Structure- and sequence-based design of synthetic single-domain antibody libraries

Area of Science:

• Biotechnology
• Immunology
• Pharmaceutical Science

Background:

• Single-domain antibody fragments, VHH, are valuable biotherapeutics derived from camelids.
• Traditional VHH generation via animal immunization is expensive and slow.
• A need exists for efficient, in vitro methods to generate VHH binders.

Purpose of the Study:

• To develop and validate a synthetic VHH library for in vitro selection of single-domain binders.
• To create a library mimicking natural VHH repertoires using structure-based design and sequencing.
• To demonstrate the library's efficacy against diverse model antigens.

Main Methods:

• Development of a synthetic VHH library incorporating structure-based design.
• Utilizing next-generation sequencing for library analysis.
• In vitro selection of VHH binders against mouse PD-1, amyloid-beta, and MrgX1 GPCR.

Main Results:

• Successful isolation of diverse VHH binders targeting multiple epitopes across all three antigens.
• Achieved high-affinity binding, with dissociation constants as low as 5 nM.
• Demonstrated functional activity of anti-mPD-1 VHH in a receptor blocking assay.

Conclusions:

• The synthetic VHH library provides an efficient and versatile platform for generating high-affinity binders.
• This approach overcomes limitations of traditional animal immunization methods.
• The isolated VHH binders show potential for therapeutic applications, including functional receptor modulation.