Engineering Agonistic Bispecifics to Investigate the Influence of Distance on Surface-Mediated Complement Activation

Area of Science:

• Immunology
• Biotechnology
• Synthetic Biology

Background:

• The human complement system, particularly the C1 complex, offers a novel pathway for targeted cell killing.
• Bispecific antibodies and nanobodies can activate C1, but their efficacy is inconsistent due to epitope, antibody type, and design variations.

Purpose of the Study:

• To investigate monomeric agonists of C1, specifically bispecific nanobodies lacking Fc domains, to explore crucial geometric parameters for C1 activation.
• To determine the impact of linker length and flexibility as a metric for antigen and epitope location on complement activation.

Main Methods:

• Utilized DNA nanotechnology and protein engineering to create bispecific nanobodies with precisely controlled linker lengths and flexibilities.
• Assessed complement activation by measuring C1 binding, C4 cleavage, C4b deposition, and downstream membrane lysis.

Main Results:

• Identified a critical range of end-to-end distances between antigen-binding sites for optimal complement activation.
• Found that variations in complement activation efficacy were primarily due to differences in C4b deposition and membrane lysis, not initial C1 activation or C4 cleavage.
• Demonstrated that linker and hinge engineering significantly impacts the potency of complement agonists.

Conclusions:

• Structural requirements for C1 binding and activation are critical for designing effective complement-based targeted cell killing strategies.
• Linker and hinge engineering represents a viable approach to enhance the potency of bispecific nanobodies for therapeutic applications.
• DNA nanotechnology and synthetic biology offer powerful tools for optimizing geometric parameters in complement activation.