There is an increasing biotechnological interest in 'arming' therapeutic
antibodies with bioactive payloads. Many antibody-
cytokine fusion
proteins (immunocytokines) have been described and some of these
biopharmaceuticals have progressed to clinical studies. Here, we describe for the first time the expression and in vivo characterization of immunocytokines based on murine IL1β and
IL6. These potent pro-inflammatory
cytokines were fused at the N-terminus or at the C-terminus of the
monoclonal antibodies F8 (specific to the alternatively-spliced extra-domain A domain of
fibronectin, a marker of
tumor angiogenesis). All immunocytokines retained the binding properties of the parental antibody and were homogenous, when analyzed by
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and size-exclusion chromatography, except for the N-terminal fusion of IL1β which revealed the presence of glycosylated species. When analyzed by quantitative biodistribution analysis using radioiodinated
protein preparations, F8 fusions with
IL6 revealed a preferential accumulation at the
tumor site for both
cytokine orientations, whereas IL1β fusions exhibited lower
tumor to organ ratios and a slower blood clearance profile. The fusion
proteins with the
cytokine payload at the C-terminus were studied in
therapy experiments in immunocompetent mice bearing F9
tumors. Immunocytokines based on IL1β resulted in 10%
body weight loss at a 5-µg dose, whereas IL6-based products caused a 5%
body weight loss at a 225-µg dose. Both F8-IL1β and F8-IL6 exhibited a <50% inhibition of
tumor growth rate, which was substantially lower than the one previously reported for F8-TNF, a closely related pro-inflammatory immunocytokine. This study indicates that
IL6 can be efficiently delivered to the
tumor neo-vasculature by fusion with the
F8 antibody. While F8-IL6 was not as potent as other F8-based immunocytokines that exhibit similar biodistribution profiles, the fusion
protein sheds light on the different roles of pro-inflammatory
cytokines in boosting immunity against the
tumor.