Design, Construction, and Validation of a Yeast-Displayed Chemically Expanded Antibody Library

Area of Science:

• Biotechnology and Molecular Biology
• Protein Engineering
• Antibody Discovery

Background:

• Current in vitro display technologies like phage and yeast display excel at antibody discovery but struggle with identifying antibodies that disrupt target functions beyond simple binding.
• There is a significant need for strategies to discover and engineer protein-based irreversible binders or inhibitory enzyme binders.
• Expanding the chemical diversity within antibody libraries is a promising approach to efficiently discover function-disrupting antibodies.

Purpose of the Study:

• To develop a yeast display-based platform for the discovery of chemically diversified antibodies.
• To construct and validate a large-scale (billion-member) antibody library incorporating noncanonical amino acids (ncAAs) for chemical conjugation.
• To explore the potential of this platform for identifying antibodies with enhanced functional properties beyond canonical binding.

Main Methods:

• Construction of a billion-member 'Clickable CDR-H3 Library' using a polyspecific orthogonal translation system for ncAA incorporation.
• Subsequent bioorthogonal click chemistry conjugation to introduce various chemical functionalities (e.g., photoreactive, proximity-reactive).
• Initial library screening using O-(2-bromoethyl)tyrosine (OBeY) for proximity-induced crosslinking, followed by flow cytometry and solution-based crosslinking experiments.

Main Results:

• Successful construction and chemical modification of the billion-member antibody library.
• Identification of OBeY-substituted clones binding to donkey IgG and protein tyrosine phosphatase 1B (PTP1B) after library enrichment.
• Flow cytometry confirmed enhanced binding retention for OBeY-substituted clones post-denaturation, though solution crosslinking results were inconclusive.

Conclusions:

• This work establishes a versatile, chemically expanded antibody library platform for antibody discovery.
• The platform demonstrates feasibility for screening chemically diverse antibody libraries, including protein-small molecule hybrids.
• This approach opens new avenues for discovering antibodies with unique functional properties, potentially leading to novel reagents, diagnostics, and therapeutics.