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0 10 20 30 40 50 600
50
100
150
200
rFVIIIa titration inFVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0%5%10%20%40%80%100%
0.5 pM TF
NPP +/- SD
0 10 20 30 40 50 600
50
100
150
200
FVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
Control
FIX-FIAV
rFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A inhibitor patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAVrFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAV
rFVIIIa
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
0
50
100
150
200
Thrombin generation eak heightin Hemophilia A patient plasma
Peak
hei
ght (
nM)
HA1 HA2 HA3 HA4
0.5 pM TF
NPP +/- SD
3%23% 13%3%
FVIIId HA2 HA3 HA40
10
20
30
40
50
Specific clotting activity (aPTT)in Hemophilia A patient plasma
FVIII
act
ivity
(%)
9% 19% 8%32%
A B
C D
E F
B FVIII-immunodepleted plasma
0 10 20 30 40 50 600
50
100
150
200
rFVIIIa titration inFVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0%5%10%20%40%80%100%
0.5 pM TF
NPP +/- SD
0 10 20 30 40 50 600
50
100
150
200
FVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
Control
FIX-FIAV
rFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A inhibitor patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAVrFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAV
rFVIIIarF
VIIIa
FIX-
FIA
VC
ontr
olrF
VIIIa
FIX-
FIA
VC
ontr
olrF
VIIIa
FIX-
FIA
VC
ontr
olrF
VIIIa
FIX-
FIA
VC
ontr
ol
0
50
100
150
200
Thrombin generation eak heightin Hemophilia A patient plasma
Peak
hei
ght (
nM)
HA1 HA2 HA3 HA4
0.5 pM TF
NPP +/- SD
3%23% 13%3%
FVIIId HA2 HA3 HA40
10
20
30
40
50
Specific clotting activity (aPTT)in Hemophilia A patient plasma
FVIII
act
ivity
(%)
9% 19% 8%32%
A B
C D
E F
C Hemophilia A inhibitor patient plasma
0 10 20 30 40 50 600
50
100
150
200
rFVIIIa titration inFVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0%5%10%20%40%80%100%
0.5 pM TF
NPP +/- SD
0 10 20 30 40 50 600
50
100
150
200
FVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
Control
FIX-FIAV
rFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A inhibitor patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAVrFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAV
rFVIIIa
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
0
50
100
150
200
Thrombin generation eak heightin Hemophilia A patient plasma
Peak
hei
ght (
nM)
HA1 HA2 HA3 HA4
0.5 pM TF
NPP +/- SD
3%23% 13%3%
FVIIId HA2 HA3 HA40
10
20
30
40
50
Specific clotting activity (aPTT)in Hemophilia A patient plasma
FVIII
act
ivity
(%)
9% 19% 8%32%
A B
C D
E F
D Hemophilia A patient plasma
FIX-FIAV rVIIa rFVIIa +FIX-FIAV
0
50
100
150
200
1 U/ml FIX-FIAVand 1 U/ml rFVIIa
Thro
mbi
n pe
ak (n
M)
FIX-FIAV 250 nM Ec
500 nMEc
250 nMEc +
FIX-FIAV
500 nMEc +
FIX-FIAV
0
100
200
300
400
500
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
0
50
100
150
200
1 U/ml FIX-FIAVand Feiba
FIX-FIAV rFVIII rFVIII +FIX-FIAV
0
100
200
300
400
500
1 U/ml FIX-FIAVand 1 U/ml rFVIII
Thro
mbi
n pe
ak (n
M)
0 1000 2000 3000 4000 50000.0
0.2
0.4
0.6
FIX (nM)
FIXa
/min
(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
Aim
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
[email protected] [email protected]
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
L 6 F
K 265 A
V 181 II 383 V
L 6 F
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
0
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
50
100
150
200
rFVIIIa titration inFVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0%5%10%20%40%80%100%
0.5 pM TF
NPP +/- SD
0 10 20 30 40 50 600
50
100
150
200
FVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
Control
FIX-FIAV
rFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A inhibitor patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAVrFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAV
rFVIIIarF
VIIIa
FIX-
FIA
VC
ontr
olrF
VIIIa
FIX-
FIA
VC
ontr
olrF
VIIIa
FIX-
FIA
VC
ontr
olrF
VIIIa
FIX-
FIA
VC
ontr
ol
0
50
100
150
200
Thrombin generation eak heightin Hemophilia A patient plasma
Peak
hei
ght (
nM)
HA1 HA2 HA3 HA4
0.5 pM TF
NPP +/- SD
3%23% 13%3%
FVIIId HA2 HA3 HA40
10
20
30
40
50
Specific clotting activity (aPTT)in Hemophilia A patient plasma
FVIII
act
ivity
(%)
9% 19% 8%32%
A B
C D
E F
ArFVIIIa titration in
FVIII-immunodepleted plasma
0 10 20 30 40 50 600
50
100
150
200
rFVIIIa titration inFVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0%5%10%20%40%80%100%
0.5 pM TF
NPP +/- SD
0 10 20 30 40 50 600
50
100
150
200
FVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
Control
FIX-FIAV
rFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A inhibitor patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAVrFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAV
rFVIIIa
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
0
50
100
150
200
Thrombin generation eak heightin Hemophilia A patient plasma
Peak
hei
ght (
nM)
HA1 HA2 HA3 HA4
0.5 pM TF
NPP +/- SD
3%23% 13%3%
FVIIId HA2 HA3 HA40
10
20
30
40
50
Specific clotting activity (aPTT)in Hemophilia A patient plasma
FVIII
act
ivity
(%)
9% 19% 8%32%
A B
C D
E FE Thrombin generation peak heightin Hemophilia A patient plasma
0 10 20 30 40 50 600
50
100
150
200
rFVIIIa titration inFVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0%5%10%20%40%80%100%
0.5 pM TF
NPP +/- SD
0 10 20 30 40 50 600
50
100
150
200
FVIII-immunodepleted plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
Control
FIX-FIAV
rFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A inhibitor patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAVrFVIIIa
0 10 20 30 40 50 600
50
100
150
200
Hemophilia A patient plasma
Time (min)
Thro
mbi
n (n
M)
0.5 pM TF
NPP +/- SD
ControlFIX-FIAV
rFVIIIa
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
rFVI
IIaFI
X-FI
AV
Con
trol
0
50
100
150
200
Thrombin generation eak heightin Hemophilia A patient plasma
Peak
hei
ght (
nM)
HA1 HA2 HA3 HA4
0.5 pM TF
NPP +/- SD
3%23% 13%3%
FVIIId HA2 HA3 HA40
10
20
30
40
50
Specific clotting activity (aPTT)in Hemophilia A patient plasma
FVIII
act
ivity
(%)
9% 19% 8%32%
A B
C D
E FF Specific clotting activity (aPTT)in Hemophilia A patient plasma
Poster No. P037
