Inhibition of cell proliferation by an anti-EGFR aptamer

Abstract

Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (K(d) = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents.

Figure 1. Binding specificity of anti-EGFR aptamers.

The N62 pool and aptamers E03, E04, and E07 were assayed in triplicate by filtration for binding to hEGFR, mEGFR, hErbB2, and hIgG1. Average values and standard deviations are shown. Binding assays were carried out either in the absence (left) or presence (right) of DTT. A no protein control was also carried through the procedure. Percent binding was relative to the total RNA added.

Figure 2. Binding constants for Aptamers E03, E04, and E07.

Binding isotherms were constructed using 0.1 nM aptamer and varying amounts of hEGFR or mEGFR. Binding assays were carried out in triplicate and the average value and standard deviation are shown. The fact that binding does not reach 100% is a function of the filtration assay, and is commonly observed. Dissociation constants were calculated following curve-fitting, as described in Materials and Methods.

Figure 3. Cellular binding and inhibition of EGF-binding by anti-EGFR aptamers.

Phycoerythrin-labeled N62 pool (green line), Aptamer E03 (pink line), Aptamer E04 (cyan line), and Aptamer E07 (orange line) were incubated with EGFR-overexpressing cells (A431); (A) and EGFR-negative cells (MDA-MB-435); (B) and analyzed on the FL2-H channel of a FACSCalibur. A no RNA control was also carried out. Alexa 488-labeled EGF (0.1 ug/ml, ca. 1.5 nM) was incubated with A431 cells (green line), and binding assessed by FACS. The interaction could be blocked by the further addition of 1 uM Aptamer E03 (cyan line), Aptamer E04 (orange line) and Aptamer E07 (dark blue line) but not unselected N62 pool RNA (pink line)(C). Counts represent number of cells counted.

Figure 4. Aptamer inhibition of EGFR phosphorylation.

The phosphorylation of EGFR was stimulated by 2 nM EGF and was detected by Western blot analysis using an anti-tyrosine phosphorylation antibody, (A, lane 2). The additions to individual reactions are shown above the gel lanes. Arrows show the position of proteins. Staining was also carried out with an anti-beta-actin antibody to ensure that similar amounts of samples were loaded. Phosphorylation is inhibited by the addition of 100 nM Ab C225 (A, lane 3), 1 uM Aptamer E07 (A, lane 4), but not unselected N62 pool RNA (A, lane 5). Aptamer 07 and pool RNA alone do not induce EGFR phosphorylation (A, lanes 6 and 7). The approximate inhibition constant for Aptamer E07 is about 300 nM (B).

Figure 5. Internalization of anti-EGFR Aptamer E07.

Phycoerythrin-labeled unselected N62 pool RNA (100 nM, green line) and PE-labeled Aptamer E07 (100 nM, cyan line) were incubated with A431 cells either on ice (left) or at 37°C (right) for 30 min. After the binding reaction, cells were exposed to Riboshredder for 10 min at 25°C (pink line and orange lines, respectively). Residual fluorescence was analyzed by FACS. Putative internalized Aptamer E07 conjugates are indicated by arrows. Counts represent number of cells counted.

Figure 6. Inhibition of A431 cell proliferation by Aptamer E07.

A431 cells were seeded into Matrigel in 48 well plates and treated with untreated or dephosphorylated Aptamer E07 or Mutant Aptamer (1 uM) every other day for a total of 6 treatments. (A) Cell colonies were released from Matrigel by Cell Recovery Solution, transferred to a 24-well plate, and imaged with an IX51 Inverted Microscope (Olympus, Center Valley, PA) under 4x objective. The inset shows which of the five micrographs go with which specific treatment parameters. (B) The nucleic acids content was measured using a CyQUANT® Cell Proliferation Assay Kit. CyQUANT® GR dye was incubated with cell lysate and exhibited strong green fluorescence when bound to cellular nucleic acids. The fluorescence is linearly correlated with the number of cells and readily detected by a plate reader. (p = 5.4E−13).