Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry.

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Conventional methods for determining bi-specific antibody (BsAb) antigen-binding affinity require purified antibodies, a time-consuming process.
• Developing efficient methods to screen and characterize novel BsAb formats is crucial for therapeutic advancements.

Purpose of the Study:

• To establish a novel, purification-free method for assessing BsAb antigen-binding affinity using bacteria display technology and flow cytometry (FCM).
• To compare the antigen-binding affinity and bioactivity of two BsAb formats: scFv2-CH/CL and Diabody-CH/CL, targeting human interleukin 1β (hIL-1β) and human interleukin 17A (hIL-17A).

Main Methods:

• Engineered Escherichia coli to display two BsAb formats (scFv2-CH/CL and Diabody-CH/CL) specific for hIL-1β and hIL-17A using a new lipoprotein A (NlpA)-based bacteria display system.
• Converted engineered bacteria to spheroplasts and incubated them with fluorescently conjugated antigens.
• Analyzed antigen-binding affinity, association, and dissociation kinetics using FCM-based assays without antibody purification.

Main Results:

• The NlpA-based bacteria display and FCM method successfully detected and quantified BsAb antigen-binding affinity without requiring expression and purification.
• Diabody-CH/CL format exhibited significantly higher antigen-binding affinity compared to the scFv2-CH/CL format for both hIL-1β and hIL-17A.
• Purified BsAbs confirmed higher relative affinities for Diabody-CH/CL, correlating with superior in vitro bioactivity and in vivo efficacy in a collagen-induced arthritis (CIA) mouse model.

Conclusions:

• NlpA-based bacteria display combined with FCM provides a rapid and efficient platform for identifying high-affinity BsAbs early in the drug development pipeline.
• The Diabody-CH/CL format demonstrates promising therapeutic potential due to its enhanced binding affinity and bioactivity.
• This purification-free screening approach accelerates the discovery of lead BsAb compounds for treating inflammatory diseases.