Deep sequencing combined with high-throughput screening enables efficient development of a pH-dependent high-affinity...

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Research

Background:

• In vitro methods for developing binding domains are established but generating high-affinity binders is time-consuming.
• Developing focused maturation libraries requires detailed understanding of binding surfaces, slowing down the process.

Purpose of the Study:

• To accelerate the development of high-affinity binding domains using deep sequencing data.
• To identify conditional binding domains using high-throughput screening.
• To develop a novel therapeutic agent targeting the cancer biomarker HER3.

Main Methods:

• Utilized deep sequencing data from initial selection to guide maturation library design.
• Employed high-throughput screening with flow cytometry and Escherichia coli display.
• Developed a high-affinity binder for HER3 with a pH-dependent release mechanism.

Main Results:

• Achieved a 100-fold increase in binding affinity for HER3 (3.3 nM at pH 7.4).
• Demonstrated a pH-dependent release mechanism, releasing in acidic endosomal conditions (pH ≈6).
• Conjugated binder to mertansine (DM1) showing specific cytotoxicity (IC50 of 2-5 nM) against HER3-expressing cells.

Conclusions:

• The presented approach efficiently develops conditional binding domains.
• This method accelerates the discovery of targeted cancer therapies.
• The developed HER3-targeting binder shows promise for therapeutic applications.