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Rational Design of EGFR Antibody Mimics Based on Knob-Socket Model

S. Sachdeva, D. Su, H. Joo, J. Tsai, B. Jasti, X. Li


University of the Pacific
Purpose
To design and characterize peptides to mimic antibody binding to Epidermal Growth Factor Receptor (EGFR) based on
Knob-Socket model.
Methods
Antibody mimic molecules were designed by mapping the Cetuximab-EGFR epitope surface and identifying the sockets.
Each socket is formed by three amino acid residues on the EGFR surface and binds with the amino acid knob residues from
the antibodys complementary binding region loops. Antibody mimics were designed by selecting the knob residues with
high probability to pack into sockets on the EGFR surface. Twenty five molecules (Pep1-Pep25) were designed and
analyzed using Molecular Operating Environment (MOE) software to determine the binding energy, total number of
interactions and preserved interactions between EGFR and designed molecules. A pair of peptides (Pep6 and Pep24) with
reverse sequence was designed to test the sequence requirement for binding and a scrambled peptide (Pep25) was created as a
control. The specific binding of antibody mimics was evaluated by observing uptake of the FITC labeled antibody mimics in
A431, MDA-MB468 cells (overexpressed-EGFR) and HEK293 (control) using confocal microscope and flow cytometry.
Equilibrium dissociation constant (KD) was determined by using surface plasmon resonance for antibody mimics binding to
EGFR and bovine serum albumin. Cell based ELISA was performed to analyze the percentage phosphorylation inhibition by
antibody mimic molecules.
Results
Pep6, Pep11, Pep22, and Pep24 showed low calculated binding energy and high number of total and preserved interactions.
Pep11 containing high probability interaction knobs F and Y for sockets VSS and ISI, respectively, exhibited the lowest KD
of 252nM. Pep6 containing lower probability reversed sequence knobs (Y and F) showed a higher KD. Pep 6 and Pep24
packed into sockets in similar fashion, showed comparable KD values. Confocal microscopy images and mean fluorescence
intensities in flow cytometry analysis displayed significantly higher cellular uptake of antibody mimics in EGFR
overexpressed cells and negligible uptake in control cells. Percentage inhibition of phosphorylation for antibody mimics was
found to be in the range of 4.400.60 to 8.130.12 as compared with 37.900.07 inhibition by Cetuximab.
Conclusion
Antibody mimics against EGFR have been successfully designed using a rational approach based on the Knob-Socket model.
These antibody mimics can specifically bind to EGFR and display partial EGFR phosphorylation inhibition.