Albumin-binding domain from Streptococcus zooepidemicus protein Zag as a novel strategy to improve the half-life of therapeutic proteins.

Recombinant antibody fragments belong to the promising class of biopharmaceuticals with high potential for future therapeutic applications. However, due to their small size they are rapidly cleared from circulation. Binding to serum proteins can be an effective approach to improve pharmacokinetic properties of short half-life molecules. Herein, we have investigated the Zag albumin-binding domain (ABD) derived from Streptococcus zooepidemicus as a novel strategy to improve the pharmacokinetic properties of therapeutic molecules. To validate our approach, the Zag ABD was fused with an anti-TNFα single-domain antibody (sdAb). Our results demonstrated that the sdAb-Zag fusion protein was highly expressed and specifically recognizes human, rat and mouse serum albumins with affinities in the nanomolar range. Moreover, data also demonstrated that the sdAb activity against the therapeutic target (TNFα) was not affected when fused with Zag ABD. Importantly, the Zag ABD increased the sdAb half-life ∼39-fold (47min for sdAb versus 31h for sdAb-Zag). These findings demonstrate that the Zag ABD fusion is a promising approach to increase the half-life of small recombinant antibodies molecules without affecting their therapeutic efficacy. Moreover, the present study strongly suggests that the Zag ABD fusion strategy can be potentially used as a universal method to improve the pharmokinetics properties of many others therapeutics proteins and peptides in order to improve their dosing schedule and clinical effects.

Biodistribution of a (67)Ga-labeled anti-TNF VHH single-domain antibody containing a bacterial albumin-binding domain (Zag).

INTRODUCTION: Small domain antibodies (sdAbs) present high potential for both molecular in vivo imaging and therapy. Owing to the low molecular weight they are rapidly cleared from blood circulation, and new strategies to extend their half-lifes are needed for therapeutic applications. We have selected a bacterial albumin-binding domain (ABD) from protein Zag to be fused to an anti-tumor necrosis factor (TNF) single variable-domain heavy-chain region antibody (VHH) to delay blood clearance, and evaluated the biodistribution profile of the fusion protein. METHODS: The anti-TNF VHH and the fusion protein VHH-Zag were conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA). The anti-TNF and albumin-binding properties of the conjugates NOTA-VHH and NOTA-VHH-Zag were assessed by enzyme-linked immunosorbent assay (ELISA). The radioconjugates (67)Ga-NOTA-VHH and (67)Ga-NOTA-VHH-Zag were obtained by reaction of (67)GaCl3 with the corresponding conjugates at room temperature. Biodistribution studies were performed in healthy female CD-1 mice. RESULTS: The immunoreactivity of the VHH-based proteins is preserved upon conjugation to NOTA as well as after radiometallation. The radiochemical purity of the radioconjugates was higher than 95% as determined by ITLC-SG after purification by gel filtration. The biodistribution studies showed that the Zag domain affected the pharmacokinetic properties of VHH, with impressive differences in blood clearance (0.028 ± 0.004 vs 1.7 ± 0.8 % I.A./g) and total excretion (97.8 ± 0.6 vs 25.5 ± 2.1 % I.A.) for (67)Ga-NOTA-VHH and (67)Ga-NOTA-VHH-Zag, respectively, at 24h p.i. CONCLUSION: The Zag domain prolonged the circulation time of VHH by reducing the blood clearance of the labeled fusion protein (67)Ga-NOTA-VHH-Zag. In this way, the anti-TNF VHH in fusion with the Zag ABD presents a higher therapeutic potential than the unmodified VHH.