Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability

J Mol Biol. 2005 Apr 8;347(4):773-89. doi: 10.1016/j.jmb.2005.01.053.

Abstract

Recombinant antibody fragments, most notably Fab and scFv, have become important tools in research, diagnostics and therapy. Since different recombinant antibody formats exist, it is crucial to understand the difference in their respective biophysical properties. We assessed the potential stability benefits of changing the scFv into the Fab format, the influence of the variable domains on the stability of the Fab fragment, and the influence of the interchain disulfide bond in the Fab fragment. To analyze domain interactions, the Fab fragment was broken down into its individual domains, several two-domain assemblies and one three-domain assembly. The equilibrium denaturation properties of these constructs were then compared to those of the Fab fragment. It was found that mutual stabilization occurred across the VH/VL and the CH1/CL interface, whereas the direct interaction between the V) and the CL domain had no influence on the stability of either domain. This observation can be explained by the different interfaces used for interaction. In contrast, the whole CH1CL and VHVL unit showed significant mutual stabilization, indicating a high degree of cooperation between the VH/VL and CH1/CL interface. The interchain disulfide bond in the Fab fragment plays an essential role in this stabilization. In addition to the effects of domain association on the thermodynamic (equilibrium) stability, Fab fragments differ from scFv fragments of similar equilibrium stability by having a very slow unfolding rate. This kinetic stabilization may increase significantly the resistance of Fab fragments against short time exposure to adverse conditions.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Disulfides / metabolism
  • Guanidine / pharmacology
  • Immunoglobulin Fab Fragments / chemistry*
  • Immunoglobulin Fab Fragments / genetics
  • Immunoglobulin Fab Fragments / isolation & purification
  • Immunoglobulin Fab Fragments / metabolism*
  • Immunoglobulin Heavy Chains / chemistry*
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Heavy Chains / isolation & purification
  • Immunoglobulin Heavy Chains / metabolism*
  • Kinetics
  • Models, Molecular
  • Protein Binding
  • Protein Denaturation / drug effects
  • Protein Engineering*
  • Protein Folding
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Thermodynamics
  • Urea / pharmacology

Substances

  • Disulfides
  • Immunoglobulin Fab Fragments
  • Immunoglobulin Heavy Chains
  • Urea
  • Guanidine