Fig. 2. Pro-IL-12 shields IL-12 activity in vitro and reduces systemic toxicity in vivo.
(A-B) Successful cleavage of pro-IL-12 by MMP14. 5μg of each pro-IL-12 fusion protein was co-cultured with or without 200ng activated MMP14 at 37°C overnight. The MMP14-digested pro-IL-12 under different conditions was analyzed by Western Blot (A) and Hek-Blue™ IL-12 reporter cell line (B).
(C) Serum IFNγ levels of C57BL/6N mice (n=9/group) bearing MC38 tumors after treatment with PBS, 33.3pmol of hetero-IL-12-Fc or pro-IL-12. Serum was collected six hours after the third injection and measured by Cytometric Bead Array (CBA).
(D) Body weight loss of C57BL/6N mice (n=9/group) inoculated with 5×105 MC38 cells and then treated with PBS, 33.3pmol of hetero-IL-12 or pro-IL-12 by i.p. injection on days 10, 13, and 16. Mouse body weight was measured every two days, starting from day 0 of the first injection.
Data indicate mean ± SEM and are repeated two or three independent experiments. Statistical analysis for C was performed using unpaired two-tailed t-tests.*P <0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.