Journal: Molecular Therapy Oncology
Article Title: A measles virus encoding a CD19/CD3 bispecific T cell engager shows enhanced preclinical anti-BCP-ALL efficacy without significant toxicity
doi: 10.1016/j.omton.2026.201127
Figure Lengend Snippet: MV-Blina infected cells secrete target-binding secBlina, which initiates ALL cell kill more efficiently than CD19/CD3-bsTE (A) Vero cells secrete secBlina. secBlina was harvested from the supernatant of infected Vero cells, followed by purification and concentration. Eluate (2 μg protein) was detected via His-tag by Western Blot in comparison to the commercial CD19/CD3-bsTE (1.8 μg). (B) Purified and concentrated secBlina binds specifically to target cells. Binding to target cells was confirmed by flow cytometry using CD3 − CD19 + REH, CD3 + CD19 - Jurkat, and CD3 − CD19 - K562 cell lines incubated with secBlina. Binding of secBlina to target cells was detected by flow cytometry via a His-tag antibody. (C) secBlina strongly binds to REH target cells. The REH cell line was first incubated with 1 μg secBlina. Increasing concentrations of CD19/CD3-bsTE were then added to displace cell-bound secBlina. Cell-bound secBlina was detected via an HA tag antibody by flow cytometry. (D) T cells in human PBMCs are activated by secBlina. Pooled PBMCs were stimulated with an (E) T cells are activated by secBlina during co-culture of PBMCs with REH cells. Pooled PBMCs were co-cultured with REH cells in a 1:1-ratio for 24 h while being treated with secBlina or the CD19/CD3-bsTE. Activated CD2 + CD69 + T cells were detected by flow cytometry. (F) MV-Blina infected leukemia cells produce and secrete secBlina. REH and Jurkat cells are shown 72 h post MV infection or Opti-MEM control. secBlina was detected by His-tag IF staining. Scale bars 50 μm. (G) secBlina effectively induces REH target cell kill compared to CD19/CD3-bsTE, whereas K562 control cells are unaffected. Target (T) cells REH (left panels) and negative control cells K562 (right panels) were incubated with secBlina (green) or CD19/CD3-bsTE (blue) for 24 h at indicated concentrations in the presence of PBMCs (effector; E) at different E:T ratios. Specific cell kill was determined by flow cytometry. Results in A, B, C, and F are shown as representatives of three independent experiments. Data are shown as mean ± SD of n = 3 (D, E) or n = 5 (G) independent experiments. Statistical analysis was performed using two-way ANOVA with Dunnett’s correction. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001.
Article Snippet: For the displacement assay, 1 × 10 6 REH cells were incubated with 1 μg secBlina for 30 min, followed by increasing concentrations of a research-grade analog of the CD19/CD3-bsTE blinatumomab (BPS Bioscience) for an additional 30 min, all at 4°C. secBlina bound to target cells was detected using rabbit anti-HA antibody (clone EPR22819-101, 1:600, ab256483, Abcam) for 30 min at 4°C, followed by a goat-anti-rabbit IgG (H&L) AF488 (1:2000, ab150077, Abcam).
Techniques: Infection, Binding Assay, Purification, Concentration Assay, Western Blot, Comparison, Flow Cytometry, Incubation, Co-Culture Assay, Cell Culture, Control, Staining, Negative Control
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