A single-chain variable fragment (scFv) is a fusion protein

Fusion protein

Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...

 of the variable regions of the heavy (V_H) and light chains

Immunoglobulin light chain

]The immunoglobulin light chain is the small polypeptide subunit of an antibody .A typical antibody is composed of two immunoglobulin heavy chains and two Ig light chains.-In humans:...

 (V_L) of immunoglobulins, connected with a short linker peptide

Peptide

Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...

 of ten to about 25 amino acid

Amino acid

Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

s. The linker is usually rich in glycine

Glycine

Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...

 for flexibility, as well as serine

Serine

Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...

 or threonine

Threonine

Threonine is an α-amino acid with the chemical formula HO2CCHCHCH3. Its codons are ACU, ACA, ACC, and ACG. This essential amino acid is classified as polar...

 for solubility, and can either connect the N-terminus of the V_H with the C-terminus of the V_L, or vice versa.
This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. The image to the right shows how this modification usually leaves the specificity unaltered.

These molecules were created to facilitate phage display

Phage display

Phage display is a method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P...

, where it is highly convenient to express the antigen-binding domain as a single peptide. As an alternative, scFv can be created directly from subcloned

Subcloning

In molecular biology, subcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality....

 heavy and light chains derived from a hybridoma

Hybridoma

Hybridoma technology is a technology of forming hybrid cell lines by fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis...

. ScFvs have many uses, e.g., flow cytometry

Flow cytometry

Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...

, immunohistochemistry

Immunohistochemistry

Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...

, and as antigen-binding domains of artificial T cell receptors.

Unlike monoclonal antibodies

Monoclonal antibodies

Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....

, which are often produced in mammalian cell cultures, scFvs are more often produced in bacteria cell cultures such as E. coli.

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

Protein G

Protein G is an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria much like Protein A but with differing specificities. It is a 65-kDa and a 58 kDa cell surface protein that has found application in purifying antibodies through its binding to the Fc region...

) used to purify antibodies. These fragments can often be purified or immobilized using Protein L

Protein L

Protein L was first isolated from the surface of bacterial species Peptostreptococcus magnus and was found to bind immunoglobulins through L chain interaction, from which the name was suggested. It consists of 719 amino acid residues...

, 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). For unknown reasons, some scFv can also be captured by Protein A

Protein A

Protein A is a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in...

.

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 V_H and two V_L 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 constant

Dissociation constant

In chemistry, biochemistry, and pharmacology, a dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into...

s 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 (BiTEs).

Examples

• Pexelizumab
  Pexelizumab
  Pexelizumab is a drug designed to reduce side effects of coronary artery bypass grafting and angioplasty, among other types of cardiac surgery. It is a single chain variable fragment of a monoclonal antibody targeted against component 5 of the complement system....
  
  , a scFv binding to component 5
  Complement component 5
  Complement component 5 is a protein that in humans is encoded by the C5 gene.Complement component 5 is involved in the complement system. It is cleaved into C5a and C5b:* C5a plays an important role in chemotaxis....
  
   of the complement system
  Complement system
  The complement system helps or “complements” the ability of antibodies and phagocytic cells to clear pathogens from an organism. It is part of the immune system called the innate immune system that is not adaptable and does not change over the course of an individual's lifetime...
  
   and used to reduce side effects of cardiac surgery
  Cardiac surgery
  Cardiovascular surgery is surgery on the heart or great vessels performed by cardiac surgeons. Frequently, it is done to treat complications of ischemic heart disease , correct congenital heart disease, or treat valvular heart disease from various causes including endocarditis, rheumatic heart...
  
  
• C6.5, a diabody targeting HER2/neu
  HER2/neu
  HER-2 also known as proto-oncogene Neu, receptor tyrosine-protein kinase erbB-2, CD340 or p185 is an enzyme that in humans is encoded by the ERBB2 gene. Over expression of this gene is correlated with higher aggressiveness in breast cancers...
  
   found in some breast cancer
  Breast cancer
  Breast cancer is cancer originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules that supply the ducts with milk. Cancers originating from ducts are known as ductal carcinomas; those originating from lobules are known as lobular carcinomas...
  
  s