Fig. 2
Liposome-conjugated anti-CD137 and IL-2-Fc retain bioactivity with lower systemic exposure than the free drugs. a Representative cryo-TEM image of IL-2-Fc-liposome (anti-CD137 liposomes were qualitatively identical). b Polyclonal T-cells from C57Bl/6 mice were activated for 2 days with anti-CD3/CD28 beads, then soluble or liposomal IL-2-Fc (10 ng/mL of protein) were added, and CD8+ and CD4+ T-cell counts were determined on days 3 and 4. c Activated T-cells were incubated with soluble anti-CD137 or Lipo-αCD137 (final αCD137 concentration: 10 μg/mL) for 6 h, and the secreted IFN-γ in the cell culture supernatant was analyzed by ELISA. d–g Groups of C57Bl/6 mice were injected s.c. with 5 × 105 B16F10 tumor cells, and then received i.v. injections of fluorescently labeled soluble IL-2-Fc, soluble anti-CD137, Lipo-αCD137, or Lipo-IL-2-Fc proteins (100 µg αCD137, 60 µg IL-2-Fc) on day 10. Pharmacokinetics of the labeled proteins were followed over time in the blood (d, e) and protein levels in the organs were compared at 4 and 24 h later (f, g). *p < 0.05, **p < 0.005, ***p < 0.0005. h, i The anti-CD137 (h) and IL-2-Fc (i) accumulation in unpigmented B16F10-Trp2KO xenograft tumors were determined at 4 and 24 h after bolus i.v injection. *p < 0.01, **p < 0.005. j Twenty-four hours after i.v. injections of fluorescently labeled free or liposomal αCD137 and IL-2-Fc, lymphocytes from peripheral blood were collected, and their association with fluorescent protein drugs were analyzed by flow cytometry. *p < 0.05, **p < 0.01. All measurements shown are mean ± s.e.m. NS not significant
