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Engineering pH-Sensitive Single-Domain Antibodies.

Tosha M Laughlin¹, James R Horn²

¹Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, USA.

Methods in Molecular Biology (Clifton, N.J.)

|February 14, 2022

Summary

This summary is machine-generated.

Researchers developed a method to create pH-dependent single-domain antibodies (sdAbs). This technique enables custom regulation of antibody binding events by introducing histidine substitutions into the antibody-antigen interface.

Keywords:

Combinatorial histidine library Linked-equilibria Linked-protonation Nanobody Phage display VHH pH switch

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Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

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Published on: January 14, 2020

Area of Science:

Biotechnology
Immunology
Protein Engineering

Background:

Antibody engineering seeks to enhance functionality beyond affinity and specificity.
pH-dependent control of antibody binding is a desirable feature for regulated molecular interactions.

Purpose of the Study:

To provide a methodology for generating single-domain antibodies (sdAbs) with pH-dependent binding capabilities.
To establish a framework for designing combinatorial histidine scanning libraries within sdAb-antigen interfaces.

Main Methods:

Conceptual framework for combinatorial histidine scanning library design.
Construction of a phage display library with up to 1 x 10^10 unique members.
Phage display protocols for selection and analysis of pH-dependent sdAb clones.

Main Results:

Successful generation of a methodology for pH-dependent sdAbs.
Demonstration of histidine scanning for modulating binding characteristics.
Establishment of protocols for isolating specific pH-dependent antibody variants.

Conclusions:

The described methodology enables the creation of custom-regulated single-domain antibodies.
Histidine scanning is an effective strategy for engineering pH-dependent antibody binding.
This work provides tools for developing novel antibody-based therapeutics and diagnostics with conditional activity.