Fusion with an albumin‐binding domain improves pharmacokinetics of an αvβ3‐integrin binding fibronectin scaffold protein
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Abstract
Fusion with an albumin-binding domain (ABD) of streptococcal protein G represents a popular approach for half-life extension of small protein therapeutics in the organism. To increase the circulation time of engineered αvβ3-integrin-binding protein (JCL) based on the 10th human fibronectin type III domain (10 Fn3), we have constructed several fusions with ABD with different orientations of the partner proteins and linker length. The recombinant proteins were expressed in Escherichia coli cells and purified by nickel-affinity chromatography. All fusion proteins bound human serum albumin (HSA) in ELISA assay; however, fusions with longer linkers demonstrated better performance. Interaction of ABD-L15 -JCL and JCL-L14 -ABD with HSA was confirmed by analytical size exclusion chromatography and pull-down assays. Surprisingly, the thermal stability of ABD-L15 -JCL was dramatically decreased in comparison with JCL and JCL-L14 -ABD proteins. Pharmacokinetic studies revealed that JCL-L14 -ABD circulated in murine blood about 10 times longer than ABD-L15 -JCL and 960 times longer than JCL. Biodistribution studies of JCL-L14 -ABD in mice revealed its increased level in blood and a decreased accumulation in liver and kidneys in comparison with JCL. Obtained results demonstrate the utility of the fusion with ABD for half-life extension of the binding proteins based on 10 Fn3.
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