Cloning, expression and in vitro characterization of F8-muIL10. (a) Schematic representation of the cloning strategy of F8-muIL10. The murine IL-10 moiety was fused by a 15-amino-acid linker (SSSSG)3 to the C-terminus of the F8 scFv antibody fragment in diabody format (five-amino-acid linker between variable heavy chain (VH) and variable light chain (VL)). SS, signal sequence. (b) Schematic representation of protein domain assembly of the noncovalent F8-muIL10 dimer. (c) SDS-PAGE analysis of purified F8-muIL10. M, molecular weight marker; NR, nonreducing conditions; R, reducing conditions. (d) Size exclusion chromatography (SEC200) of noncovalent homodimeric F8-muIL10. (e) MC/9 cell proliferation assay. F8-muIL10 and recombinant murine IL10 induced proliferation of MC/9 cells (mean ± standard deviation (SD), n = 3). (f) BIAcore analysis of F8-muIL10 on extra-domain A of fibronectin (EDA)-coated chip. (g) Quantitative biodistribution study of radioiodinated F8-muIL10. Mice bearing subcutaneous F9 tumors were injected intravenously with 15 μg radiolabeled protein (n = 3). Mice were sacrificed after 24 hours and organs were excised and radioactivity counted, expressing results as percent of injected dose per gram of tissue (%ID/g ± SD).
Doll et al. Arthritis Research & Therapy 2013 15:R138 doi:10.1186/ar4319