Abstract
The therapeutic and diagnostic efficiency of engineered small proteins, peptides, and chemical drug candidates is hampered by short in vivo serum half-life. Thus, strategies to tailor their biodistribution and serum persistence are highly needed. An attractive approach is to take advantage of the exceptionally long circulation half-life of serum albumin or IgG, which is attributed to a pH-dependent interaction with the neonatal Fc receptor (FcRn) rescuing these proteins from intracellular degradation. Here, we present molecular evidence that a minimal albumin binding domain (ABD) derived from streptococcal protein G can be used for efficient half-life extension by indirect targeting of FcRn. We show that ABD, and ABD recombinantly fused to an Affibody molecule, in complex with albumin does not interfere with the strictly pH-dependent FcRn-albumin binding kinetics. The same result was obtained in the presence of IgG. An in vivo study performed in rat confirmed that the clinically relevant human epidermal growth factor 2 (HER2)-targeting Affibody molecule fused to ABD has a similar half-life and biodistribution profile as serum albumin. The proof-of-concept described may be broadly applicable to extend the in vivo half-life of short lived biological or chemical drugs ultimately resulting in enhanced therapeutic or diagnostic efficiency, a more favorable dosing regimen, and improved patient compliance.
Highlights
These include increasing the molecular size by chemical modifications such as conjugation with polyethylene glycol [5, 6] or genetic fusion to human serum albumin (HSA)2 [7,8,9] or the Fc portion of human IgG [10]
The same enzyme-linked immunosorbent assay (ELISA) was performed with monomeric HSA (Sigma) directly coated in wells followed by detection of bound shFcRn-glutathione S-transferase (GST) in the absence or presence of albumin binding domain (ABD), hIgG1 with irrelevant specificity (hIgGIr), or monomeric HSA
ABD in Complex with Albumin Does Not Interfere with shFcRn Binding—The impact of ABD on the pH-dependent ligand binding of shFcRn was analyzed by surface plasmon resonance (SPR) and by a set of ELISA experiments
Summary
The pH-dependent interaction between albumin and FcRn must be preserved, and fourth, the ABD fusion protein must remain bound to albumin at the acidic pH of the endosome. As for the first criterion, the binding sites for ABD and FcRn on HSA are known to be distally located. The interaction site between HSA and FcRn has not been mapped in detail, but deletion studies have located the FcRn-binding site to domain III of HSA [35, 36]. We have explored the prerequisites for using the ABD as a general carrier molecule for half-life extension of short lived proteins. We have performed interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) to ensure that ABD in complex with albumin does not interfere with pH-dependent binding of FcRn to either albumin or IgG. The biodistribution and blood clearance rate of isotope-labeled ABD fusion protein were compared with that of rat serum albumin (RSA) injected simultaneously into rats, confirming a similar half-life
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