Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents

Abstract

Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab′)2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule. To demonstrate the applicability of this technology, an anti‐neuraminidase NC10 scFv‐streptavidin fusion was expressed in E. coli and the product was refolded and purified to homogeneity from 6 M guanidine hydrochloride. Analysis in a BIAcoreTM biosensor showed that the NC10 scFv moiety reacted with immobilised neuraminidase and that the core streptavidin moiety was able to bind biotinylated anti‐ferritin Fab' to produce a new model bispecific reagent which bound ferritin. Conceptually, this design principle can be applied to the creation of useful diagnostic and possibly therapeutic molecules.

Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design

“…It was also exploited for creating bispecific antibody multimers (62) (Fig. 3, 4-6 ), pretargeting in radionuclide therapy of cancer, (63) and tumor imaging.…”

“…Strept(avidin)-biotin system with very high binding affinity (K d $10 À14 -10 À15 M (61) is widely used for analytical applications. It was also exploited for creating bispecific antibody multimers (62) (Fig. 3, 4-6 ), pretargeting in radionuclide therapy of cancer, (63) and tumor imaging.…”

Designed Auto-assembly of Nanostreptabodies for Rapid Tissue-specific Targeting in Vivo

“…Although the interaction between biotin and SA is one of the strongest noncovalent association known (14), the classical reaction of chemical biotinylation produces somewhat arbitrary, nonspecifically localized linkages that lead to poorly characterized heterocomplexes. Accordingly, there has been only one report on an attempt to create bispecific antibodies, despite the wide availability of biotinylation reagents (39). We have anticipated that the association between SA and biotinylated proteins would follow precise assembly rules, generating structurally well-defined complexes that would be easy to purify if we were able to incorporate biotin residues at discrete locations on the protein scaffold and control the assembly of the varied biotinylated moieties.…”