PolyIC GE11 polyplex inhibits EGFR-overexpressing tumors

Abstract

Phage display has identified the dodecapeptide YHWYGYTPQNVI (GE11) as a ligand that binds to the epidermal growth factor receptor (EGFR) but does not activate the receptor. Here, we compare the EGFR binding affinities of GE11, EGF, and their polyethyleneimine-polyethyleneglycol (PEI-PEG) conjugates. We found that although GE11 by itself does not exhibit measurable affinity to the EGFR, tethering it to PEI-PEG increases its affinity markedly, and complex formation with polyinosine/cytosine (polyIC) further enhances the affinity to the submicromolar range. PolyIC/PPGE11 has a similar strong antitumor effect against EGFR overexpressing tumors in vitro and in vivo, as polyIC/polyethyleneimine-polyetheleneglycol-EGF (polyIC/PP-EGF). Absence of EGFR activation, as previously shown by us and easier production of GE11 and GE11 conjugates, confer polyIC/PPGE11 a significant advantage over similar EGF-based polyplexes as a potential therapy of EGFR overexpressing tumors.

Figure 1

Binding of [¹²⁵I]-EGF in A431 cells. A431 cells were incubated with [¹²⁵I]-EGF (0 – 3000 pM, 4°C, 4 h). To measure non-specific binding (NSB), a parallel set of cells was incubated with an excess of unlabeled EGF (1.5 µM). The data were analyzed using GraphPad Prism 5, yielding a B_max value of 2.77×10⁶ EGFRs/ cell and a Kd value of ~1 nM.

Figure 2

Binding of [¹²⁴I]-GE11 in A431 cells. A431 cells were incubated with [¹²⁴I]-GE11 (0 – 3000 pM, 4°C, 4 h). To measure non-specific binding (NSB), two additional sets of cells were incubated with an excess of unlabeled EGF (A) or unlabeled GE11 (B). Calculated B_max values indicated approximately 340,000 – 450,000 EGFR/ cell.

Figure 3

In vitro comparison of PolyIC/PP-EGF and PolyIC/PPGE11. U138MG (3000 cells) and 4000 cells of each other indicated cell line were seeded into 96 well plates, and grown overnight. Cells were then transfected with PolyIC, formulated with the indicated conjugate, as described (3). Survival of the cells was analyzed by Methylene blue assay 48 h after transfection. U138MG cells do not express EGFR (21); MCF7 express 0.8 – 5×10³ EGFRs/ cell. MDA-MB-231 (MDA231): 3 – 7×10⁵; MDA-MB-468 (MDA468): 2×10⁶ (22); U87MG: 1×10⁵; U87MG.wtEGFR: 1×10⁶ (23); and A431: 2 – 3×10⁶ EGFRs/ cell (24). The experiment was repeated 3 times with representative shown.

Figure 4

In vivo comparison of PolyIC/PP-EGF and PolyIC/PPGE11. PolyIC conjugates were used for treating (A) subcutaneous A431 xenografts and (B, C) intracranial U87MG tumors. Tumors were established and treated as described under materials and methods. (B, C left panels Survival plots and (B, C right panels) average survival period and percentage of increase in survival, following each treatment, with respect to untreated (UT) mice.