The Effect of EGFR Targeting Peptides isolated by us on EGFR Autophosphorylation
The effect of several peptides on the phosphorylation levels of human EGFR in A431 cell line was assessed by ELISA test: Exponentially growing A431 cells were seeded on a 96-well plate, and grown for about 3 days. Following medium exchange, solutions containing 50 mg/ml EGF and 0.2 mg/ml peptides were added. The plate was incubated for 7.5 min at 37 ºC. The medium was removed, the cells fixed, and the plate washed with Triton washing solution. The level of phosphorylates was assessed by incubation with phospho-EGFR (Tyr1045) antibody as a primary antibody, and Anti-rabbit IgG as a secondary antibody. The ability of different peptides to inhibit autophosphorylation of EGFR is presented by the decrease of the fluorescent signal (Y axis).
Accumulation of Fluorescent PEG-Peptide In Mice’s Cancer Tumors
EGFR antagonist peptide, attached to PEG, was labeled with Fluorescein, and injected intravenously to 5 Xenograft mice, bearing subcutaneous NCI-H1650 tumors (lung cancer that overexpress EGFR). The 6th mouse served as time 0, and was injected only with saline. Each mouse was subjected to anesthesia, perfusion and organ collection at a specific time point. The organs were homogenized in a volume of solution proportional to their weight, and spun down. Fluorescence of each sample was measured.
As can be seen, our peptide reached its targeted tumor cells, and accumulated there, unlike in the kidneys and the liver, where its concentration went down again.
Internalization of Fluorescent PEG-Peptide into Cancer Cells in Mice’s Tumors
The upper part of the photograph shows fluorescently labeled cancer cells taken from the tumors 1 hour and 24 hours after the injection. The lower part is a regular photograph of the same cells (no excitation that causes the fluorescence emission). In this part, cells that did not interact with the fluorescent peptide may be seen, but it looks like all, or most of the cells interacted with, and internalized the fluorescent peptide.
Efficacy Test - Killing Cancer Cells
We have synthesized a molecule that contains several copies of EGFR targeting peptide, which are bound to several copies of two different toxic peptides. We have tested the effect of this molecule on two kinds of cancer cell lines:
- A431 - Human epithelial carcinoma that contains around 100,000 copies of EGFR on each cell.
- MCF-7- Breast adenocarcinoma that contains around 3,000 copies of EGFR on each cell (a partial negative control).
- Solutions were applied to the cells in a 96 well plate, and after 48 hours the wells were photographed.
As can be seen in the photographs, at concentrations above 3 micromolar, all A431 cells lost their tissue-like structure, and died. The partial negative control - survived.