Purpose: To explore the relationship between selectivity and the drug delivery efficiency of three peptide- monomethyl auristatin E (MMAE) conjugates (PDC)1 with different affinities to epidermal growth factor receptor (EGFR).
Methods: Three peptides (L1: KLARLLT, Pep11: WSGENGPGFYDYEA and Ge11: YHWYGYTPQNVI)2-4 were synthesized using standard Fmoc solid-phase synthesis. These peptides were then conjugated with MMAE to form PDC using valine-citrulline linker. The PDC were purified using HPLC and lyophilized. ESI mass spectrometry and HPLC were used to characterize the peptides and PDC. A431 and MDA-MB-468 cells were used as EGFR-positive cancer cells. HEK293 was used as normal cells without any EGFR receptors. Cells were treated with free drug, peptides or PDC. After 72 hours incubation, the cell viability was determined by using Sulforhodamine B assay. The drug delivery efficiency was evaluated in EGFR-positive cells based on the differences in IC50 between MMAE and PDC. The selectivity was evaluated by the difference of IC50 between EGFR-positive cells and EGFR-negative cells.
Results: Three PDCs were synthesized and purified to the purity > 90%. In A431 cells, the IC50 values were: MMAE: 21.57±0.69nM, L1-MMAE: 26.69±0.77nM, Pep11-MMAE: 10.66±0.82nM and Ge11-MMAE: 0.01873±0.69nM, respectively. The IC50 in MDA-MB-468 cells were: MMAE: 28.78±0.66nM, L1-MMAE: 55.45±0.58nM, Pep11-MMAE: 16.75±0.58nM and Ge11-MMAE: 0.1396±0.55nM. Highest affinity Ge11-MMAE showed highest selectivity among all PDC with the biggest difference of IC50 between EGFR-positive cells and EGFR-negative cells. Ge11-MMAE also showed the highest drug delivery efficiency with more than 200 times higher cytotoxicity compared to MMAE in EGFR-positive cells. Pep11-MMAE increased cytotoxicity in EGFR-positive cells by more than 5 times compared to HEK 293 cells. Pep11-MMAE also enhanced the drug delivery by 2-fold compared to MMAE in EGFR-positive cells. For L1-MMAE, it did not show significantly increase of IC50 compared to MMAE in all the cell lines.
Conclusion: The results of this study indicate that PDC successfully enhance the drug delivery efficiency to the EGFR-positive cells. The higher EGFR binding affinity PDC can result in higher selectivity and drug delivery efficiency.