Groups of mice were treated daily for 6 days with fusion protein, treated with vehicle, or untreated as indicated in the legend of Fig. 6. On day 7, the animals were killed; the omenta were removed and treated with collagenase, then stained for flow cytometry as described previously with minor modifications.32 Preliminary Gefitinib nmr experiments were performed using normal omental cells, tumour cells and a reconstructed mixture of tumour cells and omental cells to establish the gates shown. Colony-forming assays were performed as described previously.33 Statistical analyses testing for significance were performed as indicated in the figure legends. We set out to create a cytokine fusion protein that could be cleaved
by a tumour cell expressed protease so that it becomes more active after cleavage. We initially tested a strategy based on steric hindrance PKC412 chemical structure by constructing
a fusion protein consisting of IL-2 and Mip1-a separated by a PSA cleavage sequence.34 We hypothesized that both immunomodulatory proteins would be largely inactive in the fusion protein because of their close proximity, but would become more active if the fusion protein could be successfully cleaved, thereby separating the two proteins. Although the fusion protein could be expressed and cleaved by PSA, IL-2 did not appear to be attenuated in the intact fusion protein and the biological activity of the IL-2 did not increase after cleavage (data not shown). Hence, simply joining two molecules, even very closely, does
not necessarily interfere with their functional activity. However, we reasoned that if we constructed a molecule in which the putative inhibitory portion of the fusion protein bound the cytokine specifically, this would be more likely to inhibit its activity. As we describe below, we used two distinct strategies to inhibit the biological activity of IL-2. The first strategy employed a cytokine receptor whereas the second used an antibody fragment (scFv). The first strategy using specific inhibition employed IL-2 and a portion of the IL-2 receptor is illustrated schematically in Fig. 1(a). We used mouse IL-2 cDNA and took advantage of aminophylline the alpha chain of the IL-2 receptor (IL-2Rα) which can bind IL-2 in the absence of the other subunits (β and γ) of the high-affinity IL-2 receptor.35 In this construct, we eliminated the transmembrane and cytoplasmic region of the IL-2Rα chain, creating a soluble form of the receptor. To increase flexibility and allow the IL-2Rα portion of the molecule to fold back and inhibit IL-2, we also introduced a repeating Gly–Ser linker consisting of (GGGGS)2 (designated 2 ×), or (GGGGS)436 (designated 4 ×), and in some cases also added a 6 × His tag. These plasmids were used to construct recombinant baculoviruses to mediate expression in insect cells as described in the Materials and methods. As shown in Fig. 1(b), we examined the fusion proteins with a capture ELISA using antibodies reactive with IL-2Rα and IL-2. Also, the immunoblot analysis in Fig.