Reformatting between VHH, scFv, Fab and IgG
The same paratope can be delivered in several formats: a single domain VHH, a single chain variable fragment, a Fab, a full IgG, or a fusion such as VHH-Fc. Reformatting is routine, and it is also where programs quietly lose comparability, because a format change alters valency, size, stability and effector function all at once.
What each format is
VHH. One domain, about fifteen kilodaltons, no light chain. Small and stable, short serum half-life.
scFv. VH and VL joined by a flexible linker, usually a repeat of glycine and serine residues. About twenty-five kilodaltons. Convenient for display and for building larger molecules, but the linker length and the chain order both affect behavior, and scFvs are more prone to aggregation and to domain swapping than Fabs.
Fab. VH-CH1 paired with the full light chain, held by an interchain disulfide. About fifty kilodaltons, monovalent, generally better behaved than an scFv, and closer to how the site sits in a full antibody.
IgG. Two Fabs on an Fc. Bivalent, long circulating half-life, and effector functions that depend on isotype and glycosylation.
Fusions. VHH-Fc, scFv-Fc, bispecifics and tandem formats, chosen to add valency, half-life or a second specificity.
Valency is the part that surprises people
A bivalent molecule binding a surface with more than one copy of its target binds with avidity, and avidity can improve apparent affinity by orders of magnitude. That is a real functional gain, and it is not a change in the intrinsic affinity of the site.
The practical consequences are constant sources of confusion:
- A monovalent Fab measured by SPR and a bivalent IgG measured on cells are not comparable numbers.
- Ranking a panel as scFvs and then shipping IgGs can reorder the panel, because avidity rescues weak binders.
- Capturing an IgG on a sensor surface creates avidity at the surface. Flowing the IgG over immobilized antigen creates it differently. The orientation of the experiment changes the kinetics you report.
The rule that avoids most of the trouble is to measure in the format you intend to ship, and to keep orientation consistent across a panel.
Other things that move
Stability and aggregation. Linker choice and domain order change scFv aggregation. Fc fusions add stability but also add interfaces.
Expression. Yields differ by format, and a construct that expressed well as a VHH can be a poor IgG.
Effector function. Isotype and glycosylation decide complement and Fc receptor engagement, which can be the point of the molecule or an unwanted liability.
Developability liabilities. Moving a paratope onto a new framework changes its neighbors, so the liability profile has to be re-run rather than carried over.
A working sequence
Decide the final format early. Screen in a format that is cheap and informative. Before any decision that costs money, reformat the shortlist into the final format, re-express it, and re-measure binding, stability and function. Treat every number generated in the screening format as a rank, not as a property of the molecule you will ship.