Background: The T-cell engager antibody blinatumomab (Blincyto)
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 PAX5, one of the most often mutated genes in
B-ALL, affecting the CD19 surface expression on lymphoblasts, and could be
explored in vitro 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 in vitro to blinatumomab and the simultaneous increase in blast
mortality and T-lymphocytes activation induced by the drug was observed at day +7
(both effects: p 0.0001 versus untreated, two-way ANOVA,
Bonferroni post-test), and was particularly pronounced in REH compared to NALM6
co-cultures (p 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,
p-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 in vitro (simultaneous blast mortality and
T-lymphocyte activation: both effects: p 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 PAX5-regulated CD19 expression is unclear
and whether it is predictive of in vivo response to therapy remains to
be established. Further dedicated studies are required to investigate these
issues in detail.