Immune Cell Phenotype and Function After Treatment With Blinatumomab For Childhood Relapsed B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

Blinatumomab is a bi-specific monoclonal antibody designed to engage and tether cytotoxic T-cells (CTL) to CD19-expressing target B cells. An ongoing phase I multicenter study in pediatric patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has shown that blinatumomab induces morphological and molecular remissions, defined as minimal residual disease (MRD) levels <10^-4, in 47% of patients [Gore L, et al. J Clin Oncol 31, 2013 (suppl; abstr 10007)]. It is presently unknown whether and to what extent blinatumomab affects T-cell phenotype and function in pediatric patients with BCP-ALL.

Eight children diagnosed with relapsed/refractory BCP-ALL at the Bambino Gesù Children’s Hospital in Rome (median age at diagnosis 5.8 years, range 0.5-14.6) received blinatumomab as continuous intravenous infusion for 28 consecutive days, followed by a 2-week drug-free period. Four out of 8 patients were given repeated treatment courses. Peripheral blood samples were collected before treatment (day 0) and weekly thereafter, for 4 consecutive weeks. Bone marrow (BM) aspirates were available on days 0 and +29 of each drug course. Peripheral blood mononuclear cells (PBMC) were labeled with appropriate combinations of fluorochrome-conjugated monoclonal antibodies to quantitate naïve/memory T cells, αβ/γδ-expressing T cells and other immune effectors with potential anti-leukemia activity, such as CD3⁺CD56⁺ natural killer (NK) T cells and CD3^-CD56⁺ NK cells. T-cell production of interferon (IFN)-γ, interleukin (IL)-4 and IL-17 was measured at the single-cell level, after short-term (4-hour) stimulation with phorbol myristate acetate (PMA) and ionomycin. The TCR-Vβ Repertoire Kit® (Beckman Coulter, Milan, Italy) allowed the flow cytometry analysis of 24 different Vβ specificities on T cells, thus covering approximately 70% of the normal human TCR-Vβ repertoire.

Peripheral blood lymphocytes reached their nadir on day +1 (median 300/µL of blood [inter-quartile range 40-380] compared with 1,080/µL of blood at baseline [inter-quartile range 360-2,310]; p=0.0037 by Mann-Whitney U test for paired data), expanded within 7 days up to 3.5-fold above baseline, and included both CD4⁺ and CD8⁺ T cells. By contrast, the frequency of both CD3⁺CD56⁺ NK T cells and CD3^-CD56⁺ NK cells remained unchanged compared to baseline. IFN-γ production by patient-derived CD4⁺ T cells exceeded that observed in CD4⁺ T cells from healthy controls by 2-fold, indicating robust T helper type 1 (Th1) polarization. The frequency of Th2/Th17 cells, defined as CD4⁺IL-4⁺ and CD4⁺IL-17⁺ cells, respectively, was not different after treatment compared to baseline. CD31 expression on recovering CD45RA⁺ naïve T cells, a surrogate phenotypic feature for recent thymic emigrants (RTEs), suggested that thymic output may contribute to T-cell expansion after blinatumomab administration. Non-significant changes in the relative proportion of TCR-αβ and TCR-γδ-expressing CD3⁺ T cells were detected after treatment (median 79.5% TCR-αβ⁺ T cells and 19.3% TCR-γδ⁺ T cells among total CD3⁺ cells) compared with baseline (median 87.4% TCR-αβ⁺ T cells and 12.2% TCR-γδ⁺ T cells among total CD3⁺ cells). Importantly, both CD3⁺CD8^bright T cells and NK cells expressed lytic granule proteins, such as perforin and granzyme-B, at levels that increased during treatment. The analysis of Vβ TCR repertoire revealed a restricted usage of single Vβ domains by BM-resident CD8⁺ T cells, but not by CD4⁺ T cells. Specifically, the sum of Vβ within CD8⁺ T cells in the BM averaged 56.7±6.2% after blinatumomab, compared with 78±5.1% in healthy controls (p=0.04; Mann-Whitney U test for unpaired data).

Blinatumomab expands both CD31⁺CD45RA⁺ thymic-naïve and memory T cells with heightened IFN-γ production and is highly effective at clearing MRD in children with BCP-ALL. Skewing of the Vβ repertoire within BM-resident CD8⁺ T cells may be consistent with clonal expansions.

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