IN VITRO BINDING SPECIFICITY AND UPTAKE OF A NOVEL PEPTIDE FOR HER2 TARGETING
Introduction/Abstract
Human Epidermal Growth Factor Receptor 2 (HER2) is a cell surface receptor tyrosine kinase and plays a role in the signal pathways leading to cell proliferation and differentiation. To create a novel peptide that specifically binds to HER2, we previously designed a peptide molecule (HER2-PEP) that consists of two peptide chains based on the binding interactions between Pertuzumab and HER2. The in vitro binding specificity and uptake of HER2-PEP in HER2 over-expressed and non-expressed cells are reported in this presentation.
Purpose
To study the binding specificity and uptake of a novel peptide in HER2 over-expressed and non-expressed cells in vitro.
Method
A novel peptide was designed to bind HER2 based on interactions in Pertuzumab-HER2 complex. The interactions between Pertuzumab and HER2 were identified by using Molecular Operating Environment (MOE) software. A two-chain peptide (HER2-PEP) with sequences of Thr-Phe-Thr-Asp-Tyr-Thr-Met-Asp-Trp-Val and Asp-Val-Asn-Pro-Asn-Ser-Gly-Gly-Ser-Ile-Tyr linked by Gly-Lys was designed and synthesized by solid phase synthesis. HER2-PEP was tagged with FITC through a 6-aminohexanoic acid linker (Ahx). Cell binding of the peptide was studied using HER2 over-expressed MDA-MB-361 cells and non-HER2 expressing HEK293 cells as control. Cells were incubated with HER2-PEP and a control peptide with sequence of Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Ahx-FITC at concentration of 10μM at 37C for 30min. Cells were then stained with AlexaFlour 594 (plasma membrane dye) and visualized under a confocal microscopy. The uptake of the peptides was quantified by determining the fluorescence intensity inside the cells. The fluorescence intensities were also determined using flow cytometry after cells were treated with HER2-PEP or control peptide.
Results
Higher intensity of fluorescence was observed on the surface of HER2 over-expressed MDA-MB-361 cells when treating with HER2-PEP, while only background fluorescence was observed when treating with control peptide. The fluorescence inside MDA-MB-361 cells when treating with HER2-PEP was about 6 fold higher than that of control peptide. For non-HER2 expressing HEK293 cells, only background fluorescence was observed on cell surface when treating with both HER2-PEP and control peptide. The fluorescence inside HEK293 cells was only 10% of MDA-MB-361 cells when treated with HER2-PEP. Flow cytometric studies showed that the geometric mean fluorescence intensity (MFI) (n=6) of MDA-MB-361 cells after treating with HER2-PEP was about 14 fold higher than that of control peptide, while no significant difference of MFI in HEK293 cells was observed between the cells treated with HER2- PEP and control peptide.
Significance
This presentation illustrated that design of antibody mimic based on antibody-antigen interactions was feasible and this antibody mimic had potential to be used for targeting HER2 over-expressed cancers.
Location
DeRosa University Center, Stockton campus, University of the Pacific
Format
Poster Presentation
IN VITRO BINDING SPECIFICITY AND UPTAKE OF A NOVEL PEPTIDE FOR HER2 TARGETING
DeRosa University Center, Stockton campus, University of the Pacific
Human Epidermal Growth Factor Receptor 2 (HER2) is a cell surface receptor tyrosine kinase and plays a role in the signal pathways leading to cell proliferation and differentiation. To create a novel peptide that specifically binds to HER2, we previously designed a peptide molecule (HER2-PEP) that consists of two peptide chains based on the binding interactions between Pertuzumab and HER2. The in vitro binding specificity and uptake of HER2-PEP in HER2 over-expressed and non-expressed cells are reported in this presentation.
