Affinity Enhancement in Discrete Multivalent MegaMolecules.

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor 2 (HER2) are crucial targets in cancer therapy.
• Nanobodies offer high specificity and affinity for target antigens.
• Multivalent interactions can significantly enhance binding avidity.

Purpose of the Study:

• To quantitatively investigate the binding properties of multivalent megamolecules functionalized with nanobodies targeting EGFR or HER2.
• To explore the impact of valency and structure on the binding affinity of these megamolecules.
• To establish megamolecules as a versatile platform for developing targeted therapeutics.

Main Methods:

• Synthesis of structurally defined multivalent megamolecules with varying numbers of nanobodies (up to six).
• Quantitative assessment of monovalent affinities of nanobodies targeting EGFR and HER2.
• Measurement of apparent binding affinities of megamolecules using biolayer interferometry.
• Synthesis and testing of reduced-valency variants to determine the contribution of valency to affinity enhancement.

Main Results:

• Engineered megamolecules demonstrated substantial improvements in apparent binding affinities, ranging from 31- to ~118,000-fold.
• A dendritic hexavalent megamolecule achieved sub-picomolar binding affinity.
• Reduced-valency variants exhibited lower binding affinities, confirming valency as the primary driver of affinity enhancement.
• Monovalent affinities of nanobodies varied by up to 2800-fold.

Conclusions:

• Multivalent megamolecules offer a powerful strategy to achieve ultra-high binding affinities for therapeutic targets like EGFR and HER2.
• The valency and spatial presentation of nanobodies on megamolecular scaffolds are critical for optimizing binding avidity.
• This research validates megamolecules as a versatile platform for designing advanced targeted therapies with picomolar or sub-picomolar binding.