Dimerization of scFv and Fab Fragments
The Fab and scFv fragments derived from filamentous phage display antibody libraries are monovalent, whereas in many in vitro and in vivo applications, multivalency of antibody molecules is a desirable property. In addition, linking two or more binding sites efficiently increases the functional avidity of antibody molecules or results in the construction of antibodies with dual specificities.
In designing strategies for dimerization of antibody fragments, several issues need to be addressed including stability and homogeneity of the dimers, resistance to proteolytic cleavage during in vivo assembly, efficient production of preferably soluble protein, easy gene engineering steps, great expression level in bacterial cells, good activity and specificity maintenance, thermal stability and the general applicability for the construction of both bivalent and bispecific recombinant antibodies. With these issues in mind, we have investigated and established a series of approaches for dimerization of both scFv and Fab fragments.
o scFv Dimerization
We have developed three approaches to generate genetically engineered, dimerized scFv antibody fragments, namely, “Diabody”, “Tandem scFv” and “Miniantibody”.
In a scFv expression vector, the cDNAs that encode the heavy-chain variable region (VH) and the light-chain variable region (VL) of an antibody are assembled via the sequence encoding a linker peptide. 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 polypeptide chains, VHA-VLB and VHB-VLA, 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. Diabodies have a rigid structure and can be expressed at high yields in bacteria.
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. We have also invented a unique linker [to bring together two scFv molecules] that permits long-half-life and low-immunogenicity in humans, two great features for developing bi-specific therapeutic antibodies.
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.
o Fab Dimerization
We are professional in producing bivalent or bispecific Fab antibodies in miniantibody format.