A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties

BACKGROUND: Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpG(C3)) as module for half-life extension. SpG(C3) is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions. METHODOLOGY/PRINCIPAL FINDINGS: Using site-directed mutagenesis, we generated a Fab-selective mutant (SpG(C3Fab)) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpG(C3Fab)RR) conferred prolonged plasma half-lives compared with SpG(C3Fab) when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpG(C3Fab)RR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics. CONCLUSIONS/SIGNIFICANCE: The half-life extension properties of SpG(C3) can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpG(C3) module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.