Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering.
By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient folding. This is particularly important when antibodies are to be used under conditions where the disulfide bonds cannot form, i.e., in intracellular applications (as "intrabodies"). We describe in detail two methods for the knowledge-based improvement of antibody stability and folding efficiency. While CDR grafting from a non-human to the most closely related human antibody framework is an established technique to reduce the immunogenicity of a therapeutic antibody, CDR grafting for stabilization implies the use of a more distantly related acceptor framework with superior biophysical characteristics. The use of such dissimilar frameworks requires particular attention to antigen contact residues outside the classical CDR definition and to residues capable of indirectly affecting the conformation of the antigen binding site. As a second alternative, the stability of a suboptimal framework can be improved by the introduction of point mutations designed to optimize key residue interactions. We describe the analysis methods used to identify such point mutations, which can be introduced all at once, while maintaining the framework features necessary for antigen binding. These rational approaches render the continued "rediscovery" of certain mutations by directed evolution unnecessary, but they can also be used in conjunction with such methods to discover even better molecules.
Ewert
Stefan
S
Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland.
Honegger
Annemarie
A
Plückthun
Andreas
A
D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D045604:Extracellular Fluid / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship
D022801:Complementarity Determining Regions