Effects of genetic fusion of factor IX to albumin on in vivo clearance in mice and rabbits

Abstract

SummaryIndividuals with haemophilia B require replacement therapy with recombinant or plasma-derived coagulation factor IX (fIX). More benefit per injected dose might be obtained if fIX clearance could be slowed. The contribution of overall size to fIX clearance was explored, using genetic fusion to albumin. Recombinant murine fIX (MIX), and three proteins with C-terminal epitope tags were expressed in HEK 293 cells: tagged MIX (MIXT), tagged mouse serum albumin (MSAT) and MFUST, in which MIX and MSAT were fused in a single polypeptide chain. Proteins MFUST and MIXT were two-to threefold less active in clotting assays than MIX. In mice, the area under the clearance curve (AUC) was reduced for MFUST compared with MSAT or plasma-derived MSA (pd-MSA); the terminal catabolic halflife (t 0AE5 ) did not differ amongst the three proteins. Two minutes after injection, >40% of the injected MFUST was found in the liver, compared with <10% of either MSAT or pd-MSA. In rabbits, the AUC for MFUST was reduced compared to MIXT, MSAT, or pd-MSA, while the t 0AE5 of the fusion protein fell between that of MIXT and MSAT or pd-MSA. Similar results were obtained with non-radioactive fused or non-fused recombinant human fIX in fIX knockout mice. The clearance behaviour of the fusion protein thus more closely resembled that of fIX than that of albumin despite a modest increase in terminal half-life, suggesting that fIX-specific interactions that are important in determining clearance were maintained in spite of the increased size of the fusion protein.

Recombinant Human Serum Albumin Dimer has High Blood Circulation Activity and Low Vascular Permeability in Comparison with Native Human Serum Albumin

“…On the other hand, Komatsu et al (33) has demonstrated the utility of an rHSA dimer, which was cross-linked by the thiol group of Cys-34 with 1,6-bis(maleimido)hexane, and its synthetic heme hybrid as an oxygen carrier displayed a longer half-life in the bloodstream and tissue distributions that were similar to rHSA. The discrepancy between the studies may probably due to species difference, as shown in the study by Sheffield et al (34). All values are mean T SD (n = 4).…”

The role of sphingosine-1-phosphate in endothelial barrier function

“…Clearance rates of serum albumin and HDL differ in vivo [147, 148] and differences in clearance of extracellular HDL–S1P and albumin–S1P might variably limit S1P responses. Though recent findings suggest that albumin–S1P turnover is greater than HDL–S1P in vivo [120], we found no significant difference in the clearance of albumin–S1P and HDL–S1P from culture medium by endothelial cells [90].…”

Cell-matrix Interactions of Factor IX (FIX)-engineered human mesenchymal stromal cells encapsulated in RGD-alginate vs. Fibrinogen-alginate microcapsules

“…Human FIX antigen was detected by ELISA assay as previously described (Wen et al 2006, 2007, Sheffi eld et al 2004, Sayyar et al 2012 using the reagents and protocol from Affi nity Biologicals Inc. (Ancaster, ON, Canada).…”