A rapid phage-free platform for antigen-specific VNAR screening with BATCH system

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Shark-derived variable new antigen receptors (VNARs) are advantageous single-domain antibodies.
• Conventional phage display methods for VNAR discovery can be complemented by alternative screening approaches.

Purpose of the Study:

• To develop and validate a phage-free bacterial two-hybrid (BATCH) platform for rapid, antigen-specific VNAR discovery.
• To streamline the identification of stable VNARs from immunized Chiloscyllium plagiosum.

Main Methods:

• A bacterial two-hybrid (BATCH) system was employed using a cDNA library from immunized Chiloscyllium plagiosum spleen.
• Screening involved dual lacZ and CmR readouts with enhanced reporter systems in BTH101 cells.
• AlphaFold3 modeling and mutagenesis were used to analyze VNAR binding and function.

Main Results:

• The BATCH platform identified two unique VNAR binders (VNAR1 and VNAR2) against Cre recombinase in 16 hours.
• Mutagenesis studies confirmed the importance of the CDR3 loop and disulfide bond for VNAR affinity and function.
• The platform demonstrated a 1- to 2-day screening phase for VNAR discovery.

Conclusions:

• The phage-free BATCH platform offers a rapid and efficient alternative to conventional methods for VNAR discovery.
• This system facilitates the identification of stable VNARs with potential therapeutic and diagnostic applications.
• The study highlights the utility of simplified in vivo selection processes for antibody engineering.