Figure 1 Induction of FGFR1 by IFN-��/�� treatment in hepatic cancer cells. Development of an anti-FGFR1 monoclonal antibody We developed novel anti-FGFR1 mAbs by immunizing BALB/c mice with an FGFR1 expression vector. Six antibodies selleckchem Crizotinib recognizing FGFR1 were isolated from the mice, two of which, designated A2C9-1 and A2D11-1, showed strong affinity in ELISAs and were characterized further. For kinetic analyses, the extracellular domain of FGFR1 was covalently coupled to a CM-5 sensor chip at low density (215 response units of FGFR1), after which we determined the Kd values for A2C9-1 and A2D11-1 to be 209 nM and 7.03 ��M, respectively (Figure S2A). Thus A2C9-1 showed the strongest affinity for FGFR1.

Flow cytometric analysis confirmed that A2C9-1 reacts with FGFR1 (Figure 2), and Western blot analysis showed the molecular weight of the ectopically expressed FGFR1 to be around 115 kDa (Figure S2B). Figure 2 Development of anti-FGFR1 mAbs. Anti-FGFR1 mAbs inhibit HCC cell growth in vitro We next examined the effects of A2C9-1 and A2D11-1 mAbs on the growth of hepatic cancer cells (Figure 3). IFN-�� showed some antitumor activity against hepatic cancer cells, and weak growth suppression was seen when A2C9-1 or A2D11-1 was added to cultures in the absence of IFN-��. On the other hand, treatment with a combination of A2C9-1 and IFN-�� significantly reduced cell survival, as compared to treatment with IFN-�� alone (P=0.01) (Figure 3). The effect of A2D11-1 in combination with IFN-�� was no greater than the effect of IFN-�� alone. Figure 3 Antitumor activity of anti-FGFR1 mAbs in combination with IFN-�� in vitro.

Effects of A2C9-1 with and without IFN-�� in a mouse xenograft tumor model We next tested the antitumor effects of an anti-FGFR1 mAb in a mouse xenograft model of human HCC (Figure 4A and B). In mice treated with only A2C9-1 or IFN-��, tumor volumes did not differ from the control group administered PBS. However, treatment with IFN-��+A2C9-1 had an inhibitory effect on tumor growth, though the suppression was not statistically significant. Finally, in mice treated with IFN-��+A2C9-1+PBMCs (peripheral blood mononuclear cells), there was a significant antitumor effect, as compared to groups treated with PBS (p=0.026), INF-�� (p=0.03), A2C9-1 (p=0.014), PBMC (p=0.022) or IFN-��+PBMCs (p=0.007). In fact, the tumor disappeared in 2 of the 4 animals tested. During the course of the experiments we detected no cytotoxicity against normal hepatocytes (data not shown). Histological analysis revealed marked infiltration by mononuclear cells of the residual Brefeldin_A tumor tissues from mice treated with IFN-��+A2C9-1+PBMCs, but no such infiltration was observed in tumor tissues from mice in the other groups (Figure S3).