Fig. 5

Binding properties of the divalent scFvF7-Fc Ab. a Binding curve of purified divalent scFvF7-Fc on recombinant LAG3. 0.1 μg of recombinant LAG3 was coated on ELISA microwells, and then different amounts of the reconstituted F7 Ab were incubated for one hour. The Ab binding was finally revealed through the incubation with an HRP-conjugated anti-human antibody. The mean values ±SEM are shown as calculated from quadruplicate wells from a representative of two independent experiments. O.D.: optical density (450 nm–620 nm). b FACS analysis on LAG3 overexpressing 293 T cells. Both parental and LAG3 engineered 293 T cells were incubated with the divalent scFvF7-Fc Ab, and then with FITC-conjugated anti-human IgGs. Data are shown from a representative of six independent experiments. c Dose-response binding of the reconstituted F7 Ab on cell membrane of activated human CD8⁺ T lymphocytes. PBMCs were isolated from the peripheral blood of healthy donors, and then the CD8⁺ T fraction was isolated by immunomagnetic selection. The cells were activated with PHA and, after six days, labeled with the indicated amounts of the divalent scFvF7-Fc Ab. As control, activated cells were labeled with the secondary Abs alone. As an additional control, unstimulated CD8⁺ T cells were labeled with the highest anti-LAG3 Ab concentration. The results shown are representative of two independent experiments