Department of Medical, Surgical and Health Sciences, University of Trieste, 34149 Trieste, Italy.
Azienda Sanitaria Universitaria Giuliano Isontina (ASUGI), 34148 Trieste, Italy.
Front Biosci (Landmark Ed). 2022 Jan 24;27(2):39. doi: 10.31083/j.fbl2702039.
The T-cell engager antibody blinatumomab (BlincytoTM) 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.
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.
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.
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.
T 细胞衔接抗体blinatumomab（blinatumomab）代表了一种有前途的挽救疗法，适用于复发/难治性 CD19+急性淋巴细胞白血病（B-ALL），尽管仍有约 20-30%的患者对治疗无反应。Blinatumomab 在 CD3+T 淋巴细胞和白血病性 CD19+B 细胞之间形成紧密的突触，导致颗粒酶 B（GzB）介导的白血病细胞特异性裂解。
本研究旨在提供证据，证明blinatumomab 反应的可变性可能具有遗传基础，是 B-ALL 中最常突变的基因之一，影响淋巴母细胞表面 CD19 的表达，并可通过细胞荧光计检测来探索，同时染色表面抗原（CD45、CD19 和 CD3）和细胞内标记物（7AAD、Syto16）。选择两种人永生化 B-ALL 细胞系（NALM6 和 REH），因为它们的 PAX5 和 CD19 蛋白水平不同，分别通过 Western blot 和流式细胞术验证。
与 NALM6 相反，REH 细胞不表达全长 PAX5 蛋白，细胞表面的 CD19 表达较少（荧光峰中位强度：9155 与 28895）。将来自健康供体的 CD3+T 淋巴细胞与 B-ALL 细胞系以效靶细胞比 1:10 共培养，模拟由于白血病细胞恶性侵袭而存在于骨髓中的条件。将共培养物暴露于 blinatumomab 中，并观察到药物在第+7 天诱导的白血病细胞死亡率和 T 淋巴细胞激活增加（两种作用：均<0.0001，双向 ANOVA，Bonferroni 后检验），在 REH 与 NALM6 共培养物中更为明显（<0.05）。令人惊讶的是，通过 ELISA 测定，在早期时间点（第+3 和第+4 天，-值<0.0001，三向 ANOVA），药物暴露的 REH 共培养物中上清液中 GzB 的每日释放量明显低于 NALM6，仅在孵育期结束时才达到可比的平台（在第+5 天）。从 B-ALL 患者骨髓抽吸物中分离的白血病和单核细胞的 5 个原发性共培养物中，只有 2 个对药物有反应（同时诱导白血病细胞死亡率和 T 淋巴细胞激活：两种作用：均<0.0001，与未处理组相比），并且在不同的药物浓度下有反应。结果与诊断样本中不成熟 CD19+B 细胞的百分比无关。
总之，细胞荧光分析可以突出 co-cultures 之间由 blinatumomab 诱导的不同反应。-调控的 CD19 表达如何以及是否影响这种差异尚不清楚，它是否能预测治疗反应仍有待确定。需要进一步的专门研究来详细探讨这些问题。
