Evaluation of a Blood Coagulation Factor IX Variant that ...uniqure.com/EAHAD_2020_FIX-FIAV.pdf ·...

0 10 20 30 40 50 600

rFVIIIa titration inFVIII-immunodepleted plasma

Time (min)

0%5%10%20%40%80%100%

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

FVIII-immunodepleted plasma

Time (min)

0.5 pM TF

NPP +/- SD

Control

FIX-FIAV

rFVIIIa

0 10 20 30 40 50 600

Hemophilia A inhibitor patient plasma

Time (min)

0.5 pM TF

NPP +/- SD

ControlFIX-FIAVrFVIIIa

0 10 20 30 40 50 600

Hemophilia A patient plasma

Time (min)

0.5 pM TF

NPP +/- SD

ControlFIX-FIAV

rFVIIIa

Thrombin generation eak heightin Hemophilia A patient plasma

HA1 HA2 HA3 HA4

0.5 pM TF

NPP +/- SD

3%23% 13%3%

FVIIId HA2 HA3 HA40

Specific clotting activity (aPTT)in Hemophilia A patient plasma

9% 19% 8%32%

B FVIII-immunodepleted plasma

0 10 20 30 40 50 600

Time (min)

0%5%10%20%40%80%100%

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

Control

FIX-FIAV

rFVIIIa

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

ControlFIX-FIAV

rFVIIIarF

HA1 HA2 HA3 HA4

0.5 pM TF

NPP +/- SD

3%23% 13%3%

FVIIId HA2 HA3 HA40

9% 19% 8%32%

C Hemophilia A inhibitor patient plasma

0 10 20 30 40 50 600

Time (min)

0%5%10%20%40%80%100%

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

Control

FIX-FIAV

rFVIIIa

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

ControlFIX-FIAV

rFVIIIa

HA1 HA2 HA3 HA4

0.5 pM TF

NPP +/- SD

3%23% 13%3%

FVIIId HA2 HA3 HA40

9% 19% 8%32%

D Hemophilia A patient plasma

FIX-FIAV rVIIa rFVIIa +FIX-FIAV

1 U/ml FIX-FIAVand 1 U/ml rFVIIa

FIX-FIAV 250 nM Ec

500 nMEc

250 nMEc +

FIX-FIAV

500 nMEc +

FIX-FIAV

1 U/ml FIX-FIAVand Emicizumab

FIX-FIAV 0.5 U/mlFeiba

0.5 U/ml Feiba +

FIX-FIAV

1 U/mlFeiba

1 U/mlFeiba +

FIX-FIAV

1 U/ml FIX-FIAVand Feiba

FIX-FIAV rFVIII rFVIII +FIX-FIAV

1 U/ml FIX-FIAVand 1 U/ml rFVIII

0 1000 2000 3000 4000 50000.0

FIX (nM)

FIX - WTFIX - FIAV

FIX was incubated with TF (500 nM) and FVIIa (100 nM) over time; the FIXa generated was determined employing chromogenic Pefachrome conversion using a PD-FIXa reference curve.

Specific FVIII clotting activity (aPTT) in FVIII-immunodepletedplasma with normal plasma concentrations of FIX-FIAV or FIX-WT(‘Wild-Type’) (5 µg/ml; 100%).

Introduction

Fully characterize and assess the biochemical properties of FIX-FIAV

Evaluation of a Blood Coagulation Factor IX Variant that Functions Independently of Factor VIII as an Alternative Treatment for Hemophilia A

Viola J.F. Strijbis1, Ka Lei Cheung1, Pavlina Konstantinova2, Ying Poi Liu2, Sander J. van Deventer2, Mettine H.A. Bos1

1Division of Thrombosis & Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine Leiden University Medical Center, Leiden, The Netherlands 2uniQure Biopharma B.V., Amsterdam, The Netherlands

Specific FVIII-independent Activity

FIX(a) Purification

FIX-FIAV Efficacy Assessment

Tissue factor (TF)-initiated thrombin generation (TG) was assessedin FVIII-immunodepleted plasma (Panels A,B) and hemophilia Apatient plasma with (80 Bethesda Units) (Panel C) or withoutinhibitors (Panels D,E). Panel A: A rFVIII reference curve (0-100%)was generated for each individual patient plasma to determine the %FVIII-independent activity of FIX-FIAV (5 µg/ml) based on thrombinpeak height. Panels B-D: Plasma was incubated with rFVIII(NovoEight; 1 U/ml) or FIX-FIAV (5 µg/ml), and the thrombin peakwas determined (Panel E). The inserts indicate the FVIII-independentactivity (%) of FIX-FIAV; the grey area represents the thrombin peak± SD in normal pooled plasma (NPP). Panel F: The % FVIII-specificclotting activity (aPTT) of FIX-FIAV (5 µg/ml) was determined usingindividual rFVIII reference curves for each plasma.

Kinetics of TF/FVIIa Activation

V.J.F.Strijbis@lumc.nl M.H.A.Bos@lumc.nl

FIX variant FVIII-independent Activity (%)

FVIII-independent Activity (U/mg)

Wild-Type < 6 < 13

FIAV 32 ± 6 56 ± 4

FIX-WT FIX-FIAV

Kcat (sec-1) 0.011 0.010

Km (nM) 548 631Previously, the molecular constraints of the 99-loop werelifted due to specific modifications in both the 99-loop(K265A), the S1 active site subpocket (V181I, I383V), andthe L6F substitution, thereby generating FIX-FIAV1. As aresult, this variant is capable of functioning independentlyof factor VIII (FVIII). Moreover, FIX-FIAV was demonstratedto ameliorate the hemophilia A phenotype both in vitroand in vivo1.1Quade-Lyssy et al. Next generation FIX muteins with FVIII-independent activityfor alternative treatment of hemophilia A J. Thromb. Haemost. 2014 11:1861.

FIX-FIAV Efficacy Assessment:• Demonstrated FIX-FIAV efficacy in hemophilia A

plasma, also in hemophilia A plus inhibitors:• Up to ~30% increase in FVIII-independent activity

based on both thrombin peak and endogenousthrombin potential in FVIII-immunodepleted plasma.• Up to 18% or 23% FVIII-independent activity in

hemophilia A patient plasma with or without aninhibitor, respectively.

• Up to 32% of FVIII-independent activity for FIX-FIAV atFIX plasma levels (5 µg/ml).• FIX-FIAV has the potential to enhance thrombin

generation in FVIII deficiency.

• During FIX activation, prolonged formation of FIX-FIAVa was observed, with no FIXa-WTa formed.

• This is consistent with delayed cleavage at position180, likely resulting from the V181I substitution inFIX-FIAV.

Factor IX-FIAV:• Shows a preserved mechanism of

activation.• Can sustain therapeutic levels of

coagulation activity in FVIII deficiency.• Has the potential to function as an

alternative treatment for hemophilia A.

• No synergistic effect was observed when combiningFIX-FIAV with physiologically relevant concentrationsof FEIBA or NovoSeven.• Hyperactivity of FIX-FIAV in a cofactor-dependent

system: 9-fold enhanced thrombin peak with rFVIII orEmicizumab.

Tissue factor (TF)-initiated (0.5 pM) thrombin generation was assessed in FVIII-immunodepleted plasma incubated with therapeutic concentrations of rFVIIa(NovoSeven®; 1 U/ml), aPCC (FEIBA; 0.5 or 1 U/ml), rFVIII (NovoEight®; 1 U/ml),or low (250 nM) to mid-range (500 nM) concentrations of the bispecific antibodyEmicizumab (Ec; Hemlibra®). The grey area indicates the thrombin peak ± SD innormal pooled plasma.

FIX Activation on Western blot

K 265 A

V 181 II 383 V

K 265 A

I 383 V

V 181 ICatalytic Residues

29% FVIII Act.

FIX-FIAV + Standard Therapy

18% FVIII Act. 23% FVIII Act.

Conclusions

Future Studies

Kinetics of:• Antithrombin Inhibition

± Heparin• FX Activation ± FVIIIa 1

Substitution Reported effect

L 6 F Improves kcat/Km for FX conversion ± FVIIIa

V 181 I Stable salt bridge with Asp194(364)

K 265 A Opens the S4 subsite

I 383 V Known functional effect in FX/FVII/PC –S1 subsite

FIX Activation

Figure adapted from Gailani D. Activation of factor IX by factor XIa. Trends. Cardiovasc. Med. 2000; 10:198.

FIX variants were incubated (0-60 min) with TF/FVIIa or FXIa. Western Blotanalysis was performed with a monoclonal antibody (FIX-AHIX) targeting the FXIheavy chain.

TF/FVIIa-dependent Activation

FXIa-dependentActivation

FIX-WT

1 10 30 60

FIX-FIAV

55kDa = FIX

38kDa = FIXα

28 kDa = FIXa/FIXaα

1 10 30 600 1 10 30 600 1 10 30 600

FIX-WT FIX-FIAV

• Stable expression in HEK cells• Purified to homogeneity

using:FIX: ion-exchange and

hydrophobic affinitychromatography

FIXa: resin IX-Selectchromatography

• FIXa was generated by incubation of FIX with FXIa (molar ratio 1:100)

WT FIAVPDWT FIAV

- 55 kDa

28 kDa -17 kDa -

FIXaFIX

The serine protease factor IXa (FIXa) serves an importantrole in coagulation by catalyzing the proteolyticactivation of factor X (FX) together with its cofactor VIIIa(FVIIIa). While FIXa displays considerable structuralhomology with other coagulation serine proteases, itsactive site is uniquely controlled by the 99-loop thatblocks access to the active site pocket. Cofactor-mediated interaction of FIXa with its substrate FXinduces a conformational change that allows for activesite engagement and substrate catalysis.

kcat / Km1

(nM-1 min-1)

+ FVIIIa - FVIIIa

WT 0.18 5 x 10-4

FIAV 0.64 2 x 10-3

1 Quade-Lyssy et al. J. Thromb. Haemost. 2014 11:1861.

0 10 20 30 40 50 600

Time (min)

0%5%10%20%40%80%100%

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

Control

FIX-FIAV

rFVIIIa

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

ControlFIX-FIAV

rFVIIIarF

HA1 HA2 HA3 HA4

0.5 pM TF

NPP +/- SD

3%23% 13%3%

FVIIId HA2 HA3 HA40

9% 19% 8%32%

ArFVIIIa titration in

0 10 20 30 40 50 600

Time (min)

0%5%10%20%40%80%100%

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

Control

FIX-FIAV

rFVIIIa

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

ControlFIX-FIAV

rFVIIIa

HA1 HA2 HA3 HA4

0.5 pM TF

NPP +/- SD

3%23% 13%3%

FVIIId HA2 HA3 HA40

9% 19% 8%32%

E FE Thrombin generation peak heightin Hemophilia A patient plasma

0 10 20 30 40 50 600

Time (min)

0%5%10%20%40%80%100%

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

Control

FIX-FIAV

rFVIIIa

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

0 10 20 30 40 50 600

Time (min)

0.5 pM TF

NPP +/- SD

ControlFIX-FIAV

rFVIIIa

HA1 HA2 HA3 HA4

0.5 pM TF

NPP +/- SD

3%23% 13%3%

FVIIId HA2 HA3 HA40

9% 19% 8%32%

E FF Specific clotting activity (aPTT)in Hemophilia A patient plasma

Poster No. P037

Evaluation of a Blood Coagulation Factor IX Variant that ...uniqure.com/EAHAD_2020_FIX-FIAV.pdf ·...

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