scFvs

Single-chain variable fragments (scFvs) are recombinant antibody fragments that consist of the variable regions of the heavy and light chains (VH and VL) connected by a flexible peptide linker. The linker, commonly (Gly4Ser)3, allows the two domains to pair intramolecularly and form an antigen-binding site similar to that of a full antibody. scFvs can be oriented as VH–VL or VL–VH and typically have a molecular weight of about 25–30 kDa.

Recombinant production is versatile: they can be expressed in bacteria (often periplasmically to facilitate disulfide bond formation), yeast, or mammalian cells. The small size enables easy genetic fusion to other proteins, including fluorescent reporters or effector domains, and enables tissue penetration. They are frequently used as targeting moieties in immunotherapies, diagnostic reagents, and research tools.

scFvs are used to construct bispecific antibodies and T cell engagers, such as tandem scFvs or scFv-Fc fusions. They can be engineered into other dimeric formats such as diabodies or tandem constructs that confer dual specificities. Fusion to an Fc region or albumin-binding domain can extend serum half-life.

Limitations include potential stability and solubility issues, rapid clearance from circulation, and immunogenicity if derived from non-human sequences. Humanization or affinity maturation, formulation optimizations, and fusion to Fc or other half-life extending moieties are common mitigation strategies.

Because of their modular nature, scFvs remain a versatile tool in basic research, diagnostics, and targeted therapies, complementing or replacing larger antibody fragments in many contexts.