Monoclonal
antibodies (mAbs) have proven to be useful for development of new therapeutic drugs
and diagnostic techniques. To overcome the difficulties posed by their complex
structure and folding, reduce undesired
immunogenicity, and improve pharmacoki- netic properties, a plethora of
different Ab fragments have been developed. These include recombinant Fab and
Fv segments that can display improved properties over those of the original
mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs,
diabodies, and nanobodies, all contain the variable Ig domains responsible for
binding to specific antigenic epitopes, allowing for specific targeting of
pathological cells and/or molecules. These fragments can be easier to
produce, purify and refold than a full Ab, and due to their smaller size they
can be well absorbed and distributed into target tissues. However, the
physicochemical and structural properties of the immunoglobulin (Ig) domain,
upon which the folding and conformation of all these Ab fragments is based,
can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to
unfolding and aggregation when produced out of the structural context of an
intact Ab molecule. When unfolded, Ab fragments may lose their specificity as
well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered
pharmacokinetic and immunogenic properties that can augment their toxicity.
Therefore, much effort has been placed in understanding the factors impacting
the stability of Ig folding at two different levels: 1) intrinsically, by
studying the effects of the amino acid sequence on Ig folding; 2)
extrinsically, by determining the environmental conditions that may influence
the stability of Ig folding. In this review we will describe the structure of the Ig
domain, and the factors that impact its stability, to set the context for the
different approaches currently used to achieve stable recombinant Ig domains
when pursuing the development of Ab fragment-based biotechnologies.