SpyMask enables combinatorial assembly of bispecific binders

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Bispecific antibodies represent a growing class of therapeutics.
• Traditional methods for bispecific antibody generation often involve complex disulfide reduction/oxidation steps or specialized formats.
• There is a need for more streamlined and versatile approaches to bispecific antibody production.

Purpose of the Study:

• To introduce SpyMask, a modular and scalable platform for bispecific antibody assembly.
• To explore the impact of scaffold geometry and binder orientation on bispecific antibody activity.
• To facilitate the discovery of novel synergistic activities in bispecific antibodies.

Main Methods:

• Development of SpyMask, a system utilizing SpyTag/SpyCatcher spontaneous amidation for modular bispecific assembly.
• Engineering of DoubleCatcher, a tandem SpyCatcher scaffold with a protease-activatable second domain.
• One-pot assembly and purification of bispecific antibodies in 96-well plates.
• Coupling of HER2-targeting binders to DoubleCatcher scaffolds with varying geometries and orientations.

Main Results:

• Successful generation of diverse bispecific antibodies using the SpyMask platform.
• Demonstration that bispecific activity is highly sensitive to binder orientation and scaffold geometry.
• Identification of unexpected anti-proliferative and pro-proliferative activities in HER2-targeting bispecifics.
• The SpyMask system allows for scalable discovery of synergistic effects.

Conclusions:

• SpyMask provides a flexible and scalable tool for the generation and discovery of bispecific antibodies.
• The modular nature of SpyMask supports the exploration of diverse formats and geometries.
• Controlling receptor organization and geometry is crucial for optimizing bispecific antibody function.