Humanization of Chicken-Derived scFv Using Yeast Surface Display and NGS Data Mining

Area of Science:

• Immunology
• Biotechnology
• Protein Engineering

Background:

• Chicken immunization is effective for generating high-affinity antibodies, especially against conserved mammalian epitopes.
• Humanization of non-human antibodies is crucial for therapeutic applications to reduce immunogenicity.
• Yeast surface display is a powerful tool for antibody selection and engineering.

Purpose of the Study:

• To establish a generic procedure for humanizing chicken-derived monoclonal antibodies.
• To generate humanized antibodies with restored affinity and improved protein characteristics.
• To explore the impact of antibody format on humanization efficiency.

Main Methods:

• Isolation of high-affinity binders against the epidermal growth factor receptor extracellular domain from immunized chickens via yeast surface display.
• Grafting of complementarity determining regions (CDRs) from chicken antibodies onto a human framework, with partial randomization of Vernier zone residues.
• Screening of a large yeast surface display library (≈300,000 variants) in single-chain variable fragment (scFv) and fragment antigen-binding (Fab) formats using next-generation sequencing.

Main Results:

• Identification of humanized antibody variants with restored binding affinity compared to parental chicken antibodies.
• Demonstration of improved protein characteristics in the humanized antibody variants.
• Next-generation sequencing provided insights into antibody format influence during humanization.

Conclusions:

• The developed high-throughput approach is effective and fast for generating multiple humanized antibodies from immunized chickens.
• The humanized antibodies generated possess potential therapeutic relevance.
• The study highlights the importance of antibody format in the humanization process.