Patient's T Cell Functionality Determines Blinatumomab-Mediated Killing of B-ALL Leukemia Cells

Introduction B cell acute lymphoblastic leukemia (B-ALL) is a severe disease caused by malignant transformation and uncontrolled proliferation of immature B lymphocytes. Blinatumomab, a Bi-specific T cell engager (BiTE®) combining the VH and VL domains of two antibodies against human CD19 and CD3, has been approved by U.S. Food and Drug Administration (FDA) for the treatment of Philadelphia chromosome negative (Ph-) relapsed or refractory B-ALL in 2014, and recently Philadelphia chromosome-positive (Ph+) B-ALL. Blinatumomab brings killer T and target B cells into close proximity, activating patients' autologous T cells to kill both normal and malignant B cells via mechanisms such as cytolytic immune synapse formation and inflammatory cytokine production. Clinical trials have shown that there are still patients refractory to blinatumomab. It is thus of great importance to understand the resistance mechanisms and search for biomarkers for patient selection.

Methods and Results In this study, we used Mass cytometry-based immunophenotyping and Luminex based multiple protein quantitation, to search for biomarkers correlated with the killing capacity of blinatumomab. First, we collected PBMCs from 12 B-ALL patients for an in vitro killing assay, focusing on the quantity and quality profiling of T cells. PBMCs were used as the effector cells and NALM-6 as the target cells. The final effector to target (E/T) ratio is 10:1. Blinatumomab were added to the co-cultured cells (final concentration 10 ng/mL) for 18hrs. A 5% specific lysis of NALM-6 was used as cut-off threshold. Samples with specific killing above 5% were classified as "positive" for blinatumomab response, and below 5% as "negative". Among the patients analyzed, 4 out of 12 patients (33.3%) showed apparent and specific lysis of NALM6 leukemia cells, while the remaining samples showed no obvious effect. This divergent killing effect of blinatumomab with the presence of PBMCs from different donors may reflect the differentiated treatment response in B-ALL patients.

Concurrently we immunophenotyped each patient's PBMCs by Mass cytometry with a panel of antibodies for surface lineage markers and intracellular effector molecules and cytokines /chemokines. Unsupervised clustering showed that a panel of T-cell associated biomarkers highly significantly correlated with B-ALL cell response to blinatumomab (p=0.001, R=0.839). This panel of biomarkers included T cell markers of CD45, CD3 and CD4; cytolytic proteins of Perforin and GranzymeB; cytokines of IL-2, INFγ and TNFα; and chemokine CCL4 (also known as MIP-1b).

In a Luminex analysis examining multiple proteins accumulating in the culture supernatant from the in vitro killing assay, the top biomarkers identified by Mass cytometry also showed significant response to blinatumomab at different time points (12, 24 and 48 hrs). They included TNF-α, CCL4, IL-2, INFγ and Granzyme B. Furthermore, released CCL4 at 24 and 48 hrs and INFγ at 48 hrs significantly correlated with blinatumomab mediated cytotoxicity.

Conclusion In summary, the immunophenotyping and multiplexed protein measurement by Mass cytometry and Luminex assay identified key biomarkers that may correlate with blinatumomab-mediated killing of B-ALL leukemia cells. The biomarker panel reflected the importance of primed T cell activation, associated key cytokine/chemokine as well as the consequent cytolytic protein release in the overall response to blinatumomab. Thus, in vitro biomarker profiling focusing on T cell status with these key molecules may facilitate patient selection and predict the response to the immunotherapy.