Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes

Area of Science:

• Biotechnology
• Chemical Biology
• Protein Engineering

Background:

• Site-specific protein conjugation is crucial for advancing antibody-based technologies like antibody drug conjugates and bispecific antibodies.
• Incorporating non-natural amino acids into antibodies offers site specificity and enables bio-orthogonal chemistry.
• Current methods face challenges due to variable efficiency in incorporating non-natural amino acids at different antibody sites.

Purpose of the Study:

• To develop a robust and efficient method for synthesizing chemically functionalized antibodies with non-natural amino acids at defined positions.
• To identify new sites for efficient chemical conjugation in antibody fragments (Fab).
• To engineer a novel antibody format, Variabodies, using the identified conjugation sites.

Main Methods:

• Utilized codon reassignment technology for the synthesis of antibodies incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys).
• Employed X-ray crystallography to analyze the structure and accessibility of the incorporated azido group in a Fab variant.
• Engineered Variabodies by chemically connecting two Fab molecules at newly identified sites.

Main Results:

• Successfully synthesized chemically functionalized antibodies with o-Az-Z-Lys at defined positions with high efficiency.
• Identified multiple new sites in Fab-constant domains suitable for efficient chemical conjugation.
• Created Variabodies, a novel antibody format, with some variants demonstrating agonistic activity.

Conclusions:

• The developed approach significantly enhances the availability of antibodies for chemical conjugation.
• This method facilitates the identification of new conjugation sites and the engineering of novel antibody formats.
• The findings may contribute to the development of new therapeutic antibodies with diverse functionalities.