The smallest antibody-derived binding structures are the separate variable domains. Earlier, isolated domains were not of
practical interest because of poor solubility and low affinity compared to the parent antibody.8
However, discoveries were made later that certain types of organisms, the camelids and cartilaginous fish, possessed high
affinity single V-like domains mounted on an Fc equivalent domain structure as part of their immune system.9,10 The V-like domains (called VhH in camelids and V-NAR in sharks) typically display long surface loops, which allow penetration
of cavities of target antigens. They also stabilize isolated VH domains by masking hydrophobic surface patches.11
Human V domain variants have been designed using selection from phage libraries and other approaches that have resulted in
stable, high binding VL- and VH-derived domains.
Engineering the monovalent structures (Fab, scFv, V-domain) in multivalent structures can increase functional affinity (avidity).
Such multispecific molecules allow direct association of two or more different targets engineered into dimeric, trimeric,
or tetrameric conjugates, either chemically of genetically.12,13
Other antibody-derived structures include Genentech's one-armed antibodies and Genmab's Unibody, which omit parts of the IgG
structure to give specific therapeutic benefits.14.15
Mammalian cell expression has been used extensively to produce full-length IgGs that have been appropriately glycosylated.
However, for antibody fragments, which lack the Fc region with its N-linked glycans, microbial systems are the most effective
production system for the following reasons:16
- easy and rapid strain generation
- short fermentation time
- simple fermentation media
- robustness and scalability of microbial fermentation
- no virus testing requirements or viral reduction steps.
These factors can reduce therapeutic costs by streamlining development timelines and reducing manufacturing CoGs.
Fragment production with microbial systems has been demonstrated with E. coli, yeasts, and fungi. Filamentous fungi has been reported for antibody fragment expression but is not a recommended system
because of high risks of product proteolysis and high culture viscosities.17 From a regulatory perspective, a fungal system lacks the history of use in therapeutic proteins, which exists for E. coli and yeasts.
With yeast systems, correctly folded product is secreted into the culture medium.18–20 The disadvantages of yeast expression compared with E. coli expression include longer fermentation time, the potential for N glycosylation that will be nonauthentic and high in mannose,
and the potential for proteolytic clipping by host proteases.
Fragment production in E. coli is preferably achieved through secretion into the oxidizing periplasmic space, which results in an authentically formed antibody
fragment. The technology has been demonstrated not just for single-chain containing fragments but also for more complex structures
such as Fabs. In the latter case, both H and L chains express separately into the periplasm where self assembly and refolding
of the Fab takes place.
Thus, E. coli is a highly suitable and versatile expression system with many desirable features for antibody fragment production, including
significant regulatory experience for therapeutic protein production.