A Single-Framework Synthetic Antibody Library Containing a Combination of Canonical and Variable...

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Synthetic antibody libraries offer a powerful approach to generate antibodies with tailored properties.
• The trastuzumab framework provides a stable scaffold for antibody engineering.
• Human epidermal growth factor receptor-3 (HER3) is implicated in various cancers.

Purpose of the Study:

• To design, construct, and validate a large-scale phage-displayed antigen-binding fragment (Fab) library.
• To select high-affinity antibodies against the extracellular domain of HER3 (HER3-ECD).
• To evaluate the therapeutic and diagnostic potential of selected antibodies.

Main Methods:

• Engineering a phage-displayed Fab library with fixed CDRs L1, L2, H1, H2 and diversified CDRs L3, H3.
• Utilizing custom trinucleotide phosphoramidite mixes for biased CDR diversification.
• Performing selections against HER3-ECD and analyzing CDR diversity via next-generation sequencing.
• Overexpressing and purifying selected antibody formats (Fab and IgG) for characterization.

Main Results:

• The library comprised approximately 7.6 billion unique Fabs with high encoding accuracy.
• Selection against HER3-ECD yielded a lead candidate, Her3-3.
• Fab HER3-3 exhibited high affinity (K_D = 2.14 nm) for HER3-ECD and recognized cell-surface HER3.
• HER3-3 IgG bound HER3 but did not inhibit cell proliferation.
• Fab HER3-3 demonstrated selective accumulation in HER3-positive xenografts in vivo.

Conclusions:

• The engineered synthetic antibody library is effective for generating specific binders against therapeutic targets.
• Fab HER3-3 shows promise as a diagnostic imaging agent for HER3-expressing tumors.
• Further development of HER3-targeting antibodies for therapeutic applications warrants investigation.