was then used to assess cytotoxicity, T-cell activation, TCR signaling (phospho [p-] mTOR, p-MEK, p-S6, p-p38 MAPK), and T-cell cytokine responses (interleukin-2 [IL2], interferon-gamma [IFN-γ], tumor necrosis factor-alpha [TNF-α]). Early TCR signaling was assessed via flow cytometry for p-ZAP70 in T-cells cocultured with B-ALL cells (E:T = 1) and blinatumomab (10 ng/ml) for 1 hour. All assays were optimized for low cell numbers. Results: Eleven patients were enrolled (median age 5 years, range 1-21 years); 55% had high-risk B-ALL or mixed phenotype acute leukemia. Most patients (91%) were MRD negative prior to blinatumomab; 82% were in first CR. Thirty-six percent received blinatumomab due to toxicities precluding chemotherapy. T-cells showed a decline in cytotoxic function with continued blinatumomab exposure (mean % lysis of CD19+ cells induced by T-cells from baseline vs mid vs end blood samples = 71% vs 56% vs 53%, n = 10 patients, ANOVA p=0.011). However, there was inter-patient heterogeneity in the temporal profile of cytotoxic function. Three patients showed sustained cytotoxicity throughout the 28-day cycle; these patients had higher baseline T-cell function (mean % CD19+ lysis by baseline T-cells for patients with sustained vs unsustained cytotoxicity = 93% vs 63%, p=0.003). The proportion of stem memory (p=0.027) and regulatory T cells (p=0.053) decreased, while naïve, central memory, effector memory, effector, and activated T-cell subsets were unchanged with continuous blinatumomab exposure (n=11 patients, T-cells from baseline vs mid vs end). Exhaustion marker analysis showed that the % TIM3+ T-cells increased with blinatumomab exposure (p=0.003, baseline vs mid or end), while the expression of PD1, LAG3, CTLA4, and TIGIT did not change. T-cells showed a decrease in both early (p-ZAP70) and late (p-mTOR, p-MEK, p-S6) TCR signaling events as well as IFN-γ production during the blinatumomab cycle (p=0.011, baseline vs mid or end). Conclusions: Loss of T-cell function including impaired proximal TCR signaling was seen with continuous blinatumomab exposure in the setting of pediatric CD19+ leukemia in CR. Patients with robust baseline T-cell function exhibited sustained cytotoxicity throughout the 28-day blinatumomab cycle. These findings suggest that a shorter duration of blinatumomab or treatment-free intervals may improve response durability. Further studies are needed to investigate baseline T-cell function as a biomarker for informing dosing schedules. RNA sequencing is ongoing to identify mechanisms of T-cell dysfunction.