title: Characterization of single point mutation in the gamma-carboxyglutamic acid domain of recombinant clotting Factor IX and its therapeutic potential
title: Charakterisierung einer einzelnen Punktmutation in der Gamma-Carboxyglutaminsäure-Domäne des rekombinanten Gerinnungsfaktors IX und ihr therapeutisches Potenzial
titleType: AlternativeTitle
Creators: name: Knoll Machado, Steffi
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: Medical sciences Medicine
Medizin
Hemophilia B
Faktor 9
rekombinantes Protein ,
factor 9
Hämophilie B
Gerinnungsfaktor IX

description: Hemophilia B (HB) is an inherited bleeding disorder caused by deficiency of FIX. The severity is classified based on the FIX activity level in the plasma. The trough levels of FIX activity in the circulation above 5% is considered the relevant parameter to maintain hemostasis. Even though rFIX products with extended halflife have proven to be highly efficient in reducing annual bleeding rates in HB patients, there is an emerging concept that in addition to circulating FIX, extravascular (EV) FIX that is not circulating in plasma and is therefore not 
measurable, contributes to hemostasis. A single point mutation near the N-terminus of rFIX has been described as a determinant for the binding of FIX to extravascular space comprising endothelial cells and components of extracellular matrix. In this thesis the biological significance of rFIX in the extravascular space (EVS) was investigated using recombinant FIX proteins with a substitution at position 5, known to either enhance (rFIXK5R) or reduce (rFIXK5A) binding to EVS, more specifically collagen IV. Albumin fusion extends the half-life of rFIX and is a standard of care for prophylactic therapy. To understand the impact of albumin fusion in EV distribution and hemostatic efficacy of rFIX, albumin fused and non-fused rFIX protein K5 variants were generated and thoroughly characterized in vitro and in vivo. A functional characterization of the rFIX variants was conducted in a FIX one stage clotting assay and in a modified FIX chromogenic activity assay, mimicking the tenase complex in vitro. The pharmacokinetic (PK) profile was evaluated in HB (FIX ko) mice after intravenous and subcutaneous administration. Plasma levels of the rFIX proteins and the immunohistochemical evaluation of the liver samples confirmed published data that rFIX carrying a K5A and K5R mutation have an impact on PK behavior and tissue sequestration. The albumin fused and non-fused rFIXK5R was found in lower levels in the plasma when administered via both intravenous and subcutaneous routes and rFIXK5R was found to be retained in the tissue. Nevertheless, collagen IV alone seems not to be the binding partner in the extravascular compartment as revealed in the surface plasma resonance experiments. The efficacy studies revealed that the ability of FIX to bind to components of the EVS has an impact on the hemostatic response in HB mouse models. Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding to components of the EVS (rFIXK5R) confers prolonged hemostatic efficacy while the duration of efficacy of rFIXK5A was relevantly shorter after intravenous administration of the 
recombinant proteins. These results can be explained by an additional local accessible rFIXK5R at the site of injury, even at later timepoints. We showed that, after subcutaneous administration, only the group injected with rFIXK5A significantly reduced the blood loss in tail clip bleeding model. The decreased binding of rFIXK5A to EVS and the observed greater plasma exposure after subcutaneous administration potentially contributed to the hemostatic efficacy. These results further support that (1) maintaining a steady trough level of FIX is crucial for efficacy and (2) the modulation of the FIX binding property to extravascular can be an important therapeutic strategy for subcutaneous delivery.