Background: Half-life extension has become increasingly important for therapeutic proteins. Results: Fusion of different bacterial immunoglobulin-binding domains to small recombinant antibodies prolonged their half-life to varying extents. Conclusion: Fusion of domain C3 of streptococcal protein G showed the best effects, thus representing a promising module for half-life extension of small-sized therapeutics. Significance: This study further established immunoglobulin-binding domains as suitable half-life extension modules.

Binding of a therapeutic protein to a long-circulating plasma protein can result in a strongly extended half-life. Among these plasma proteins, albumin and immunoglobulins are of special interest because of their exceptionally long half-life, which is to a great extent determined by recycling through the neonatal Fc receptor (FcRn). Many strategies have been established employing reversible binding to albumin, e.g. using an albumin-binding domain from streptococcal protein G. We show here that the half-life of a recombinant antibody molecule can also be prolonged by fusion to a single immunoglobulin-binding domain (IgBD) from staphylococcal protein A. This domain (domain B, SpA(B)) is composed of 56 amino acid residues and was fused to the C-terminus of a bispecific single-chain diabody (scDb). The scDb-SpA(B) fusion protein was produced in HEK293 cells and retained its antigen-binding activity as shown by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, the fusion protein was capable of binding to human and mouse IgG in a pH-dependent manner. In mice, the terminal half-life of the fusion protein was improved from ∼1-2 h of the unmodified scDb to 11.8 h. Although the fusion protein did not reach the long half-life seen for IgG, our results established the applicability of a single bacterial IgBD for half-life extension purposes.

Recombinant bispecific antibodies have many different applications; especially promising is their therapeutic potential due to their ability to retarget an effector molecule or a cell to a disease-related target structure. In the last years, many formats have been developed: two commonly used are the bispecific diabody and the tandem scFv. In this chapter, the cloning, bacterial production, purification, and characterization of the two antibody formats are described in detail.

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