Fig. 4. Engineering of an albumin-binding site in CMPX-321A to increase its serum half-life.

(A) By grafting only the short albumin-binding helices of F. magna protein G (red) on α-helix C of CMPX-321A, the resulting CPAB CMPX-383B is capable of binding albumin at the expense of only a 3.6-kDa increase in molecular weight. (B) Fitting of a 1:1 Langmuir binding model (red) to the BLI data traces (black) of the interaction CMPX-383B to immobilized glutathione S-transferase (GST)–_ΔNΔCMCL-1, plotted as the spectral shift in function of time. Experiment was performed in duplicate. (C) Alphabody concentrations of CMPX-321A (blue) and the serum half-life extended Alphabody CMPX-383B (orange) in the sera of, respectively, three mice and two mice treated with a single intravenously administered bolus injection of 20 mg/kg. (D) Confocal microscopy experiments on HeLa cells in the presence of 10 μM MSA show that substitution of Gly⁴⁰ by alanine (right and middle columns, respectively) allows a faster cellular uptake. After the indicated incubation time, cells were washed and probed for the presence of the Alphabody (green). The resulting immunofluorescence image was overlaid with a Ruby stain for the nuclei (red). Scale bar, 20 μm.