Cytofluorimetric assay to investigate variability in blinatumomab response.

Background: The T-cell engager antibody blinatumomab (BlincytoT⁢M) represents a promising rescue therapy for relapsed/refractory CD19+ acute lymphoblastic leukemia (B-ALL), although ~20-30% of patients still do not respond to treatment. Blinatumomab creates a tight synapsis between CD3+ T-lymphocytes and leukemic CD19+ B-cells, resulting in a granzyme B (GzB)-mediated specific lysis of leukemic cells.

Methods: Aim of the study was to provide evidence that variability in blinatumomab response could have a genetic basis in , one of the most often mutated genes in B-ALL, affecting the CD19 surface expression on lymphoblasts, and could be explored by means of a cytofluorimetric assay, staining both surface antigens (CD45, CD19 and CD3) and intracytoplasmic markers (7AAD, Syto16). Two human immortalized B-ALL cell lines (NALM6 and REH) were chosen for their different PAX5 and CD19 protein levels, as verified by western blot and flow cytometry, respectively.

Results: In contrast to NALM6, REH cells do not express the full-length PAX5 protein and show less CD19 on the cell surface (fluorescence peak median intensity: 9155 versus 28895). Co-cultures of CD3+ T-lymphocytes from healthy donors and B-ALL cell lines were seeded at an effector-to-target cell ratio of 1:10 for simulating the condition existing in the bone marrow due to the malignant invasion of blast cells. Co-cultures were exposed to blinatumomab and the simultaneous increase in blast mortality and T-lymphocytes activation induced by the drug was observed at day +7 (both effects: < 0.0001 versus untreated, two-way ANOVA, Bonferroni post-test), and was particularly pronounced in REH compared to NALM6 co-cultures ( < 0.05). Surprisingly, daily release of GzB in supernatants, measured by an ELISA assay, was significantly lower in drug-exposed REH co-cultures compared to NALM6 at early time-points (days +3 and +4, -value < 0.0001, three-way ANOVA), reaching a comparable plateau only towards the end of the incubation period (at day +5). Only 2 out of 5 primary co-cultures of leukemic and mononuclear cells isolated from bone marrow aspirates of B-ALL patients (age: median 10.7 years, interquartile range (IQR) 3.4; males: 60%) responded to the drug (simultaneous blast mortality and T-lymphocyte activation: both effects: < 0.0001 versus untreated) and at different drug concentrations. Results were unrelated to the percentages of immature CD19+ B-cells in the diagnostic samples.

Conclusions: In conclusion, cytofluorimetric analysis can highlight the different response induced by blinatumomab among co-cultures. Whether and how this difference is affected by -regulated CD19 expression is unclear and whether it is predictive of response to therapy remains to be established. Further dedicated studies are required to investigate these issues in detail.