Bispecific targeting of EGFR and uPAR in a mouse model of head and neck squamous cell carcinoma

Abstract

Objectives: To investigate the efficacy of the bispecific targeted toxin, dEGFATFKDEL, on head and neck carcinoma cell lines in vitro and in vivo.

Materials and methods: A deimmunized bispecific anti-cancer agent was constructed to simultaneously target both the overexpressed EGF receptor on carcinomas and the urokinase receptor (uPAR), that is found on the endothelial cells of the neovasculature within tumors. Flow cytometry assays were performed to determine the level of EGFR expressed on a variety of carcinoma lines. These lines were then tested in tritiated leucine incorporation assays to determine the efficacy of dEGFATFKDEL. Human vein endothelial primary cells were also tested to determine the effectiveness of the ATF portion of the molecule that binds uPAR. Furthermore, mouse studies were performed to determine whether dEGFATFKDEL was effective at inhibiting tumor growth in vivo.

Results: UMSCC-11B and NA, two head and neck squamous cell carcinomas, highly expressed EGFR. Both the carcinoma lines and the human vein endothelial cells were inhibited at sub-nanomolar concentrations by dEGFATFKDEL. The tumor studies showed that the tumors treated with dEGFATFKDEL were significantly inhibited whereas the negative control and untreated tumors progressed. In a separate in vivo study involving another carcinoma line, MDA-MB-231, the effectiveness of dEGFATFKDEL was confirmed. No toxicity was seen at the doses used in either of these mouse studies.

Conclusions: This bispecific agent is effective in a mouse model of head and neck squamous cell carcinoma. Further study of this reagent for use in the treatment of carcinomas is warranted.

Figure 1

Flow cytometry dot plot of the head and neck carcinoma line UMSCC-11B probed with either FITC conjugated anti-EGFR antibody or anti-uPAR antibody. Cells were also probed with the negative control HD37-FITC (an anti-CD19 antibody).

Figure 2

The activity of dEGFATFKDEL against the carcinoma lines was determined by Leucine and Thymidine incorporation assays. UMSCC-11B, NA-SCC, MDA-MB-231, and Caco-2 Leucine incorporation assays showed that dEGFATFKDEL was selectively active while the negative control in each case had no effect (a–d). HUVEC primary cells were also tested and were selectively inhibited by dEGFATFKDEL as well. However, dEGFATFKDEL had no effect on the EGF and uPAR negative B cell lymphoma line Raji, while a positive control targeted toxin specific against CD22 and CD19 had activity (f).

Figure 3

Mouse tumor model of UMSCC-11B show dEGFATFKDEL is effective at inhibiting tumor growth. As seen in (a) tumor growth was inhibited in dEGFATFKDEL treated mice as compared with the no treatment mice. Student t test analysis was performed for dEGFATFKDEL treated tumors compared with untreated tumors on each day. The tumor size between groups became significantly different on day 23 (p<0.05). Body weight was measured in (b) and shows that treated mice did not have any significant weight loss due to drug toxicity throughout the study.

Figure 4

Real time images of representative mice from the UMSCC-11B study are shown. Mice treated with dEGFATFKDEL exhibited tumor reduction over time shown by the corresponding decrease in luminescence. Control treated and untreated mice, however, had continually increasing luminescent levels.

Figure 5

A second mouse carcinoma model was used to confirm the results from the UMSCC-11B study. MDA-MB-231 tumors treated with dEGFATFKDEL were inhibited, while untreated tumors grew normally over time. Student t tests were performed on every day. The dEGFATFKDEL treated tumors were statistically different (p<0.05) from the untreated controls on day 35, and on every subsequent day.