Development of a high affinity, non-covalent biologic to add functionality to Fabs

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Monoclonal antibody (mAb) functionalization traditionally involves chemical derivatization or genetic manipulation.
• These methods often lead to challenges with protein heterogeneity, stability, and solubility.
• A non-covalent intermediate could offer an alternative approach to functionalize mAbs, potentially avoiding these issues.

Purpose of the Study:

• To engineer a high-affinity, non-covalent intermediate for monoclonal antibody (mAb) functionalization.
• To enhance the affinity of a peptide binder (meditope) by engineering its binding site within the Fab region of trastuzumab.
• To assess the specificity and utility of the engineered construct for adding new functionalities to mAbs.

Main Methods:

• Engineered a binding site for a peptide (meditope) within the Fab fragment of trastuzumab.
• Constructed a fusion protein linking the meditope to Protein L via a peptide linker.
• Assessed binding affinity using dissociation constant (KD) measurements and specificity via flow cytometry.

Main Results:

• The meditope-Protein L construct achieved a dissociation constant (KD) of approximately 180 pM, representing a 7000-fold increase in affinity.
• The construct demonstrated high specificity for the engineered trastuzumab, confirmed by flow cytometry.
• Fusion of a bulky Green Fluorescent Protein (GFP) to the construct did not impede binding to cell surface antigens.

Conclusions:

• A specific, high-affinity, non-covalent intermediate has been successfully engineered for mAb functionalization.
• This approach overcomes limitations associated with chemical derivatization and genetic manipulation of mAbs.
• The engineered construct provides a versatile platform for rapidly adding new functionalities to monoclonal antibodies.