Mutational approaches to improve the biophysical properties of human single-domain antibodies

Monoclonal antibodies are a remarkably successful class of therapeutics used to treat a wide range of indications. There has been growing interest in smaller antibody fragments such as Fabs, scFvs and domain antibodies in recent years. In particular, the development of human V-H and V-L single-domain antibody therapeutics, as stand-alone affinity reagents or as warheads for larger molecules, are favored over other sources of antibodies due to their perceived lack of immunogenicity in humans. However, unlike camelid heavy-chain antibody variable domains (V(H)Hs) which almost unanimously resist aggregation and are highly stable, human V(H)s and V(L)s are prone to aggregation and exhibit poor solubility. Approaches to reduce V-H and V-L aggregation and increase solubility are therefore very active areas of research within the antibody engineering community. Here we extensively chronicle the various mutational approaches that have been applied to human VHS and V(L)s to improve their biophysical properties such as expression yield, thermal stability, reversible unfolding and aggregation resistance. In addition, we describe stages of the V-H and V-L development process where these mutations could best be implemented. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved.