Figure 2.

Preclinical characterization of mIL12-7NP2. (A) Schematic representation of mIL12-7NP2; (B) sodium dodecyl-sulfate polyacrylamide gel electrophoresis, 10% gel in non-reducing (NR) and reducing (R) conditions of purified mIL12-7NP2; (C) size exclusion chromatogram of mIL12-7NP2; (D) interferon-gamma (IFN-γ) induction assay by mIL12-7NP2 in freshly isolated BALB/c splenocytes. Cells were resuspended at 5×10⁶ cells/mL and incubated for 5 days at 37°C and 5% CO₂ with or without 0.1 pM of the interleukin-12 (IL-12) derivatives. IFN-γ was measured in cultured supernatants by sandwich ELISA; (E–F) quantitative biodistribution analysis of radioiodinated mIL12-7NP2 in BALB/c nude mice bearing SKRC52 renal cell carcinoma (E) or HT1080 (F) fibrosarcoma. Results are expressed as percentage of injected dose per gram of tissue (%ID/g±SEM; n=4–5); (G) microscopic fluorescence analysis of tumor-targeting performance of mIL12-7NP2 in CT26-hFAP tumor and organs from BALB/c mice. Cryosections were stained with ProteinA-AlexaFluor 488; cell nuclei were stained with DAPI (blue). 20× magnification, scale bars=100 µm; (H) therapeutic performance of mIL12-7NP2 in BALB/c nude mice bearing SKRC52-hFAP human renal cell carcinoma. Data represent mean tumor volume±SEM, n=6 mice per group; (I) therapeutic performance of mIL12-7NP2 in BALB/c mice bearing CT26-hFAP colon carcinoma. Data represent mean tumor volume±SEM, n=3-5 mice per group. CR, complete response; hFAP, human fibroblast activated protein; IP-10; IFN-inducible protein 10; i.v., intravenous; TNF, tumor necrosis factor. *P<0.05; **p<0.01; ***p<0.001; ****p<0.0001.