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How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project
Published on: February 11, 2019
Engineering antibodies for stability and efficient folding.
1Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, Zürich, Switzerland. honegger@bioc.uzh.ch
Handbook of Experimental Pharmacology
|December 12, 2007
Summary
Antibody variable domains exhibit diverse stability. Engineering can improve stability and folding efficiency of antibody fragments like scFv and Fab, preserving antigen binding.
Area of Science:
- Biochemistry
- Immunology
- Protein Engineering
Background:
- Antibody variable domains display a wide range of intrinsic thermodynamic stability.
- Single-chain variable fragments (scFv) often show limited stability despite domain mutual stabilization.
- Fab fragments and full-sized antibodies exhibit slow unfolding kinetics, contributing to apparent stability despite low thermodynamic stability.
Purpose of the Study:
- To investigate the thermodynamic stability and kinetic stabilization of antibody variable domains.
- To explore methods for engineering improved stability and folding efficiency in antibody fragments.
- To ensure preservation of antigen-binding specificity and affinity during stability engineering.
Main Methods:
- Thermodynamic stability analysis of antibody variable domains.
- Kinetic unfolding studies of antibody fragments (scFv, Fab, full-sized antibodies).
- Engineering strategies including point mutations and domain grafting for stability enhancement.
Main Results:
- Most engineered scFv fragments show poor to moderate stability without specific engineering.
- Disulfide-linked C(L)/C(H) heterodimers in larger antibody formats have limited thermodynamic stability but slow unfolding kinetics.
- Fab fragments benefit from kinetic stabilization, appearing more stable than their thermodynamics suggest.
- Engineering approaches like point mutations and framework grafting can enhance stability and folding efficiency while maintaining antigen specificity and affinity.
Conclusions:
- Intrinsic thermodynamic stability varies significantly among antibody variable domains.
- Kinetic stabilization plays a crucial role in the observed stability of antibody fragments and full-sized antibodies.
- Antibody variable domains can be successfully engineered for enhanced stability and folding efficiency without compromising antigen-binding properties.
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