Fusion with an albumin‐binding domain improves pharmacokinetics of an αvβ3‐integrin binding fibronectin scaffold protein

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.