Related Experiment Video
Updated: Mar 3, 2026
07:59
A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
15.7K
Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A
Cindy Zer1, Kendra N Avery1, Kassondra Meyer1
1Department of Molecular Medicine, Beckman Research Institute at City of Hope.
Protein Engineering, Design & Selection : PEDS
|April 22, 2017
Summary
Researchers grafted a peptide binding site, called meditope, onto an anti-CEA antibody (M5A). This enables non-covalent attachment of imaging agents for cancer diagnostics without affecting antibody function.
Area of Science:
- Bioconjugation
- Antibody Engineering
- Cancer Diagnostics
Background:
- A cyclic peptide, meditope, binds to cetuximab's Fab region.
- Meditope binding sites can be engineered onto other antibodies, like trastuzumab.
- This enables non-covalent attachment of imaging agents to antibodies.
Purpose of the Study:
- To engineer the meditope binding site onto the M5A antibody, an anti-CEA antibody.
- To identify key amino acid substitutions responsible for meditope binding.
- To assess the impact of these substitutions on antibody-antigen recognition.
Main Methods:
- Sequential amino acid substitutions in the M5A antibody's Fab region.
- Reverse substitution experiments to confirm key residues.
- Affinity measurements using single-digit micromolar affinity.
Main Results:
- Specific substitutions (Pro40Thr, Gly41Asn, Phe83Ile, Thr85Asp) recreated the meditope binding site in M5A.
- Pro40 was found to abrogate peptide binding when other substitutions were present.
- All 13 substitutions did not interfere with M5A's antigen recognition.
Conclusions:
- The engineered meditope binding site in M5A retains high affinity for the peptide.
- Key residues have been identified for fine-tuning meditope binding site affinity.
- This strategy facilitates the development of novel antibody-based imaging agents for cancer diagnostics.
