Conclusions
The described products may represent useful research tools for the study of the anti-arthritic properties of TNF blockade and of IL10-based immunocytokines in syngeneic immunocompetent models of arthritis.
Methods
Two fusion proteins (termed muTNFR-Fc and F8-muIL10) were cloned, expressed in chinese hamster ovary (CHO) cells, purified and characterized. Biological activity of muTNFR-Fc was assessed by its ability to inhibit TNF-induced killing of mouse fibroblasts, while F8-muIL10 was characterized in terms of muIL10 activity, of binding affinity to the cognate antigen of F8, the alternatively-spliced EDA domain of fibronectin, by quantitative biodistribution analysis and in vivo imaging. The therapeutic activity of both fusion proteins was investigated in a collagen-induced mouse model of arthritis. Mouse plasma was analyzed for anti-drug antibody formation and cytokine levels were determined by bead-based multiplex technology. The association of F8-IL10 proteins with blood cells was studied in a centrifugation assay with radiolabeled protein.
Results
Both fusion proteins exhibited excellent purity and full biological activity in vitro. In addition, F8-muIL10 was able to localize on newly-formed blood vessels in vivo. When used in a murine model of arthritis, the two proteins inhibited arthritis progression. The activity of muTNFR-Fc was tested alone and in combination with F8-huIL10. The chimeric version of F8-IL10 was not better then the fully human fusion protein and showed similar generation of mouse anti-fusion protein antibodies. Incubation studies of F8-muIL10 and F8-huIL10 with blood revealed that only the fully human fusion protein is not associated with cellular components at concentrations as low as 0.2 μg/ml, thus facilitating its extravasation from blood vessels. Conclusions: The described products may represent useful research tools for the study of the anti-arthritic properties of TNF blockade and of IL10-based immunocytokines in syngeneic immunocompetent models of arthritis.