An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics

Abstract

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.

Programmable half-life and anti-tumour effects of bispecific T-cell engager-albumin fusions with tuned FcRn affinity

“…The LiTE 26 cDNA construct was synthesised (Genscript) with unique restriction sites between each protein domain i.e. the EgA1 nanobody, the OKT3 scFv and subcloned into vectors containing wild-type HSA and variants with high FcRn binding affinity (HB) or removed FcRn binding affinity (NB) 45 . The vectors further contained CMV enhancer and promoter supporting expression in HEK293E cells.…”

“…Binding kinetics of human FcRn for purified Albu-LiTE constructs was measured by Bio-Layer Interferometry (BLI) on an Octet Red96e system (ForteBio/Molecular Devices). Recombinant HSA variants without fusion protein corresponding to WT, no or high FcRn affinity were used as controls 45 . Briefly, biotinylated soluble human-FcRn (Immunitrack, # ITF02) was immobilised on streptavidin-coated biosensors (ForteBio/Molecular Devices) in PBS pH 7.4 supplemented with 0.01% Tween-20 at a 8.75 nM concentration.…”

Biochemical, molecular and clinical aspects of coagulation factor VII and its role in hemostasis and thrombosis

“…77 Recently, an engineered albu-min-fused rFVIIa molecule showed enhanced transcellular transport upon intranasal delivery and extended plasma half-life in transgenic mice. 78 The development of non-substitutive therapy for the hemophilias, such as the use of anti-antithrombin RNA interference or of aptamer and monoclonal antibodies directed against TFPI, have raised expectations for improvement of the quality of life in individuals with FVII deficiency. 75 Based on adeno-associated viral-mediated expression of FVII 79 , gene therapy has produced sustained correction of severe FVII deficiency in dogs.…”

Extended plasma half-life of albumin-binding domain fused human IgA upon pH-dependent albumin engagement of human FcRnin vitroandin vivo

“…76 These mice can be preloaded with human albumin without any sign of immunogenicity. 16,77,80 The set of in vitro and in vivo systems used in this study provides a guide to preclincical testing of drugs indirectly targeting human FcRn.…”

“…ABD-fused proteins may also associate with human albumin variants that have been engineered for enhanced half-life via improved pH-dependent FcRn binding. [79][80][81] One such variant harbors three mutations (E505Q/T527M/K573P; QMP) that was recently shown to have 2-fold extended half-life compared to wild-type (WT) albumin in human FcRn transgenic mice. 80 First, we addressed if the IgA1 ABD variants could bind the engineered QMP variant and subsequently engage human FcRn.…”