Single-chain variable fragment

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ScFv
)
Rotating scFv fragment with highlighted complementarity determining regions (CDRs)
The two possible structures of a single-chain variable fragment, with the antigen binding sites including the N-termini on the left and the C-termini on the right. The linker peptides are shown as arrows.

A single-chain variable fragment (scFv) is not actually a

immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.[1] The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.[2]
This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.[3] The image to the right shows how this modification usually leaves the specificity unaltered.

These molecules were created to facilitate

artificial T cell receptors
(chimeric antigen receptor).

Unlike

E. coli.[3]

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.[4][5][6]

Bivalent and trivalent scFvs

Structure of divalent (top) and trivalent (bottom) scFvs, tandem (left) and di-/trimerisation format (right)

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.

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.[10] 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.[11]

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.[12][13] The furthest developed of these are bispecific tandem di-scFvs, known as bi-specific T-cell engagers
(BiTE antibody constructs).

Examples

References

  1. PMID 3045807
    .
  2. doi:10.18452/15246. {{cite journal}}: Cite journal requires |journal= (help
    )
  3. ^
    PMID 16796389
    .
  4. ^
    S2CID 121351794
    .
  5. PMID 9186782
    .
  6. PMID 31011559
    .
  7. PMID 16760193
    .
  8. PMID 15109810
    .
  9. PMID 8341653
    .
  10. ^
    PMID 9652755
    .
  11. S2CID 20213440
    .
  12. PMID 11388794
    .
  13. ^ Kellner, C (2008). Entwicklung und Charakterisierung bispezifischer Antikörper-Derivate zur Immuntherapie CD19-positiver Leukämien und Lymphome [Development and characterisation of bispecific antibody derivatives for the immunotherapy of CD19-positive leukaemia and lymphoma] (Thesis) (in German and English). Erlangen-Nürnberg: Friedrich-Alexander-Universität.
  14. PMID 15331798
    .
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