Single-chain variable fragment

Purification

Single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites (e.g., protein G) cannot be used to purify antibodies. These fragments can often be purified or immobilized using protein L, since protein L interacts with the variable region of kappa light chains. More commonly, scientists incorporate a six histidine tag on the c-terminus of the scFv molecule and purify them using immobilized metal affinity chromatography (IMAC). Some scFv can also be captured by protein A if they contain a human VH3 domain.

Bivalent and trivalent scFvs

Divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. Another possibility is the creation of scFvs with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Consequently, diabody drugs could be dosed much lower than other therapeutic antibodies and are capable of highly specific targeting of tumors in vivo. Still shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

All of these formats can be composed from variable fragments with specificity for two different antigens, in which case they are types of bispecific antibodies. The furthest developed of these are bispecific tandem di-scFvs, known as bi-specific T-cell engagers (BiTE antibody constructs).

Examples

• Pexelizumab, a scFv binding to component 5 of the complement system and designed to reduce side effects of cardiac surgery
• C6.5, a diabody targeting HER2/neu found in some breast cancers
• Brolucizumab, a scFV binding to VEGF-A and used to treat wet age-related macular degeneration
• G6A, a scFV binding to human Hsp70.1 and can be used to target this protein as a potential marker in vast cancers.

References

References

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6. (2019-02-21). "Purification of a Novel Anti-VEGFR2 Single Chain Antibody Fragmentand Evaluation of Binding Affinity by Surface Plasmon Resonance". Advanced Pharmaceutical Bulletin.
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8. (2004). "A revival of bispecific antibodies". Trends in Biotechnology.
9. (July 1993). ""Diabodies": small bivalent and bispecific antibody fragments". Proceedings of the National Academy of Sciences of the United States of America.
10. (1998). "Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu". British Journal of Cancer.
11. (1999). "Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding". FEBS Letters.
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13. (2008). "Entwicklung und Charakterisierung bispezifischer Antikörper-Derivate zur Immuntherapie CD19-positiver Leukämien und Lymphome". Friedrich-Alexander-Universität.
14. (2004). "Preliminary report of the effects of complement suppression with pexelizumab on neurocognitive decline after coronary artery bypass graft surgery". Stroke: A Journal of Cerebral Circulation.