Chelator-Free Radiometal Labeling Inside Engineered Affibodies

Area of Science:

• Biotechnology
• Protein Engineering
• Radiochemistry

Background:

• Affibody proteins are stable three-helix bundles engineered for specific protein binding.
• Current methods use bifunctional linkers to attach chelators for metal incorporation in affibodies.
• Metal-affibody conjugates are used in cancer imaging and therapy.

Purpose of the Study:

• To develop a linker-free method for metal incorporation into affibody proteins.
• To create stable and functional affibody-metal complexes for biomedical applications.
• To evaluate the potential of bismuth-loaded affibodies for HER2-targeted cancer therapy.

Main Methods:

• Engineered a triple cysteine motif within the affibody core to bind metal ions.
• Tested binding and stability of various metal-affibody complexes (Bi(III), Pb(II), In(III), Ga(III)).
• Assessed complex integrity against glutathione and strong chelators.
• Evaluated the binding affinity of a bismuth-loaded affibody to the HER2 receptor.
• Demonstrated targeted delivery of bismuth-213 to HER2-overexpressing cancer cells.

Main Results:

• A simple triple cysteine motif enabled direct and stable binding of Bi(III), Pb(II), In(III), and Ga(III).
• Metal uptake was instantaneous at room temperature and physiological pH.
• Affibody-metal complexes remained intact for one week at 4°C and resisted glutathione.
• The bismuth-affibody complex showed high stability against strong chelators.
• Bismuth-loaded affibody retained HER2 binding affinity and selectively delivered bismuth-213 to cancer cells.

Conclusions:

• Engineered affibodies can directly bind and retain therapeutic or imaging metals without linkers.
• This linker-free approach offers a stable and efficient platform for developing targeted radiopharmaceuticals.
• Bismuth-loaded affibodies show promise for targeted alpha therapy of HER2-overexpressing cancers.