Bivalent and Bispecific Single Chain

Antibody
Bivalent and Bispecific Single Chain Antibody

Antibody is not a stranger to everyone, even a new born baby. It is the black hole on
the biological world, as there is always something new that attracts biologists and
researchers.

Since the first success achieved in single chain antibody fragmentscFvresearch

by Bird and Huston in the 1988, tremendous achievements have been made in the
research and development of single domain antibody technology.

However, in terms of application, there are still problems remain unsettled. The scFv
fragments derived from phage display antibody libraries usually have short half-life
and less affinity. However, multivalency of antibody molecules turns out to be a
desirable property in many in vitro and in vivo applications. After years of experience
and great efforts, scientists and researchers from Creative Biolabs have carried out a
series of approaches for bivalent and bispecific scFv and Fab construction, which may
greatly contribute to the current situation.
According to its newest data, there are three approaches of generating genetically
engineered and dimerized scFv antibody fragments, miniantibody, diabody and
Tandem scFv.

Miniantibody

Bivalent or bispecific (scFv) 2, the so-called miniantibody, is produced by the

combination of two scFv molecules with two modified dimerization domains. Leucine
zippers are utilized to mediate dimerization of scFv in a miniantibody form. It
constructed dimerization cassettes which allow the conversion of scFv antibodies
from all its phage display libraries to bivalent or bispecific antibodies. During this
procedure, either Fos or Jun leucine zippers are fused to scFv proteins. Two cysteine
residues were engineered in the Fos and Jun zipper domains to produce
disulfide-stabilized homodimers, which usually leads to efficient production of stable,
secreted homodimers that are able to retain their specificity as assessed in a number of
assays.

Diabody

Diabody is a non-covalent dimer of single-chain scFv, fragments that consists of the

heavy chain variable (VH) and light chain variable (VL) regions connected by a small
peptide linker. 1415 amino acid residues common linkers are long enough to span
the distance between the N- termini and C-termini of the variable domains in a scFv.
However, utilizing linkers of 312 amino acid residues in length can lead to the
formation of diabody.
When two ScFvs linked with the short linkers are expressed in the same cells,
dual-functional antigen-binding sites will be formed through crossover pairing of the
variable light-chains and heavy-chains. The bi-specific diabody, which is constructed
with heterogeneous scFvs, is also an important and commonly used form of
recombinant bi-specific antibody. A distinct feature of diabody is that it has a rigid
structure and can be expressed at high yields in bacteria. See figure 2.

Tandem scFv

Tandem scFv (taFv) is produced by connecting two scFv molecules with a short linker.
This form of scFv has a very flexible structure and is comparatively easy to be
generated. Both bacterial expression and refolding and eukaryotic expression are able
to produce tandem scFvs. With years of experiences, Creative Biolabs has
successfully constructed over 100 tandem scFvs.

It is worth mentioning here that bispecific T cell engager (BiTE) is a unique form of
tandem scFv. In a BiTE molecule, one of the scFvs binds to T cells via CD3 receptor
and the other to a tumor cell via a tumor specific molecule. As this procedure brings
together the T cell and cancer cell, it has great possibilities in cancer therapy.

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