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

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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 Poster No. P037 Evaluation of a Blood Coagulation Factor IX Variant that Functions Independently of Factor VIII as an Alternative Treatment for Hemophilia A Introduction FIX-FIAV Efficacy Assessment FIX Activation rFVIIIa titrationrFVIIIa titration in in A AFVIII-immunodepletedrFVIIIaFVIII-immunodepletedrFVIIIarFVIIIa titrationtitration titration plasma inin in plasmaB FVIII-immunodepletedB FVIII-immunodepleted plasma plasma A A FVIII-immunodepleted plasma B B FVIIIFVIII-immunodepleted-immunodepleted plasmaplasma The serine protease factor IXa (FIXa) serves an important A200 AFVIIIFVIII-immunodepletedFVIII-immunodepleted200 -immunodepletedFVIII-immunodepleted plasmaplasma plasma B200 BFVIII-immunodepletedFVIII-immunodepleted200 FVIII-immunodepleted plasmaplasma plasma 0.5 pM TF0.5 pM TF 29% FVIII Act. 0.5 pM TF 0.5 pM TF 200200 200200 200200 200 role in coagulation by catalyzing the proteolytic ) ) 0.5 pM TF ) ) 0.50.5 pMpM 0.5TFTF pM TF 0.50.5 pMpM 0.5 TFTF pM TF 150 150 NPP +/- SD0.5 pM TF 150 150 NPP +/- SD NPP +/- SD NPP +/- SD M M M M ) ) n ) n n n ) ) ) ) ) ( ( ( ( 150 150 NPP +/- SD NPP +/- SD 150 M 150150 M NPP +/- SD 150 NPP +/- SD M 150 NPP +/- SD M NPP +/- SD 150 M NPP +/- SD M NPP +/- SD activation of factor X (FX) together with its cofactor VIIIa M M 100% 100% rFVIIIa n rFVIIIa n n n n n n i i i n n i n ( n ( n ( ( ( ( ( ( b b b 80% b 80% 100 100 100 100 100% rFVIIIa n n 100%100% FIX-FIAVrFVIIIa FIX-FIAV n rFVIIIa n 100% n n i rFVIIIa n i n i m m i m i m i i i 40% 40% b b 80% 100 b (FVIIIa). While FIXa displays considerable structural 100 o o b 80% o b o b 80% 100 100 100 b 100 b 100 80% 100 FIX-FIAV r r r Control r 20% 20% FIX-FIAVFIX-FIAVFIX-FIAVControl m m m m 40% m m h h h m h m 40%40% 40% o o o o o o 10% T T 50 T 50 10% o T 50 o 50 r r Control r r Control 20% r r Control r r 20%20% 20% Control h h h h homology with other coagulation serine proteases, its h h 5% h h 5% 10% T T 50 50 10% T T 50 50 10% T T 50 50 10% T T 50 0% 0% 50 5%5% 5% 0 0 5% 0 0 active site is uniquely controlled by the 99-loop that 0%0% 0% 0 100 2010 3020 4030 50 40 60 50 0% 60 0 10 0 20 10 30 2040 3050 4060 50 60 00 00 00 0 blocks access to the active site pocket. Cofactor- 00 101000 202010Time 30(min)3020Time404030 (min)505040 606050 60 00 10100 Time202010 (min)30302020Time40403030 (min)50504040 60605050 6060 TimeTime (min)(min)Time (min) TimeTime (min)(min)TimeTime (min)(min) mediated interaction of FIXa with its substrate FX C HemophiliaC Hemophilia A inhibitor A inhibitor patient plasmapatient plasmaD HemophiliaD Hemophilia A patient Aplasma patient plasma C Hemophilia A inhibitor patient plasma D Hemophilia A patient plasma CC200HemophiliaHemophiliaHemophiliaC200Hemophilia AAA inhibitorinhibitor A inhibitor patientpatient patient plasmaplasmaplasma plasmaDD200 DHemophiliaHemophilia200 Hemophilia AA patientpatient AA patientpatient plasmaplasma plasmaplasma 0.5 pM TF0.5 pM TF 0.5 pM TF 0.5 pM TF induces a conformational change that allows for active 200200 18%200200 FVIII Act. 200200 200 23% FVIII Act. 0.5 pM TF ) ) ) ) 0.50.5 pMpM 0.5TFTF pM TF 0.50.5 pMpM 0.5 0.5TFTF pMpM TFTF 150 150 NPP +/- SD 150 150 NPP +/- SD NPP +/- SD M M NPP +/- SD M M ) ) n site engagement and substrate catalysis. n ) n ) n ) ) ) ) ( ( ( ( 150 150 NPP +/- SD NPP +/- SD M 150150 M 150 NPP +/- SD 150150 150 NPP +/- SD Figure adapted from Gailani D. Activation of factor IX by factor XIa. Trends. M NPP +/- SD M NPP +/- SD NPP +/- SD M M NPP +/- SD M M n n n n n n rFVIIIa rFVIIIa n i n rFVIIIa i rFVIIIa i i n n n n ( ( ( ( ( ( ( ( b b b b 100 100 100 100 Cardiovasc. Med. 2000; 10:198. n n rFVIIIa n n n n n n FIX-FIAV rFVIIIa i i FIX-FIAV rFVIIIarFVIIIa rFVIIIaFIX-FIAV rFVIIIa i m i m FIX-FIAV m m rFVIIIa i i i rFVIIIa i b b 100 o 100 b b o o o b b b 100100 b 100 100100 100 r r r K 265 A r FIX-FIAV ControlFIX-FIAVControlFIX-FIAV m m ControlFIX-FIAV FIX-FIAVFIX-FIAV m m FIX-FIAVControlFIX-FIAV m m m m h h h h o o o o o o o o T T 50 T 50 T 50 50 r r r r Control r r r r Control ControlControl Control h h ControlControl Control h h h h h h T T 50 50 T T 50 50 T T T K 265 A T 50 50 50 50 0 0 0 0 Kinetics of TF/FVIIa Activation I 383 V 0 100 2010 3020 4030 50 40 60 50 60 0 10 0 20 10 30 2040 3050 4060 50 60 V 181 I 00 00 00 0 I 383 V 00 101000 20201010Time 30(min)3020Time404030 (min)505040 606050 60 00 10100 Time202010 (min)30302020Time40403030 (min)50504040 60605050 6060 TimeTime (min)(min)Time (min) TimeTime (min)(min)TimeTime (min)(min) FIX was incubated with V 181 I ThrombinThrombinThrombin generation generation eakpeak height height eak height Specific SpecificclottingSpecific activity clottingclotting (aPTT) activityactivity (aPTT)(aPTT) FIX - WT E F 0.6 Catalytic E EThrombininThrombinin HemophiliaHemophiliaThrombinThrombinin Hemophilia generationgeneration AA patient generationpatient A eakeak patientplasma plasma heightheight eak plasma heightF inF SpecificSpecificHemophiliaSpecificinin HemophiliaclottingHemophiliaclotting A patient clottingclotting activityactivity AAplasma patientpatientactivityactivity (aPTT)(aPTT) plasmaplasma (aPTT)(aPTT) FIX - FIAV TF (500 nM) and FVIIa EE EEin Hemophiliainin HemophiliaHemophilia A patient A patient plasma plasma FF Fin Hemophiliain Hemophilia A patient AA patientpatient plasma plasmaplasma ) Residues 200 in200 Hemophilia A patient plasma 50 in 50Hemophilia A patient plasma M (100 nM) over time; the n 0.5 pM TF0.5 pM TF ( 200200 200200 5050 50 ) ) 0.4 n ) FIXa generated was ) 0.5 pM 0.5TF pM TF 40 40 i M M 0.5 pM TF % 150 150 NPP +/- SDNPP +/- SD % ) ( n ( n L 6 F ) ) ) ) m ) ) ( ) ( 40 / 40 40 M 40 y determined employing y M M t M t 150 % t % NPP +/- SD % t 150 % 150 NPP +/- SD a NPP +/- SD ( 150 n NPP +/- SD i i ( n ( n h ( n 30 h 30 ( ( ( ( v v X y g g i t i y y y t I t i t i t t t t t t 100 100 i chromogenic h i i 30 i e 0.2 h h 30 e 30 c h 30 c F Substitution Reported effect v v v g v i h g g h a a i g i i i i t i 20 i 20 t 100 t t 100 I 100 I 100 e c k e I e k I e c c c Pefachrome conversion I I h a a a h h L 6 F Improves k /K for FX conversion ± FVIIIa a a h a cat m 20 V 20 20 V 20 I e e I I 50 I k 50 I k I k I k F I F I I P I P I a 10 10 a a 3% 23% 13% 3% a 3% 23% 13% 3% using a PD-FIXa V V e V 181 I Stable salt bridge with Asp194(364) V V e e 50 e 5050 50 0.0 F F F P F P 32%10 9% 19% 8% P 32% 9% 19% 8% P 3%3% 3%3%23%23% 23%13%13% 13%3%3% 3% 1010 10 K 265 A Opens the S4 subsite 0 0 0 0 0 1000 2000 3000 4000 5000 reference curve. l l l l 32% 9% 19% 8% l l l l 32%32% 32%9%9% 9%19%19% 19%8%8% 8% a a a a a a a a V V V V V V V V I I I I o o o o I I I I o o o o FVIIId HA2 HA3 HA4 I I I I FVIIId HA2 HA3 HA4 I I I I r r r r r r r r I A I A I A I A A I A I A I A 00 I 0 t t t t 0 t t t 0 t I I I I 0 I I I I l l l 0 l 0 l l l l l l l l l l l I 383 V Known functional effect in FX/FVII/PC – l a a a a V V V V FIX (nM) V V V V V V V V a a a a n n n n a a a a V V V V n n n n F F F F a a a a I I I I V V V V F F F F o o o o V V V V I I I I o o o L 6 F o I I I I o o o o I I I I FVIIId HA2 HA3 HA4 o o o o I I I I - - - - - - - - FVIIId HA2 HA3 HA4 F F F F I I I I F F F F r r r r FVIIId HA2 HA3 HA4 I I I I FVIIId HA2 HA3 HA4 r r r r o o o o I I I I I A I A I A I A o o o o r r r r I A I A I A I A r r r r I A I A I A I A t t t t r r r r I A I A I A I A r r r r I I I I t t t t I I I I t t t t X X X X I I I I t t t t X X X X I I I S1 subsite I V V V V I I I I C C C C I I I I C C C C V V V V n n n n V V V V F F F F V V V V n n n n F F F F n n n n F F F F n n n n F F F F - - - - F F F F F F F F - - - - F F F F F F F F - - - - o o o o - - - - F F F F o o o o F F F F o o o o r r r r o o o o r r r r r r r r X X X X r r r r X X X X X X X X X X X X I I I I C C C C FIX-WT FIX-FIAV I I I I C C C C I I I I C C C C I I I I C C C C F F F F F F F F F F F F F F F F HA1 HA1HA2 HA2HA3 HA3HA4 HA4 K (sec-1) 0.011 0.010 Previously, the molecular constraints of the 99-loop were HA1 HA2 HA3 HA4 cat Tissue HA1HA1factorHA1HA2HA2(TF)HA2-initiatedHA3HA3 HA3HA4HA4thrombinHA4 generation (TG) was assessed K (nM) 548 631 lifted due to specific modifications in both the 99-loop in FVIII-immunodepleted plasma (Panels A,B) and hemophilia A m (K265A), the S1 active site subpocket (V181I, I383V), and patient plasma with (80 Bethesda Units) (Panel C) or without the L6F substitution, thereby generating FIX-FIAV1.
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