Fusion of engineered albumin with factor IX Padua extends half‐life and improves coagulant activity

Abstract

The short half-life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)-mediated recycling of the chimera. However, patients would greatly benefit from further FIX-HSA half-life extension. In the present study, we designed a FIX-HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, which resulted in improved HSA release. Second, insertion of the FIX R338L (Padua) substitution conferred hyperactive features (sevenfold higher specific activity) as for FIX Padua alone. Furthermore, we exploited an engineered HSA (QMP), which conferred enhanced human (h)FcRn binding [dissociation constant (K D ) 0Á5 nM] over wild-type FIX-HSA (K D 164Á4 nM). In hFcRn transgenic mice, Padua-QMP displayed a significantly prolonged half-life (2Á7 days, P < 0Á0001) versus FIX-HSA (1 day). Overall, we developed a novel FIX-HSA protein with improved activity and extended half-life. These combined properties may result in a prolonged functional profile above the therapeutic threshold, and thus in a potentially widened therapeutic window able to improve HB therapy. This rational engineering of both partners may pave the way for new fusion strategies for the design of engineered biotherapeutics.

F9 missense mutations impairing factor IX activation are associated with pleiotropic plasma phenotypes

“…The relative impacts of FIXag levels versus retained activation peptide on FIX PK were indirectly disentangled by linear regression analysis that suggested activation site mutations as independent predictors, which warrants further studies. These F9 ‐related features would permit us to better estimate the improved pharmacokinetic profile of currently used and novel long‐acting FIX molecules 61,62 …”

“…These F9 ‐related features would permit us to better estimate the improved pharmacokinetic profile of currently used and novel long‐acting FIX molecules. 61 , 62 …”

Translational readthrough at <i>F8</i> nonsense variants in the factor VIII B domain contributes to residual expression and lowers inhibitor association

“…For comparison analysis, a second version of the FVIII-GL construct was produced by cloning the Gaussia sequence including the signal peptide (SP) downstream of hF8 (Supplementary Figure S1G). Further, a glycine-serine (GS) linker, known to improve secretion efficiency of chimeric proteins containing coagulation factors by separating fusion partners, 43,44 was added between hF8 and Gaussia sequences by self-annealing oligonucleotides with flanking PmlI restriction sites to obtain the pCMV6XL4-hF8-linker-GL expression plasmid (Supplementary Figure S1H). Finally, the pCMV6XL4-hF8-Stop-GL plasmid (Supplementary Figure S1I), designed as negative control, was produced by reintroducing the hF8 NTC between hF8 and Gaussia.…”

“…We then evaluated expression levels at different time points of FVIII-GL, which showed time-dependent secreted luciferase activity (Supplementary Figure S3B). Subsequently, based on the knowledge of the effects of linker sequences on fusion protein features, 43,44 we compared the expression at 48 hours of FVIII-GL, in which FVIII and Gaussia are directly fused, with that of FVIII-linker-GL, containing an interposed glycine-serine (GS) linker. The secretion efficiency of the two chimeric proteins was similar (Supplementary Figure S3C), which prompted us to select the direct FVIII-GL fusion protein as scaffold for the insertion of the designed nonsense/missense changes.…”

Gene Therapy in Hemophilia: Recent Advances

“…Another possibility is to modify the transgene itself to boost the efficiency of the coagulation factor once it has expressed itself. In this regard, Lombardi et al [ 73 ] recently applied protein fusion engineering to design a FIX-HSA (human serum albumin) variant that lengthens the half-life of the FIX molecule. This combination could be used as a transgene in gene therapy protocols in the context of HB.…”