Abstract
BackgroundHalf-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 (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions.Methodology/Principal FindingsUsing site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) 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 (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics.Conclusions/SignificanceThe half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.
Highlights
With more and more small recombinant protein therapeutics advancing into the clinic, a major task in protein engineering is to overcome the limitation of the rapid clearance from a PLOS ONE | DOI:10.1371/journal.pone.0139838 October 2, 2015Fab-Selective Immunoglobulin-Binding DomainThere are no further patents, products in development or marketed products to declare
SpGC3 and SpGC3Fab were fused C-terminally to a 55 kDa bispecific single-chain diabody directed against Carcinoembryonic antigen (CEA) and CD3 endowed with a hexahistidyl-tag at the very C-terminus
Fusion of moieties with affinity for IgG or albumin to therapeutic proteins allows binding to the serum protein in vivo resulting in an extended half-life
Summary
With more and more small recombinant protein therapeutics advancing into the clinic, a major task in protein engineering is to overcome the limitation of the rapid clearance from a PLOS ONE | DOI:10.1371/journal.pone.0139838 October 2, 2015Fab-Selective Immunoglobulin-Binding DomainThere are no further patents, products in development or marketed products to declare. Immunoglobulins and albumin are serum proteins that show the longest circulation time at all and are interesting fusion or interaction partners [7,8]. Their astonishing long circulation time is mediated by the binding, after intracellular uptake, to the neonatal Fc receptor (FcRn) under acidic conditions in a recycling endosome [9,10,11]. Fusion of the Fc part or albumin is known to elongate the half-life of therapeutic proteins, and transient interactions through IgG- and albumin-binding peptides and protein domains are able to mediate a long circulation time [1,12,13]. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions
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