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Bivalent and Bispecific ScFv/Fab

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Advances in antibody engineering technology are enabling the designation of various engineered antibody forms for specific purposes in cancer diagnosis and immune therapy. With years of experience and great deal of researches, scientists from Creative Biolabs have investigated and established a series of approaches for bivalent and bispecific scFv/ Fab construction. The Fab and scFv fragments derived from phage display antibody libraries usually have short half-life and less affinity, whereas in many in vitro and in vivo applications, multivalency of antibody molecules is a desirable property. Linking two or more binding sites efficiently not only increases the functional avidity of antibody molecules, but also make the construction of bispecific antibodies (antibodies with dual specificities) possible.

scFv Dimerization (bivalent and bispecific)
We have developed three approaches to generate genetically engineered, dimerized scFv antibody fragments, namely, "Diabody", "Tandem scFv" and "Miniantibody".

Diabody is a non-covalent dimer of single chain Fv (scFv) fragment that consists of the heavy chain variable (VH) and light chain variable (VL) regions connected by a small peptide linker. Common linkers of 14–15 amino acid residues are long enough to span the distance between the N- and C-termini of the variable domains in a scFv. However, using linkers of 3–12 amino acid residues in length will result in the formation of diabody.

When two scFvs linked by the short linkers are expressed in the same cells, dual-functional antigen-binding sites are formed by crossover pairing of the variable light-chains and heavy-chains. The bispecific diabody, which is constrcted from heterogeneous scFvs, is also an important and commonly used form of recombinant bispecific antibody (BsAb). Diabodies have a rigid structure and can be expressed at high yields in bacteria.

Schematic diagram of Diabody Figure 1. Schematic diagram of Diabody and Tandem scFv

Tandem scFv
Tandem scFv (taFv) is produced by connecting two scFv molecules through a short linker. This format has a very flexible structure and is the simplest to be generated. Both bacterial expression plus refolding and eukaryotic expression are available for production of tandem scFvs. With years of experience, we have successfully constructed over 100 tandem scFvs. Creative Biolabs will be pleased to work on your requirements and design a proper strategy to construct the exact tandem scFv (bivalent or bispecific) you need.

It is worth mentioning that bispecific T cell engager (BiTE) is a very special form of tandem scFv. In a BiTE molecule, one of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule, which bring the T cell and cancer cell together and thus show great potentials in cancer therapy.


Bivalent (or bispecific) (scFv)2, so-called miniantibody, is produced by association of two scFv molecules through two modified dimerization domains. Leucine zippers are employed to mediate dimerization of scFv in a miniantibody form. We constructed dimerization cassettes that allow the conversion of scFv antibodies from all our phage display libraries to bivalent or bispecific antibodies. In this procedure, either Fos or Jun leucine zippers were fused to scFv proteins. Two cysteine residues were engineered in the Fos and Jun zipper domains to produce disulfide-stabilized homodimers. This approach usually results in efficient production of stable, secreted homodimers that retain their specificity as assessed in a number of assays. Also, Fos/Jun heterodimer instead of Fos/Fos or Jun/Jun homodimer formation allows production of bispecific (scFv)2 antibodies.

Fab Dimerization

We are professional in producing bivalent or bispecific Fab antibodies in miniantibody format.

Schematic diagram of bispecific antibody formats Figure 2. Schematic diagram of bispecific antibody formats

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