User:Jing Jing Gong/Notebook/Designing an affibody to target CEA

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Project Overview
For this project, we will be constructing a new type of molecule called affibodies that have high affinity for carcinoembryonic antigen (CEA), a marker present in the serum of individuals with colon cancer. By successfully utilizing the affibody as a tool for the detection of CEA, diagnosis of colon cancer could be made easier and less invasive.

Background & Goals
Colon cancer is the fifth most common cancer in the United States and the third leading cause of death in the western world. Most commonly, colorectal cancer is diagnosed by colonoscopy, an invasive procedure that uses an endoscope to pass through the anus, rectum, and colon. Not only is the procedure uncomfortable, or even painful, patients are required to eat a clear-liquid diet for up to three days preceding the procedure. However, it has been discovered that carcinoembryonic antigen (CEA) is present at elevated levels in patients with colon cancer patients. Thus, this molecule could be used a molecular marker to detect colon cancer in a much easier manner. Recently, the development of molecules called affibodies have caused heightened interest in the scientific community. Affibodies are small, engineered, protein affinity molecules that are highly stable and soluble, making them ideal tools for in vivo usage. Thus, we propose the use of an affibody molecule with affinity to CEA as an early diagnostic tool for detecting colon cancer. We model our project on the development of a HER2 affibody, which have high affinity to the receptor and may be used in tumor targeting applications and therapies for breast cancer. Through the use of phage display technology, we hope to isolate a variant that is highly affinitive to CEA and could be used for in vivo diagnostic purposes.

Project Details & Methods
For this project, we expect to use phage-display technology to select for an affibody with high affinity for CEA. This experiment would involve first creating a phagemid library through phage selection, where the variable region of the 58-amino acid affibody would be randomly mutagenized and tested. Through this process, we can determine several predominant variants that we feel may have CEA binding affinity. We will amplify the amount of these variants we have through E. Coli production and affinity purification, possibly through the use of a His tag. Finally, we will analyze the affinity of our molecules to our target, CEA, through biosensor analysis. Finally, we perform appropriate cellular tests using colorectal cancer cells and a radiolabeled known antibody such as the monoclonal antibody arcitumomab.

Resources
Jonsson, A., Wallberg H., Herne N., Stahl S., Frejd F. Y. Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro. Biolotechnology applied biochemistry 54 93-103, 2009.

This study investigated the design and selection process of affibody molecules that target human TNF-α (tumour necrosis factor-α) using phage-display technology. TNF-α, a proinflammatory cytokine, is involved inflammatory diseases, and thus affibodies that bind to TNF-α can potentially serve as a diagnostic marker for such dieases. In this study, three affibody molecules were found to have affinity for TNF-α. Specifically, ZTNF-α:185, showed high affinity for mouse and dog TNF-α and subnanomolar affinity for human TNF-α.

Lofblom, J., Feldwisch J., Tolmachev V., Carlsson J., Stahl S., Frejd F. Y. Affibody Molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Letters, 2010.

This review article describes the background, applications, and potential of affibody molecules, specifically as tools for imaging, diagnosis, and therapy. It discusses a proposed nomenclature for these newly engineered proteins and provides an overview of the currently known HER2-specific affibodies.

Wikman, M., Steffen A. C., Gunneriusson E., Tolmachev V., Adams G. P., Carlsson J., Stahl S. Selection and characterization of HER2/neu-binding affibody ligands. Protein Engineering, Design & Selection 17 455-462, 2004.

In this experiment,HER2/neu (human epidermal growth factor receptor 2 )-specific affibodies were selected using phage display technology. HER2/neu is a receptor that is found to be at high levels of expression in breast and ovarian cancers. Two affibody variants that showed affinity for the extracellular domain of HER2/neu (HER2-ECD) were isolated, but these affibodies did not bind to other non-targeted proteins. One specific variant also showed binding with nanomolar affinity to the HER2-ECD molecule at a different binding site than the antibody that also binds to this ligand.