H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
H a e r n a t o l o g i c a HAEMATOL/2019/235549
Version 3
No v e l aptamer t o v on W i l l e b r a n d f a c t o r A 1 domain ( T AGX‑0004) show s t o t a l i n h i b i t i o n o f thrombus f o r mation supe r i o r t o ARC 1 7 7 9 and
compa r a b l e t o c a p l a c i z u mab
K a z u y a S aka i , T a t s u h i k o Some y a , K a o r i Harada, Hideo Y a g i , Taei M a t s u i , and Masanori Matsumoto
D i s c l o s u r e s : M. M. i s a member o f an a d v i s o r y board o f S a n o f i . T . S . and K.H . are
employees o f TAGCyx B i o t e c h n o l o g i e s , I n c . Other a u t h o r s d e c l a r e no competing f i n a n c i a l i n t e r e s t s .
Con t r i b u t i o n s : K . S , T . S , K.H , and T.M performed experiments and wrote t h e paper . H.Y
designed t h e study and analyzed t h e d a t a . M . M designed t h e r e s e a r c h and wrote t h e
p a p e r .
H a e m a t o l o g i c a HAEMATOL/2019/235549 Ve r s i o n 3
H a e m a t o l o g i c a A r t i c l e
Novel aptamer t o von羽1 i l l e b r a n dfactor Al domain (TAGX ・ 0 0 0 4 )
shows t o t a l i n h i b i t i o n of thrombus formation superior to ARCl 779
and comparable t o caplacizumab
Kazuya S a k a i , 1 T a t s u h i k o S o m e y a , 2 K a o r i H a r a d a 2 , H i d e o Y a g i , 1 T a e i M a t s u i , 3 and M a s a n o r i Matsumoto 1
1 D e p a r t m e n t o f Blood T r a n s f u s i o n M e d i c i n e
ラNaraMed i c a l U n i v e r s i t y , K a s h i h a r a , J a p a n ;
. .
TAGCyx B i o t e c h n o l o g i e s I n
ふTokyo , J a p
組3 C l i n i c a l L a b o r a t o r y M e d i c i n e , F 可 i t aH e a l t h U n i v e r s i t y S c h o o l o f M e d i c a l S c i e n c e s , T o y o a k e , J a p a n
Running t i t l e : Novel DNA a p t a m e r t a r g e t i n g VWFAl domain
C o r r e s p o n d e n c e :
M a s a n o r i Matsumoto , MD, P h D
P r o f e s s o r o f D e p a r t m e n t o f Blood T r a n s f u s i o n M e d i c i n e Nara M e d i c a l U n i v e r s i t y
840 S h i j y o ‑ c h o , K a s h i h a r a c i t y , Nara 6 3 4 ‑ 8 5 2 2 , J a p a n
Word c o u n t s : A b s t r a c t s 249 words T e x t body 3402 words Number o f r e f e r e n c e s : 3 0
Number o f
白伊1 ・ e s : 6
Number o f s u p p l e m e n t a r y 五 l e : I
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
ABSTRACT
von W i l l e b r a n d f a c t o r (VWF) i s a b l o o d g l y c o p r o t e i n t h a t p l a y s an i m p o r t a n t r o l e i n p l a t e l e t t h r o m b u s f o r m a t i o n t h r o u g h i n t e r a c t i o n b e t w e e n i t s Al domain and p l a t e l e t g l y c o p r o t e i n l b . ARCl 7 7 9 , an a p t a m e r t o t h e VWF Al d o m a i n , was e v a l u a t e d i n a c l i n i c a l
出a lf o r a c q u i r e d t h r o m b o t i c t h r o r n b o c y t o p e n i c p u r p u r a ( a T T P ) . S u b s e q u e n t l y , c a p l a c i z u m a b , a n anti‑VWF A 1 domain n a n o b o d y , was a p p r o v e d f o r aTTP i n E u r o p e and t h e U n i t e d S t a t e s . We r e c e n t l y d e v e l o p e d a n o v e l DNA a p t a m e r , TAGX‑0004, t o t h e VWF Al d o m a i n ; i t c o n t a i n s a n a r t i f i c i a l b a s e a n d d e m o n s t r a t e s h i g h a
伍n i t yf o r VWF.
To c o m p a r e t h e e f f e c t s o f t h e s e t h r e e a g e n t s on VWF Al, t h e i r a b i l i t y t o i n h i b i t i i s t o c e t i n
幽o r b o t r o c e t i n ‑ i n d u c e d p l a t e l e t a g g r e g a t i o n u n d e r s t a t i c c o n d i t i o n s w
出a n a l y z e d , a n d t h e i n h i b i t i o n of t h r o m b u s f o r m a t i o n u n d e r h i g h s h e a r s
仕e s swas
i n v e s t i g a t e d i n a m i c r o c h i p f l o w chamber s y s t e m . I n b o t h a s s a y s , TAGX ・ 0 0 0 4showed
s t r o n g e r i n h i b i t i o n t h a n ARCl 7 7 9 , a n d had c o m p a r a b l e i n h i b i t o r y e f f e c t s t o
c a p l a c i z u m a b .
百i eb i n d i n g s i t e s o f TAGX
・0004a n d ARCl 7 7 9 were a n a l y z e d wi t h
s u r f a c e p l a s m o n r e s o n a n c e p e r f o n n e d u s i n g a l a n i n e s c a n n i n g m u t a g e n e s i s o f t h e VWF
Al d o m a i n . An e l e c t r o p h o r e t i c m o b i l i t y s h i f t a s s a y showed t h a t R1395 and R1399 i n
t h e Al domain bound t o b o t h a p t a m e r s . R 1 2 8 7 , K l 3 6 2 , a n d Rl392 c o n t r i b u t e d t o
ARCl 7 7 9 b i n d i n g , a n d F1366 was e s s e n t i a l f o r TAGX‑0004 b i n d i n g . Smface p l a s m o n
r e s o n a n c e a n a l y s i s o f t h e b i n d i n g s i t e s o f c a p l a c i z u m a b i d e n t i f i e d f i v e amino a c i d s i n
t h e VWF Al domain ( K l 3 6 2 , Rl392 , R l 3 9 5 , R l 3 9 9 , a n d Kl406 ) . 百 i e s er e s u l t s
s u g g e s t e d
仕i a t TAGX‑0004 p o s s e s s e d b e t t e r p h a r m a c o l o g i c a l p r o p e r t i e s t h a n
c a p l a c i z m n a b i n v i t r o a n d m i g h t be s i m i l a r l y p r o m i s i n g f o r aTTP t r e a t m e n t .
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
I n t r o d u c t i o n
von W i l l e b r a n d f a c t o r (VWF) i s a l a r g e m u l t i m e r i c p l a s m a g l y c o p r o t e i n t h a t p l a y s a n e s s e n t i a l r o l e i n t e t h e r i n g c i r c u l a t i n g p l a t e l e t s t o damaged e n d o t h e l i a l c e l l s . 1 I n t e r a c t i o n b e t w e e n t h e VWF Al domain and t h e p l a t e l e t g l y c o p r o t e i n ( G P ) l b r e c e p t o r l e a d s t o r a p i d d e v e l o p m e n t of VWF‑rich p l a t e l e t t h r o m b i , t h u s p r e v e n t i n g
白r t h e rb l e e d i n g . 2 A l t h o u g h n o r m a l a m o u n t s o f VWF a r e u s u a l l y h e l p f u l i n h e m o s t a s i s r e a c t i o n s , s e v e r a l VWF‑mediated d i s e a s e s , s u c h a s a c u t e c o r o n a r y syndrome
工,4c e r e b r a l i n f a r c t i o n , 5 and t h r o m b o t i c t h r o m b o c y t o p e n i c pu
中u r a ( T T P ) , 6 d e m o n s t r a t e r e m a r k a b l y e l e v a t e d plasma l e v e l s o f VWF. I n h i b i t i n g V
京T F a c t i v i t y by b l o c k i n g i t s Al domain h a s r e c e n t l y been d e m o n s t r a t e d t o be
印 刷r a c t i v et h e r a p e u t i c t a r g e t i n t h e s e a r t e 1 i a l t h r o m b o s e s . 1 ‑ 1 0
C a p l a c i z u m a b ( A b l
戸i x ) , an anti‑VWF Al humanized n a n o b o d y , 1 1 ' 1 2 was a p p r o v e d by t h e E u r o p e a n M e d i c i n e s Agency i n 2018 and by U n i t e d S t a t e s Food a n d Drug Adm i n i s t r a t i o n i n 2019 f o r t r e a t m e n t o f a c q u i r e d TTP ( a T T P ) . The a nti‑VWF Al a p t a m e r ARC 1 7 7 9 ( A r c h e m i x ) was d e v e l o p e d a s a n a n t i t h r o m b o t i c a g e n t f o r u s e i n p a t i e n t s w i t h a T T P , a n d i t showed s u f f i c i e n t i n h i b i t o r y e f f e c t s on VWF a c t i v i t y w i t h o u t any s e v e r e b l e e d i n g e v e n t s i n a r a n d o m i z e d c o n t r o l t r i a l . 1 3 However, ARCl 7 7 9 h a s n o t y e t b e e n a p p r o v e d f o r t h e t r e a t m e n t o f VWF‑mediated t h r o m b o s i s . We r e c e n t l y g e n e r a t e d a n o v e l DNA a p t a m e r , TAGX‑0004
,出a t c o n t a i n s t h e a r t i f i c i a l h y d r o p h o b i c b a s e 7 ‑ ( 2
司t h i e n y l ) i m i d a z o [ 4 , 5
・b ] p y r i d i n e( D s ) ; t h i s a p t a m e r t a r g e t s huma n VWF A l w i t h veηhigh a
自n i t y ( K v = 6 1 . 3 pM) a n d s p e c i
白c i t y . 1 4
I n t h i s s
旬d y ,we compared TAGX
・0 0 0 4 ,ARCl 7 7 9 , and c a p l a c i z u m a b i n t e r m s of
t h e i r i n h i b i t o r y e f f e c t s on VWF a c t i v i t y , by p e r f o n n i n g c o n v e n t i o n a l p l a t e l e t
a g g r e g a t i o n a s s a y s , r i s t o c e t i n ‑and b o t r o c e t i n ‑ i n d u c e d p l a t e l e t a g g r e g a t i o n a s s a y s ( R I P A
and B I P A , r e s p e c t i v e l y ) , and s h e a r ‑ s t r e s s ‑ i n d u c e d a g g r e g a t i o n a s s a y s u s i n g t h e
ト
l a e m a t o l o g i c aHAEMA TOυ2019/235549 V e r s i o n 3
m i c r o c h i p f l o w chamber s y s t e m ( 下 TAS R ) . 1 5To a s s
回st h e a
伍n i t yand b i n d i n g s i t e s of t h e two a p t a m e r s , we p e r f o n n e d b i o p h y s i c a l i n t e r a c t i o n a n a l y s i s a n d a l a n i n e s c a n n i n g mutagen e s i s o f t h e VWF Al d o m a i n . I n add i t i o n , we a n a l y z e d t h e b i n d i n g s i t e s o f c a p l a c i z u m a b u s i n g s u r f a c e p l a s m o n r e s o n a n c e ( S P R ) .
時
1 e t h o d s
D e t a i l e d o f m a t e r i a l s a n d m e t h o d s a r e p r e s e n t e d i n t h e Su p p l e m e n t a l i n f o n n a t i o n .
Sourc e s of n u c l e o s i d e , o l i g o n u c l e o t i d e s , n a n o b o d y ,
,αnd p r o t e i n
The a i i i f i c i a l n u c l e o s i d e t r i p h o s p h a t e ( d D s T P ) w a s s y n t h e s i z e d a s d e s c r i b e d p r e v i o u s l y 1 . ・ 6 1 7 百 i eo l i g o n u c l e o t i d e s TAGX‑0004 and ARC l 7 7 9 ( w i t h o u t p o l y e t h y l e n e g l y c o l [ P E G ] ) w e r e s y n t h e s i z e d by a nd p u r c h a s e d f r o m Ge n eDes i gn , I n c . ( O s a k a , J a p a n ) . The o l i g o n u c l e o t i d e s e q u e n c e ofTAGX‑0004 ( a s R n ‑ D s D s ‑ 5 l m h 2 ) 1 4 and t h a t o f ARCl 7 7 9 1 8 were r e p o r t e d p r e v i o u s l テ Thea n t i ‑ h u m a n VWF nanobody c
叩l a c i
却mab ( a s TAB
・2 3 4 )was p u r c h a s e d
企omC r e a t i v e ‑ B i o l a b s ( N Y , USA ) . The r e c o mbin a n t human VWF Al domain p r o t e i n was p u r c h a s e d f r o m U‑P r o t e i n E x p r e s s B V ( U t r e c h t , N e t h e r l a n d s ) , a n d u s e d f o r t h e m e a s u r e m e n t of b i n d i n g a
伍n i t y t o a n t i
『human V W F Al a g e n t s u s i n g SPR.
A l a n i n e ‑ s c a n n i n g m u t a g e n e s i s
Based on p r e v i o u s r e p o r t s t h a t d e s c r i b ed t h e i n t e r a c t i o n b e t w e e n VWF A 1 a nd
G P i b , 1 9 b o t r o c e t i n , 2 0 o r ARC1172 , 2 1 we d e s i g n e d 1 6 a l a n i n e ‑ s u b s t i t u t e d m u t a n t s o f t h e
human V
玖l F Al domain ( R l 2 8 7 , K1312 , R l 3 3 4 , R13 3 6 , Kl34 8 , K 1 3 62 , Fl366 , Kl371 ,
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
El376
ラR l 3 9 2 ,R l 3 9 5 , R l 3 9 9 , Kl406, Kl423, Rl426 , a n d K l 4 3 0 ) . T h e s e m u t a n t p r o t e i n s were g e n e r a t e d u s i n g a c e l l
−企e ee x p r e s s i o n s y s t e m ( T a i y o Nippon S a n s o Co
中o r a t i o n ,Tokyo , J a p a n ) .
Pl α : t e l e t A g g r e g a t i o n T e s t ( P A T )
PAT was p e r f o t m e d w i t h PRP313M a g g r e g o m e t e r (TAIYO I n s t r u m e n t s INC, O s a k a , J a p a n ) . A g g r e g a t i o n i n d u c i n g s u b s t a n c e s a n d t h e i r f i n a l c o n c e n t r a t i o n w e r e a s f o l l o w s ; r i s t o c e t i n ( 1 . 5 m g / m L ) , b o t r o c e t i n ( 1 . 0
トi g / m L ) , c o l l a g e n ( 4
問/ mL ) , e p i n e p h r i n e ( 1 . 0
×1 0 ‑ 4 M) and a d e n o s i n e d i p h o s p h a t e (ADP) ( 1 . 0
×1 0 ‑ 5 M ) .
T o t a l t h r o m b u s f o r m a t i o n a n a l y s i s s y s t e m ( T ‑ T A S )
The
下TAS R( Z a c r o s , F u j i m o r i Kogyo Co . L t d . , T o k y o , J a p a n ) i s a m i c r o ‑ c h i p f l o w ‑ c h a m b e r d e v i c e u s e d t o v i s u a l l y a n d q u a n t i t a t i v e l y a n a l y z e t h r o m b u s f o n n a t i o n i n whole b l o o d s a m p l e s u n d e r v a r i o u s b l o o d f l o w c o n d i t i o n s . 1 5 Whole b l o o d s a m p l e s w i t h e a c h a n t i
S u b s e q u e n t l y , t h e t h r o m b u s f o r m a t i o n i n t h e c a p i l l a r i e s a n d a n i n c r e a s e i n f l o w p r e s s u r e were o b s e r v e d .
E l e c t r o p h o r e s i s m o b i l i t y s h i f t a s s a y (EMSA)
百 1 eb i n d i n g a b i l i t i e s o f t h e two a p t a m e r s t o t h e human VWF A l doma i n a n d i t s
a l a n i n e ‑ s u b s t i t u t e d m u t a n t s were a n a l y z e d by EMSA. Each a p t a m e r ( f i n a l c o n c e n t r a t i o n
o f 1 0 0 nM) was mi x e d w i t h VWF A 1 ( f i n a l c o n c e n t r a t i o n o f 0 t o 800 nM) i n b i n d i n g
bu
島ra n d i n c u b a t e d a t 3 7 °C f o r 3 0 m i n
ラt h e n s u b j e c t e d t o 8% n a t i v e P A GE i n 0 . 5 x
TBE b u f f e r f o r 50 mi n a t room t e m p e r a
旬r e( 2 0 0 V/ cm) . The aptamer‑VWF Al
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
c om p l e x e s were d e t e c t e d a s a s h i f t band , a n d t h e b and p a t t e r n s were d e t e c t e d by SYER Go l d . The d i s s o c i a t i o n r a t e ( K n ) was c a l c u l a t ed by S c a t c h a r d p l o t a n a l y s i s .
Su r : f i α c e pl α smon
陀s o n a n c e
C o m p e t i t i o n a s s a y s w i t h t h e two a p t a m e r s were p e 1 f o r m e d by SPR a n a l y s i s u s i n g B i a c o r e T200 (GE H e a l t h c a r e UK L t d . , L i
枕l e C h a l f o n t , UK), a s d e s c r i b e d p r e v i o u s l y . 1 4
We a l s o p e r f o r m e d SPR a n a l y s i s t o i n v e s t i g a t e t h e b i n d i n g s i t e o f t h e VWF Al domain t o c a p l a c i z u m a b .
S t r u c t u r e mod e l s of t h e VWF Al d
側 側Thr e e ‑ d i m e n s i o n a l ( 3 D ) s t m c t u r e m o d e l s o f t he VWF A l domain were v i s u a l i z e d w i t h t h e PyMOL Mo l e c u l a r Graph i c s S y s tem (DeLano S c i e n t i f i c , San C a r l o s , USA) .
E t h i c a l s t a t e m e n t
T h i s s t u d y was a p p r o v e d by t h e e t h i c s c 0 1 m n i t t e e o f Nara M e d i c a l U n i v e r s i
句' ・
W r i t t e n i n f o r m e d c o n s e n t w a s o b t a i n e d
企0血t h ei n d i v i d u a l who d o n a t e d p l asma f o r u s e i n t h i s s t u d y .
R e s u l t s
I n h i b i t o r y e f f e c t s on pl α ' . t e l e t aggreg
瓜i o n
The i
出i b i t o r ye
能c t so f TAGX
・0 0 0 4 ,ARCl 779 , a n d c a p l a c i z u m a b l U l d e r s t a t i c
c o n d i t i o n s w e r e a n a l y z e d b y P A T . F i g u r e 1 s h ows r
叩r e s e n t a t i v er e s u l t s o f t h e a n a l y s e s
of t h e t h r e e け pe s o f p l a s m a . A l l t h r e e a g e n t s showed i n h i b i t i o n a c t i v i t i e s i n b o t h RIPA
ト
l a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
and BIPA . H o w e v e r , t h e i n h i b i t o r y e f f e c t s on p l a t e l e t a gg r e g a t i o n d i f f e r e d b e t w e e n t h e t h r e e a g e n t s . I n RIPA , t h e 80% maximal i n h i b i t o r y c o n c e n t r a t i o n ( I C s o ) o f T AG X‑0004 was 50 nM , w h e r e a s t h a t o f ARCl 779 w a s 5 0 0 nM an d t h a t o f c a p l a c i z u m a b was 5 0 nM; t h u s TAGX‑0004 b l o c k e d VWF f u n c t i o n v i a t h e A l domain a p p r o x i m a t e l y 1 0 t
出1 e smore p o t e n t l y t h a n ARCl 779 , and e x h i b i t e d a s
出世a r p o t e n c y
出c a p l a c i z u m a b . I n BIPA , t h e I C s o o f TAGX
田0004was 5 0 nM , whe r e a s t h a t o f ARCl 7 7 9 was 5 0 0 nM and t h a t o f c a p l a c i z u r n a b was 5 0 nM, r e s p e c t i v e l y ; t h u s TAGX ‑ 0004 b l o c k e d VWF f u n c t i o n a p p r o x i m a t e l y 1 0 t i m e s more p o t e n t l y t h a n ARC l 779 s h o w i n g a s i m i l a r p o t e n c y a s c a p l a c i z u m a b . No n e o f t h e t h r e e a g e n t s i n h i b i t e d p l a t e l e t a g g r e g a t i o n i n d u c e d by c o l l a g e n , e p i n e p h r i n
久o r AD P ( d a t a n o t s h o v ; 佃 ).
I n h i b i t o r y e f f e c t s on t hrombus f o r m a t i o n
To a s s e s s t h e i n h i b i t o r y e f f e c t o f t h e t h r e e a g e n t s o n p l a t e l e t t h r o m b u s f o n n a t i o n , we p e r f o m 1 e d
下TAS. I n t h i s s t u d y , compl e t e i n h i b i t i o n was d e f i n e d a s a n i n c r e a s e i n f l o w p r e s s u r e f r o m b a s e l i n e o f no mo r e t h a n 1 0 kPa . TAG X ・ 0004 , ARC1 7 7 9 , a n d c a p l a c i z u m a b w e r e a n a l y z e d t h r e e t i m e s e a c h , a nd F i g u r e 2 show s r e p r e s e n t a t i v e f l ow p r e s s u r e c u r v e s . Both T AGX‑0004 and c a p l a c i z u m a b p r e v e n t e d t h r o m b u s o c c l u s i o n und e r t h e 臼 owc o n d i t i o n a t 5 0
叫v i . I n c o n
仕a s t , ARCl 779 d i d n o t demon s t r a t e c o m p l e t e i n h i b i t i o n , e v e n a t a c o n c e n t r a t i on o f 1 0 0 0 nM . T h e s e r e s u l t s i n d i c a t e d t h a t T AGX ・ 0004 i n h i b i t e d p l a t e l e t t h r ombus f o n n a t i o n a t l e a s t 20 t i m e s more p o t e n t l y t h a n ARC 1 7 7 9 u n d e r h i g h s h e a r s t r e s s , a n d w i t h a s i m i l a r po t e n c y a s c a p l a c i z u m a b .
B i o p h y s i c a l i n t e r αc t i o n α n a l y s i s w i t h EMSA
Bi op h y s i c a l i n t e r a c t i o n a n a l y s i s w i t h EMSA was p e r f o nn e d f o u r t i m e s f o r e a c h
ト
l a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
a p t a m e
工F i g u r e 3 s h o w s t h a t t h e a
節 目t y ofTAGX‑0004 t o VWF Al domain (Kn= 2 . 2
士
0 . 9nM [ n = 4 ] ) was a p p r o x i m a t e l y 1 6
明白I dh i g h e r t h a n t h a t o f ARCl 7 7 9 (Kn = 3 5 . 5
士 山nM [ n =4]) .
A l a n i n e s c a n n i n g mut α gen
四i s w i t h E MSA
F i g u r e 4 shows t h e r e s u l t s o f EMSA u s i n g 1 6 a l a n i n e ‑ m u t a t e d VWF Al w i t h TAGX ‑ 0 0 0 4 ( A ) o r ARC 1 779 ( B ) . F i r s t , we a n a l y z e d n i n e m u t a n t s a s s ho wn i n t h e l e
食p a n e l s . B a s e d on t h e r e s u l t s o f t h i s i n i t i a l a n a l y s i s , we t h e n a n a l y z e d a d d i t i o n a l s e v e n mu t a n t s a s shown i n t h e r i g h t p a n e l s . Of t h e s e , 1 0 VWF A l m u t a n t s (Kl312A , R1334 A , R1336A , K1348A, K1371A, E 1 3 7 6 A , K1406 A , Kl 4 23A , R1 4 26 A , a n d K 1 4 30A) f o r m e d s h i f t e d b a n d s r e p r e s e n t i n g c o m p l e x e s b e t w e e n a n a p t a mer and VWF A l
ヲa s w e l l a s b e t w e e n w i l d 勾 pe (WT) VWF Al a n d e a c h a p t a m e
工h t h e r e m a i n i n g s i x mut a n t s , t h e i n t e n s i t y o f t h e comp l e x e d b a n d s was d e c r e a s e d , a n d t h e i n t e n s i t y o f t h e unbound a p t a r n e r b a n d s was i n c r e a s e d . Of t h e s e mutan t s , R l395A a n d Rl399A showed d e c r e a s e d i n t e n s i t y i n b o t h TAGX ‑ 0004 a ndARCl 779 band c ompl e x e s
Wh i l e t h e b i n d i n g a 百 i n i t y t o ARCl 7 7 9 was s i g n i f i c a n t l y d e c r e a s e d i n R l 2 8 7 A,
K1362 A , a n d Rl392A, t h a t t o TAGX
・0004 was d e c r e a s e d i n Fl366A. T h e s e f i n d i n g s
s u g g e s t e d t h a t R1395 a nd R l 3 9 9 w e r e e s s e n t i a l r e s i d u e s f o r b i n d i n g t o b o t h a p t a r n e r s ,
and t l 1 a t Rl287 , K1362
, 組dRl392 p r o b a b l y c o n t r i b u t e d t o b i n d i n g t o on l y ARC1779 ,
w h i l e Fl3 6 6 was r e q u i r e d f o r b i n d i n g t o TAG X0004 . Our d a t a i n d i c a t e d t h a t b o t h
a p t a m e r s bound t o t h e V WF A l dom a i n , e v e n t h o u g h t h e r e s i d u e s n e c e s s a η お r t h i s
b i n d i n g w e r e pa r t i a l l y d i f f e r e n t , a n d t h e s e d i
宜e r e n c e s mi g h t a f f e c t t h e i r V
京市i n h i b i t o r y
a c t i v i t y .
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
C o m p e t i t i o n α SS αy w i t h B i a c o r e
To c o m p a r e t h e b i n d i n g s i t e s o f t h e a p t a m e r s t o t h e VWF Al d o m a i n , a c o m p e t i t i o n a s s a y was p e r f o n n e d by SPR u s i n g B i a c o r e . B i o t i n y l a t e d TAGX ・ 0004 was i m m o b i l i z e d on t h e s e n s o r c h i p SA. T h e n , VWF Al and a c o m p e t i t o r a p t a m e r (TAGX‑0004 o r ARCl 7 7 9 ) a s a n a n a l y t e was i n j e c t e d i n t o t h e c h i p . The r e s u l t s o f a s e l f ‑ c o m p e t i t i o n a s s a y u s i n g TAGX
・0004showed t h a t t h e amount o f VWF Al b i n d i n g t o t h e c h i p d e c r e a s e d d e p e n d i n g on t h e c o n c e n t r a t i o n o f c o m p e t i t o r TAGX ‑ 0 0 0 4 ( S u p p l e m e n t a r y F i g u r e 1 ) . ARC1779 a l s o d o s e ‑ d e p e n d e n t l y d e c r e a s e d t h e r e s p o n s e u n i t v a l u e .
百i e s e r e s u l t s i n d i c a t e d t h a t TAGX
・0004and ARC 1 7 7 9 bound t o t h e o v e r l a p p e d r e g i o n on t h e VWF Al d o m a i n . M o r e o v e r , t h e comp
巴t i t i v e a b i l i t y of ARCl 779 a g a i n s t i m m o b i l i z e d TAGX‑0004 on t h e c h i p s u r f a c e was l o w e r t h a n t h a t o f TAGX‑0004 a g a i n s t TAGX‑0004. T h e s e r e s u l t s were c o n s i s t e n t w i t h t h o s e o f EMSA and i n d i c a t e d t h a t t h e b i n d i n g a
伍n i t yofTAGX ・ 0004 f o r VWF A 1 was h i g h e r t h a n t h a t o f ARC 1 7 7 9 .
VWF Al domain b i n d i n g s i t e of c a p l a c i z u m αb
We p e r f o r m e d SPR a n a l y s i s t o i n v e s t i g a t e t h e VWF Al domain b i n d i n g s i t e o f
c a p l a c i z u m a b . A f t e r i m m o b i l i z i n g c a p l a c i z u m a b on t h e s e n s o r c h i p CMS, 1 6
a l a n i n e
司s u b s t i t u t e dm u t a n t s o f VWF Al w e r e a n a l y z e d ( F i g u r e 5 ) . The r e l a t i v e b i n d i n g
amount o f W T was d e
宣neda s 100%. The b i n d i n g amount o f e a c h m u t a n t was e x p r e s s e d
a s t h e r e l a t i v e r a t i o compared t o t h a t of WT. 京 司 1 e na r e l a t i v e r a t i o o f 80% was s e t a s t h e
c u t ‑ o f f , f i v e m u t a n t s were j u d g e d a s p o s i t i v e . T h e s e r e s u l t s i n d i c a t e d t h a t t h e s e f i v e
r e s i d u e s (Kl362 , R 1 3 9 2 , R l 3 9 5 , R1399 , a n d K1406) were l i k e l y t o b e t h e b i n d i n g s i t e s
of c a p l a c i z u m a b .
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
C o m p a r i s o n of VWF Al dom α i n b i n d i n g s i t e s of TAGX 0 0 0 4 , ARCJ 7 7 9 , and
C
叩l a c i z u m a b
F i g u r e 6 shows 3D s t r u c
旬r em o d e l s o f t h e VWF Al domain.τb e amino a c i d r e s i d u e s shown i n r e d i n d i c a t e t h e e s s e n t i a l r e s i d u e s f o r b i n d i n g t o e a c h a p t a m e r o r c a p l a c i z u m a b . The r e s i d u e s shown i n y e l l o w were n o t i n v o l v e d i n b i n d i n g . Of t h e 1 6 a l a n i n e
引i b s t i t u t e d VWF Al m u t a n t s , EMSA p r e d i c t e d t h a t t h r e e amino a c i d s ( f 1 3 6 6 , Rl395
ラa n dRl 3 9 9 ) w e r e b i n d i n g s i t e s f o r TAGX‑0004, a s shown i n r e d i n F i g u r e 6 l e f t . The r e m a i n i n g 1 3 amino a c i d r e s i d u e s a r e shown i n y e l l o w . As f o r ARC 1 7 7 9
ラf i v e amino a c i d r e s i d u e s ( R l 2 8 7 , Kl362, R l 3 9 2 , R 1 3 9 5 , a n d R1399) p l a y e d a n i m p o r t a n t r o l e i n b i n d i n g t o VWF Al , a s shown i n r e d i n F i g u r e 6 m i d d l e . Of
仕i e s e , R1395 a n d R1399 w e r e amino a c i d s s h a r e d by b o t h a p t a m e r s . F u r t h e r , u s i n g B i a c o r e we i d e n t i f i e d f i v e amino a c i d r e s i d u e s i n t h e VWF Al domain ( K . 1 3 6 2 , R 1 3 9 2 , R 1 3 9 5 , R 1 3 9 9 , a n d Kl406) t h a t w e r e b i n d i n g s i t e s t o c a p l a c i z u m a b , a s shown i n r e d i n F i g u r e 6 r i g h t . Of t h e s e , Kl406 was u n i q u e t o c a p l a c i z u m a b . 百 i ed i
庄e r e n c e s i n VWF A 1 domain b i n d i n g s i t e s s u g g e s t e d t h a t t h e s e a p t a m e r s o r nanobody m i g h t ha ve u n i q u e e f f e c t s on p l a t e l e t a g g r e g a t i o n and t h r o m b o s i s f o r m a t i o n .
D i s c u s s i o n
VWF p l a y s a p i v o t a l r o l e i n t h e i n i t i a l p h a s e o f p l a t e l e t t h r o m b u s f o r m a t i o n u n d e r h i g h s h e a r s t r e s s t h r o u g h t h e i n t e r a c t i o n b e t w e e n i t s Al domain a n d p l a t e l e t GPib . ' 1 2
T h e b i n d i n g s i t e of t h e V
玖T F A l do m a i n i s u s u a l l y c r y p t i c and p r e v e n t s s p o n t a n e o u s
b i n d i n g t o p l a t e l e t s . f 五 g hs h e a r s
仕e s s o f b l o o d f l o w i n d u c e s c o n f o r m a t i o n a l c h a n g e s i n
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
VWF a n d e x p o s e s t h e VWF Al d o m a i n . T h e r e f o r e , t h e a s s o c i a t i o n b e t w e e n V
玖T F Al a n d p l a t e l e t GPib u s u a l l y o c c u r s u n d e r h i g h s h e a r s t r e s s . 2
h l h i b i t i o n o f VWF Al domain b i n d i n g t o p l a t e l e t GPib c o u l d p r e v e n t t h e d e v e l o p m e n t o f p l a t e l e t t h r o m b u s f o r m a t i o n t h a t c a u s e s c a r d i a c i n f a r c t i o n , c e r e b r a l i n f a r c t i o n
ラandTTP . TIP r e s u l t s
企omt h e f o r m a t i o n o f p l a t e l e t t h r o m b i i n t h e m i c r o v a s c u l a t u r e d u e t o a d e f i c i e n c y o f ADAMTSl 3 , a VWF‑cleaving p r o t e a s e . 2 2 ' 2 3
百i e r e f o r e ,p r e v e n t i n g VWF Al b i n d i n g t o p l a t e l e t GPib c o u l d be a p r o m i s i n g t h e r a p e u t i c t a r g e t f o r T T P . F o r t h i s pu
中 田 久c a p l a c i z u m a bwas e v a l u a t e d f o r i t s a n t i ‑ t h r o m b o t i c e 旺 e c t si n a p h a s e 2 c l i n i c a l t r i a l ( t h e TITAN s ぬ d y ) 1 1a n d a p h a s e 3 c l i n i c a l t r i a l ( t h e HERCULES s t u d y ) . 1 2 T h e s e s t u d i e s r e p m t e d a t h e r a p e u t i c e f f e c t of c a p l a c i z u m a b on aTTP , i n c l u d i n g f a s t e r r e c o v e 1 y of p l a t e l e t c o u n t s
ヲf e w e rp l a s m a e x c h a n g e s e s s i o n s , a n d s h o r t e r h o s p i t a l s t a y s . B l e e d i n g e v e n t was r e p o r t e d a s a c o 1mnon a d v e r s e e v e n t i n p a t i e n t s t r e a t e d w i t h c a p l a c i z u m a b c o m p a r e d w i t h p a t i e n t s w i t h o u t i t . 1 2 I n t h e TITAN and HERCULES s t u d y , 3 6 a n d 7 2 p a t i e n t s o f aTTP were t r e a t e d w i t h c a p l a c i z u r n a b a n d 1 9 ( 5 4 % ) a n d 46 ( 6 5 % ) p a t i e n t s had b l e e d i n g e v e n t s , r e s p e c t i v e l y . The m o s t common a d v e r s e e v e n t s w e r e e p i s t a x i s and g i n g i v a l b l e e d i n g , n e i t h e r o f whic h g e n e r a l l y r e q u i r e d t r e a t m e n t .
Nu c l e i c a c i d a p t a r n e r s a r e s i n g l e ‑ s t r a n d DNA o r RNA m o l e c u l e s t h a t c a n f o r m 3D
s t r u c t u r e s c a p a b l e o f s p e c i f i c a l l y b i n d i n g t o p r o t e i n s o r o t h e r c e l l u l a r t a r g e t s . They a r e
s u p e r i o r t o e x i s t i n g a n t i b o d y p r o d u c t s i n t e r m s o f t h e i r s p e c i f i c i t y
ラmanu 血 c
加出i g c o s t ,
r e l a t i v e l y s m a l l s i z e , a n d n o n ‑ i m r n u n o g e n i c i ち ' ・ P e g a p t a n i b ( M a c u g e n R , P
宣z e r ) i s t h e
o n l y a p t a r n e r a p p r o v e d by FDA i n 2004 f o r t h e t r e a
加1 e n to f wet a g e ‑ r e l a t e d m a c u l a r
d e g e n e r a t i o n b l o c k i n g v a s c u l a r e n d o t h e l i a l g r o w t h f a c t o r . 2 4 As o f J u n e 2019 , t h e r e a r e
no a p t a r n e r f o r t h e p a t i e n t s w i t h c o a g u l a t i o n o r t h r o m b o t i c d i s o r d e r i s a p p r o v e d .
H a e m a t o l o g i c a HAEMATOU2019/235549 V e r s i o n 3
H o w e v e r , some a p t a m e r s t a r g e t i n g c o a g u l a t i o n f a c t o r s , ARC 1 7 7 9 a g a i n s t VWF A l , NUl 7 2 (ARCA B i o p h a r m a ) a g a i n s t f a c t o r I l a , a n d REG 1 ほ EG2( R e g a d o B i o s c i e n c e s ) a g a i n s t f a c t o r
医a ,s t e p p e d i n t o c l i n i c a l t r i a l s . 1 3 ' 2 5 ‑ 2 7 ARC1779 was d e v e l o p e d t o t a r g e t t h e VWF Al domain a nd was e v a l u a t e d i n a p h a s e 2 c l i n i c a l t r i a l i n p a t i e n t s w i t h aTTP . 1 3 U n f o r t u n a t e l y , t h e r e c r u i h n e n t o f p a t i e n t s i n t h i s t r i a l was t e r m i n a t e d w i t h o u t c o m p l e t i n g t h e s t u d y d u e t o s p o n s o r ‑ r e l a t e d f i n a n c i a l i s s u
巴s , b u t s e v e n p a t i e n t s w i t h a TTP r e c e i v e d c o m b i n e d t h e r a p y w i t h i n t r a v e n o u s ARCl 7 7 9 i n j e c t i o n s a n d p l a s m a e x c h a n g e . 1 3
百i et r i a l showed no s e v e r e a d v e r s e e v e n t s s u c h a s b l e e d i n g , e v e n i n p a t i e n t s w i t h a TIP who h a d s e v e r e t h r o m b o c y t o p e n i a . 1 3 A s t u d y i n h e a l t h y v o l u n t e e r s c o n f i r m e d t h a t t h i s a p t a m e r had an a n t i t h r o m b o t i c e
民c ta n d d i d no t c a u s e s e v e r e b l e e d i n g e v e n t s . 2 8
TAGX‑0004 i s a n o v e l DNA a p t a m e r t h a t t a r g e t s t h e V WF Al domain and c o n t a i n s D s , a n a r t i f i c i a l n u c l e i c a c i d . TAGX ‑ 0 004 was o b t a i n e d u s i n g m o d i f i e d SELEX ( s y s t e m i c e v o l u t i o n o f l i g a n d s by e x p o n e n t i a l e n r i c h m e n t ) m e t h o d s i n c o r p o r a t i n g t h e Ds b a s e . 1 4 S i n c e t h e Ds b a s e h a s no c o m p l e m e n t a r y ba s e i n n a t u r e , Dsc o n t a i n i n g DNA a p t a m e r s c a n h a v e un
ムu e3 D s t r u c
印r
巴s .
百1 eh i g h h y d r o p h o b i c i t y o f t h e D s b a s e may c o n t r i b u t e t o s i g n i f i c a n t l y h i g h b i n d i n g a
西国t yt o t a r g e t p r o t e i n s
圃h f a c t , t h e r e s u l t s of EMSA s h o w e d t h a t t h e TAGX‑0004 b i n d i n g a f f m i t y f o r VWF (Kn= 2 . 2
土0 . 9 nM) was 1 6
・f o l dh i g h e r t h a n t h a t o f ARC 1 779 (Kn= 3 5 . 5
土1 . 5 nM) ( F i g u r e 3 ) . Howe v e r , t h e Kn v a l u e s i n t h i s s t u d y m i g h t be unde r e s t i m a t e d i n c o m p a r i s o n w i t h p r e v i o u s r e p o r t s ; t h e Ko v a l u e o f TAGX‑0004 was shown t o b e 6 1 . 3 pM b y a SPR a s s a y , 1 4 a n d t h e Kn v a l u e of ARCl 779 was 2 nM i n a R I ‑ l a b e l e d a s s a y . 1 8 P r e v i o u s r e s u l t s a l s o i n d i c a t e d t h a t t h e b i n d i n g a
缶n i t yofTAGX ‑ 0 004 w a s much h i g h e r t h a n t h a t ofARCl 7 7 9 .
The b i n d i n g a
伍n i t yo f a p t a m e r s t o V
玖7 F Al domain m i g h t a f f e c t t h e i n t e r a c t i o n
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
b e t w e e n VWF and p l a t e l e t s . A l t h o u g h b o t h TAGX‑0004 a n d ARC1779 i n h i b i t e d t h e p l a t e l e t a g g r e g a t i o n s w h i c h w e r e i n d u c e d by r i s t o c e t i n a n d b o t r o c e t i n , t h e minimum c o n c e n h ・ a t i o n o f TAGX
・0004 n e c e s s a r y f o r i n h i b i t i o n was s i g n i 五 c a n t l y l o w e r t h a n t h a t o f ARCl 7 7 9 i n b o t h RIPA a n d BIPA ( F i g u r e 1 ) . PAT a n a l y s i s r e v e a l e d t h a t TAGX‑0004 c o u l d b l o c k VWF f u n c t i o n v i a t h e Al domain a t l e a s t 1 0 t i m e s more s
むo n g l y t h a n ARC1779.
下TAS a l s o r e v e a l e d t h a t TAGX‑0004 was s u p e r i o r t o ARCl 7 7 9 a t i n h i b i t i n g VWF f u n c t i o n ( F i g u r e 2 ) . I n a d d i t i o n , c o m p a r
吋w i t h c a p l a c i z u m a b , TAGX‑0004 showed e q u a l l y e
旺e c t i vei n h i b i t i o n a g a i n s t t h r o m b o s i s f o ロ n a t i o n u n d e r v a r i o u s b l o o d f l o w c o n d i t i o n s . M o r e o v e r , TAGX
・0004 h a s a u n i q u e m i n i ‑ h a i r p i n DNA s t r n c
加r e t h a t o f f e r s b e n e f i t s i n p h a r m a c e u t i c a l a p p l i c a t i o n s , 1 4 s p e c i f i c a l l y by c o n f e r r i n g r e s i s t a n c e t o d e g r a d a t i o n by n u c l e a s e s . T h i s s h u c t u r e s h o u l d e x t e n d t h e h a l f こ l i f e o f t h i s m o l e c u l e i n ν i νo .
As d e s c r i b e d a b o v e r e g a r d i n g t h e a d v e r s e e f f e c t s o f c a p l a c i z u m a b t r e a t m e n t , t h e r e
a r e s t i l l c o n c e r n s r e g a r d i n g b l e e d i n g c a u s e d by anti‑VWF a n t a g o n i s t s . A l t h o u g h
a
凶i‑VWF a g e n t s h a v e d e m o n s t r a t e d s u p e r i o r s a f e 匂 rp r o f i l e s i n
血i sr e g a r d c o m p a r e d
w i t h a n t i ‑ p l a t e l e t a g e n t s , we s h o u l d p r e p a r e m e a s u r e s t o q u i c k l y t r e a t b l e e d i n g t h a t
o c c u r s d u r i n g a p t a m e r t r e a t m e n t . We s p e c u l a t e t h a t i f a n e u t r a l i z i n g a g e n t c o m p r i s i n g a
DNA s e q u e n c e t h a t i s p a r t i a l l y c o m p l e m e n t a r y t o t h a t o f a s p e c i 五 c a p
匂meri s p r e p a r e d ,
i t may s e r v e a s an e f f e c t i v e a n t i d o t e a s i n t r o d u c e d i n t h e l i t e r a t u r e . 2 9 I n
也e c a s e o f
TAGX
・0 0 0 4 ,s u c h p a r t i a l DNA s e q u e n c e may c o n t a i n p
戸r o l e ‑ 2 ‑ c a r b a l d e h y d e ( P a ) , t h e
c o m p l e m e n t a r y b a s e p a i r t o D s . 1 6 I n t h e p a s t d e v e l o p m e n t o f a n t i t h r o m b o t i c a p t ame r
t h e r a p e u t i c s , s i m i l a r a p p r o a c h e s o f a n t i d o t e w e r e t a k e n t o n e u t r a l i z e t h e u n w a n t e d e f f e c t
of a p t a m e r , t h o u g h s u c h a p t a m e r s a
回y e tt o be no t a p p r o v e d . 2 6 The s p e c i f i c a n t i d o t e
c o u l d c o n t r i b u t e t o c o n t r o l s e v e r e b l e e d i n g compared w i t h c a p l a c i zumab .
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
A l a n i n e m u t a g e n e s i s a n a l y s i s of t h e VWF Al domain was p e r f o r m e d i n t h i s s t u d y t o d e t e r m i n e t h e b i n d i n g s i t e s of TAGX
・0004 , ARCl 7 7 9 , a n d c a p l a c i z u m a b . The b i n d i n g s i t e s o f ARCl 7 7 9 , b u t n o t TAGX‑0004 o r c a p l a c i z u m a b , were r e p o r t e d p r e v i o u s l y . 2 1 Our r e s u l t s showed t h a t t h e b i n d i n g s i t e s of t h e s e t h r e e a g e n t s o n l y p a r t i a l l y c o r r e s p o n d e d e a c h o t h e r ( F i g u r e 6 ) . Huang e t a i 2 1 r e p o r t e d t h a t 1 8 amino a c i d r e s i d u e s i n t h e VWF Al domain were b i n d i n g s i t e s f o r ARCl 1 7 2 , which h a s t h e same f u n d a m e n t a l s t r n c
旬r ea s ARCl 7 7 9 . T h a t s t u d y d i d n o t i d e n t i f y Rl399 a s a b i n d i n g s i t e , even t h o u g h i n t h e p r e s e n t s t u d y i t was a common b i n d i n g s i t e f o r a l l t h r e e a g e n t s . Chen e t a l i d e n t i f i e d Rl399 a s o n e of t h e key amino a c i d s f o r t h e e f f e c t ofVWF t o h e m o s t a s i s and t h r o m b o s i s . 3 0 I n t e r e s t i n g l y , a h y d r o p h o b i c amino a c i d r e s i d u e ( F l 3 6 6 ) i s r e q u i r e d t o b i n d t o TAGX‑0004 , wh i c h h a s two h y d r o p h o b i c Ds b a s e s . Ma t s u n a g a e t a l p r e v i o u s l y r e p o r t e d t h a t t h e number of Ds b a s e ( 0
司2 )s t r o n g l y c o r r e l a t e d w i t h b i n d i n g a
伍n i t yt o VWF Al 1 4 and we c o n f i r m e d t h a t Ds
−合e eTAGX‑0004 cou l d n o t i n h i b i t t h r o m b u s f o 1 m a t i o n on
下TAS( d a t a n o t s h o w n ) . T h e s e r e s u l t s i n d i c a t e d t h a t t h e Ds b a s e i n t e r a c t s d i r e c t l y t o t h e VWF Al domain and t h e r e f o r e Fl366 a p p e a r s t o be an e s s e n t i a l and u n i q u e b i n d i n g s i t e o f TAGX ・ 0004 t o t h e VWF‑Al domain w i t h h y d r o p h o b i c i n t e r a c t i o n ( e . g . p i ‑ s t a c k i n g ) . Of n o t e , a l a n i n e s c a m 1 i n g o f c a p l a c i z u m a b showed t h a t l i k e ARCl 779 and TAGX‑0004 , b o t h Rl395 and Rl399 were n e c e s s a r y t o b i n d t h e VWF Al domain ( F i g u r e 6 ) .
Due t o t h e method l i m i t a t i o n s , t h e a f f i m 匂 f between t h e VWF A l domain and
TAGX‑0004 o r ARCl 7 7 9 was n o t a n a l y z e d by t h e SPR m e t h o d . S i n c e b o t h
o l i g o n u c l e o t i d e s and t h e s e n s o r c h i p a r e n e g a t i ve l y c h a r g e d , t h e KD va l u e was
u n d e r e s t i m a t e d when we i m m o b i l i z e d t h e VWF Al p r o t e i n on t h e s e n s o r c h i p and
a p p l i e d TAGX ‑ 0 004 o r ARCl 779 a s a n a n a l y t e . Th e r e f o r e , we p e r f o n n e d a l a n i n e
H a e m a t o l o g i c a HAEMATOυ2019/235549 V e r s i o n 3
s c a n n i n g by EMSA f o r DNA a p t a m e r s . I n a d d i t i o n , t h e b i n d i n g s i t e o f c a p l a c i z u m a b t o VWF Al domain was n o t a n a l y z e d w i t h EM S A , which w
出u s e dt o a s s e s s TAGX‑0004 a n d ARCl 7 7 9 , s i n c e t h i s t e c h n i q u e i s n o t s u i t a b l e f o r t h e a n a l y s i s o f p r o t e i n s s u c h a s n a n o b o d i e s a n d a n t i b o d i e s . S e c o n d l y , ARC1779 was o r i g i n a l l y P E G y l a t e d . H o w e v e r , we u s e d ARCl 779 wi t h o u t PEG i n t h i s s t u d y b e c a u s e we f o u n d no o b v i o u s d i f f e r
四i c e s b e t w e e n ARCl 7 7 9 w i t h a n d w i t h o u t PEG u s i n g RIPA , B I P A , T ‑ T A S , a n d EMSA ( d a
ぬn o t s h o w n ) .
I n a d d i t i o n , c a p l a c i z umab was u s e d i n c l i n i c a l s i u d y a n d a p p r o v e d a s b i v a l e n t nanobody which h a s
同r o b i n d i n g s i t e s t o t h e t a r g e t . On t h e o t h e r hand ,
同r o a p t a m e r s u s e d i n t h i s s t u d y were f o 1 m e d a s mono v a l e n t . I t i s known t h a t d i r e c t c o m p a r i s o n o f a n a
血n i t yo f m o n o v a l e n t e n t i 1 y w i t h a n a v i d i t y o f b i v a l e n t e n t i t y i s n o t s t r a i g h t f o r w a r d . N e v e r t h e l e s s , i t i s o f i n t r i g u i n g t h a t TAGX‑0004 h a s shown c o m p a r a b l e i n h i b i t o r y e
百e c t a g a i n s t c a p l a c i z u m a b i n t h e s t u d i e s we p e r f o r m e d , and how b i v a l e n t form of TAGX‑0004, which i s n o t a v a i l a b l e t h o u g h , b e h a v e s i n t h e same e x p e r i m e n t s
T r e a t m e n t w i t h c a p l a c i z u m a b h a s d e m o n s t r a t e d a r a p i d d e c r e a s e o f VWF r i s t o c e t i n c o f a c t o r a s s a y i n p a t i e n t s w i t h a TTP a n d t h e l e v e l s o f V W F a n t i g e n a n d f a c t o r V I I I wer e a l s o
回n s i e n t l y r e d u c e d w i t h c a p l a c i z u m a b t r e a t m e n t compared w i t h p l a c e b o d u e t o an i n c r e a s e d c l e a r a n c e o f t h e c a p l a c i z u m a b ‑ VVIF c o m p l e x . J I Whether o r n o t TAGX
・0004s h o u l d d e m o n s t r a t e s e q u i v a l e n t e f f e c t i s y e t t o s t u d y u s i n g i n v i v o m o d e l . N e v e r t h e l e s s , j u d g i n g 企 om t h e b i n d i n g a b i l i t y ofTAGX‑0004 t o plasma
幽d e r i v e dhuman VWF c o n f i r m e d w i t h EMSA ( d a t a n o t s h o w n ) , i t i s l i k e l y t h a t t h e
叩t a mershows s i m i l a r e 旺 e c ta s c a p l a c i z u m a b i n aTTP p a t i e n t .
I n c o n c l u s i o n , we confamed t h e p o t e n c y o f TA GX ‑ 0 0 0 4
白r p r e v e n t
也gp l a t e l e t
t h r o m l
ヲu sf o r m a t i o n i n v i t r o . E p i t o p e mapping o f t h e b i n d i n g s i t e s o f TAGX‑0004
ト
l a e m a t o l o g i c aHAEMATOL/2019/235549 V e r s i o n 3
compared w i t h ARCl 7 7 9 a n d c a p l a c i z u m a b p r o v i d e d u s w i t h i n f o r m a t i o n on e a c h m o l e c u l es e
白c a c yand c h a r a c t e r i s t i c s . C a p l a c i z u m a b i s now coming i n t o u s e a s a
r r s t ‑ l i n e t h e r a p y f o r a T T P . H o w e v e r , t h e r e a r e s t i l l c h a l l e n g e s t o b e o v e r c o m e w i t h t h i s a g e n t , s u c h a s b l e e d i n g a d v e r s e e v e n t s a n d h i g h c o s t . 3 1 TAGX‑0004 h a s t h e p o t e n t i a l t o o ve rc om e t h e s e p r o b l e m s a n d c o u l d be d e v e l o p e d a s a p r o m i s i n g d r u g n o t o n l y f o r a T T P , b u t a l s o f o r v a r i o u s VWF‑mediated t h r o m b o t i c d i s o r d e r s s u c h a s a c u t e c o r o n a
可S
戸i d r o m eand c e r e b r a l i n f a r c t i o n .
Funding
T h i s work was s u p p o r t e d by r e s e a r c h g r a n t s
企omt h e M i n i s t r y o f Hea l t h , L a b o u r , and
W e l f a r e o f J a p a n .
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
R e f e r e n c e
I . S a d l e r J E . B i o c h e m i s t r y and g e n e t i c s o f von Wi l l e b r a n d f a c t o r . Annu Rev B i o c h e m . 1 9 9 8 ; 6 7 : 3 9 5 ‑ 4 2 4 .
2 . R u g g e r i ZM. Von W i l l e b r a n d f a c t o r , p l a t e l e t s and e n d o t h e l i a l c e l l i n t e r a c t i o n s . J Thromb H a e m o s t . 2 0 0 3 ; 1 ( 7 ) : 1 3 3 5
・1 3 4 2 .
3 . M o n t a l e s c o t G , P h i l i p p e F , A n k r i A, e t a l . E a r l y i n c r e a s e of von Wi l l e b r a n d f a c t o r p r e d i c t s a d v e r s e outcome i n u n s t a b l e c o r o n a i y a r t e r y d i s e a s e : ben e f i c i a l e
百e c t sof e n o x a p a r i n . F r e n c h I n v e s t i g a t o r s o f t h e ESSENCE T r i a l . C i r c u l a t i o n . 1 9 9 8 ; 9 8 ( 4 ) : 294 ・ 2 9 9 .
4 Ray KK, Morrow DA, Gibson C M
ラMurphyS , An
加an E M , Br
加nwaldE . P r
吋i c t o r s o f t h e r i s e i n vWF a f t e r ST e l e v a t i o n m y o c a r d i a l i n f a r c t i o n : i m p l i c a t i o n s f o r t r e a t m e n t s t r a t e g i e s and c l i n i c a l outcome : An ENTIRE‑TIM I 23 s u b s t u d y . Eur H e a r t J . 200 5 ; 2 6 ( 5 ) : 4 4 0 ‑ 4 4 6 .
5 . Bath PM, B l a n n A, Sm i t h N, B u t t e r w o r t h R J . Von W i l l e b r a n d f a c t o r
,巳s e l e c t i n and f i b r i n o g e n l e v e l s i n p a t i e n t s w i t h a c u t e i s c h a e m i c and h a e m o r r h a g i c s t r o k e , and t h e i r r e l a t i o n s h i p w i t h s t r o k e s u b ‑ t y p e and f u n c t i o n a l o u t c o m e . P l a t e l e t s . 1 9 9 8 ; 9 ( 3 ‑ 4 ) : 1 5 5 ‑ 1 5 9 .
6 . Moake J L , Rudy CK, T r o l l J H , e t a l . U n u s u a l l y l a r g e plasma f a c t o r V I I I : v o n W i l l e b r a n d f a c t o r m u l t i m e r s i n c h r o n i c r e l a p s i n g t h r o m b o t i c t h r o m b o c y t o p e n i c pu
中u r a . N E n g l J Med. 1 9 8 2 ; 3 0 7 ( 2 3 ) : 1 4 3 2 ‑ 1 4 3 5 .
7 . Gragnano F , S p e r l o n g a n o S , G o l i a E , e t a l . The R o l e o f von W i l l e b r a n d F a c t o r i n V a s c u l a r I n f l a m m a t i o n : From P a t h o g e n e s i s t o T a r g e t e d T h e r a p y . M e d i a t o r s I n f l a m m . 2 0 1 7 ; 2 0 1 7 : 5 6 2 0 3 1 4 .
8 . S i l l e r ‑ M a t u l a JM, Kmmphuber J , J i l r n a B . Pham1
〜a c o k i n e t i c , ph a
ロnacod
戸i a m i c and c l i n i c a l p r o f i l e o f no v e l a n t i p l a t e l e t d r u g s t a r g e t i n g v a s c u l a r d i s e a s e s . B r J P h a r m a c o l . 2 0 1 0 ; 1 5 9 ( 3 ) : 5 0 2 ‑ 5 1 7 .
9 . Bae ON. T a r g e t i n g von W i l l e b r a n d f a c t o r a s a n o v e l a n t i ‑ p l a t e l e t t h e r a p y ; a p p l i c a t i o n o f ARCl 779 , an A n t i ‑ v
玖T F a p t a m e r , a g a i n s t t h r o m b o t i c r i s k . Arch Phann R e s . 2 0 1 2 ; 3 5 ( 1 0 ) : 1 6 9 3
『1 6 9 9 .
1 0 . Mat s u i T , H o r i A
うHamako J , e t a l . Mutant b o t r o c e t i n ‑ 2 i n h i b i t s v on Wi l l e b r a n d f a c t o r ‑ i n d u c e d p l a t e l e t a g g l u t i n a t i o n . J 百 romb H a e m o s t . 2 0 1 7 ; 1 5 ( 3 ) : 5 3 8 ‑ 5 4 8 .
1 1 . P e y v a n d i F , S c u l l y M, Kremer Ho v i n g a J A , e t a l . C a p l a c i z u m a b f o r Acqu i r e d T h r o m b o t i c 百 r r o m b o c y t o p e n i c Pu
甲 山a . N E n g l J Med. 2 0 1 6 ; 3 7 4 ( 6 ) : 5 1 1 ‑ 5 2 2 .
1 2 . S c u l l y M, C a t a l a n d SR , P e y v a n d i F , e t a l . C a p l a c i z u m a b T r e a t m e n t f o r A c q u i r e d Thrombot i c
百r r o m b o c y t o p e n i cP u r p u r a . N E n g l J Med. 2 0 1 9 ; 38 0 ( 4 ) : 335 ‑ 346 .
1 3 . C a t a l a n d S R , Peyv and i F , Mannucci PM , e t a l . I n i t i a l e x p e r i e n c e from a
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
d o u b l e ‑ b l i n d , p l a c e b o ‑ c o n t r o l l e d , c l i n i c a l outcome s t u d y of ARCl 779 i n p a t i e n t s w i t h t h r o m b o t i c t h r o m b o c y t o p e n i c pu
中u r a . Am J H e m a t o l . 2 0 1 2 ; 8 7 ( 4 ) : 4 3 0
・432 .
1 4 . Matsunaga K I , Kimoto M, H i r a o I . H i g h ‑ A f f i n i t y DNA Aptamer G e n e r a t i o n T a r g e t i n g von W i l l e b r a n d F a c t o r Al‑Domain by G e n e t i c A l p h a b e t E x p a n s i o n f o r S y s t e m a t i c Evo l u t i o n o f L i g a n d s by E x p o n e n t i a l Emichment Using Two Types of L i b r a r i e s Composed of F i v e Di
妊e r e n tB a s e s . J A m Chem S o c . 2 0 1 7 ; 1 3 9 ( 1 ) : 3 2 4 ‑ 3 3 4 .
1 5 . Hosokawa K, 0
加i i s h iT , Kondo T , e t a l . A n o v e l a u t o m a t e d m i c r o c h i p flow‑chambe r s y s t e m t o q u a n t i t a t i v e l y e v a l u a t e thrombu s f o r m a t i o n and a n t i t h r o m b o t i c a g e n t s u n d e r blood f l o w c o n d i t i o n s . J Thromb Haemost. 2 0 1 1 ; 9 ( 1 0 ) : 2 0 2 9
・2 0 3 7 .
1 6 . H i r a o I , Kimoto M, M i t s u i T , e t a l . An u n n a t u r a l h y d r o p h o b i c b a s e p a i r s y s t e m : s i t e ‑ s p e c i f i c i n c o r p o r a t i o n of n u c l e o t i d e a n a l o g s i n t o DNA and RNA. Nat M e t h o d s . 2006 ; 3 ( 9 ) : 7 2 9 ‑ 7 3 5 .
1 7 . Yamashige R , Kimoto M, Takezawa Y , e t a l . H i g h l y s p e c i f i c u n n a t u r a l b a s e p a i r s y s t e m s a s a t h i r d b a s e p a i r f o r PCR a m p l i f i c a t i o n . N u c l e i c A c i d s R e s . 2012 ; 4 0 ( 6 ) : 2 7 9 3 ‑ 2 8 0 6 .
1 8 . D i e n e r J L , D a n i e l L a g a s s e HA, Duerschmied D, e t a l . I n h i b i t i o n of von Wi l l e b r a n d f a c t o r ‑ m e d i a t e d p l a t e l e t a c t i v a t i o n and t h r o m b o s i s by t h e a n t i ‑ von Wi l l e b r a n d f a c t o r Al‑domain a p t a m e r ARC1779. J Thromb Haemost.
2 0 0 9 ; 7 ( 7 ) : 1 1 5 5
調1 1 6 2 .
1 9 . Huiz i n g a EG, T s u j i S , Romijn RA, e t a l . S t r u c t u r e s of g l y c o p r o t e i n I b a l p h a and i t s complex w i t h von W i l l e b r a n d f a c t o r Al d o m a i n . S c i e n c e . 2 0 0 2 ; 2 9 7 ( 5 5 8 4 ) : 1 1 7 6 ‑ 1 1 7 9 .
2 0 . M a t s u s h i t a T , Meyer D , S a d l e r J E . L o c a l i z a t i o n of von w i l l e b r a n d f a c t o r ‑ b i n d i n g s i t e s f o r p l a t e l e t g l y c o p r o t e i n l b and bo
仕o c e t i nby c h a r g e d ‑ t o ‑ a l a n i n e s c a n n i n g m u t a g e n e s i s . J B i o l Chem. 2 0 0 0 ; 2 7 5 ( 1 5 ) : 1 1 0 4 4 ‑ 1 1 0 4 9 .
2 1 . Huang RH , Fremont DH, D i e n e r J L , Schaub RG, S a d l e r J E . A s t r u c t u r a l e x p l a n a t i o n f o r t h e a n t i t h r o m b o t i c a c t i v i t y of ARCl 1 7 2 , a DNA a p t a m e r t h a t b i n d s von W i l l e b r a n d f a c t o r domain Al. S
佐UC
加r
巴.2 0 0 9 ; 1 7 ( 1 1 ) : 1 4 7 61 4 8 4 .
22 . F u r l a n M, R o b l e s R , G a l b u s e r a M, e t a l . von W i l l e b r a n d f a c t o r ‑ c l e a v i n g p r o t e a s e i n t h r o m b o t i c t h r o m b o c y t o p e n i c pu
中u r a and t h e h e m o l y t i c ‑ uremic s
戸d r o m e . N Eng l J Med. 1998 ; 3 3 9 ( 2 2 ) : 1 5 7 8 ‑ 1 5 8 4 .
2 3 . T s a i HM, L i a n EC. A n t i b o d i
巴s t o von W i l l e b r a n d f a c t o r ‑ c l e a v i n g p r o t e a s e i n a c u t e t h r o m b o t i c t h r o m b o c y t o p e n i c pu
中u r a .N Engl J Med. 1 9 9 8 ; 3 3 9 ( 2 2 ) : 1 5 8 5
・1 5 9 4 . 2 4 . Gragoudas E S , Adamis A P , Cunningham ET, J r . , F e i n s o d M, Guyer DR, Gro up VISiONCT. P e g a p t a n i b f o r n e o v a s c u l a r a g e ‑ r e l a t e d m a c u l a r d e g e n e r a t i o n . N E n g l J Med. 2 0 0 4 ; 3 5 1 ( 2 7 ) : 2805
帽2 8 1 6 .
25 . Gomez‑Outes A , S u a r e z ‑G e a ML, Le cum b e r r i R , Rocha E , Pozo‑Hernandez C ,
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
V a r g a s ‑ C a s t 1 i l l o n E . New p a r e n t e r a l a n t i c o a g u l a n t s i n d e v e l o p m e n t . τ ' h e r Adv C a r d i o v a s c D i s . 2 0 1 1 ; 5 ( 1 ) : 3 3 ‑ 5 9 .
2 6 . V a v a l l e J P , Cohen M G . The REG l a n t i c o a g u l a t i o n s y s t e m : a n o v e l a c t i v e l y c o n t r o l l e d f a c t o r IX i n h i b i t o r u s i n g RNA a p t a m e r t e c h n o l o g y f o r t r e a t m e n t o f a c u t e c o r o n a r γ s y n d r o m e . F u t u r e C a r d i o l . 2 0 1 2 ; 8 ( 3 ) : 37 1 ‑ 3 8 2 .
2 7 . Ponce A T , Hong KL. A M i n i ‑ R e v i e w : C l i n i c a l D e v e l o p m e n t and P o t e n t i a l of A p t a m e r s f o r T h r o m b o t i c E v e n t s T r e a t m e n t a n d M o n i t o r i n g . B i o m e d i c i n e s . 2 0 1 9 ; 7 ( 3 ) . 2 8 . G i l b e r t J C , Defeo
・F r a u l i n iT , H u t a b a r a t RM, e t a l . F i r s t ‑ i n ‑ h u m a n e v a l u a t i o n o f a n t i von W i l l e b r a n d f a c t o r
血e r a p e u t i ca p t a m e r ARCI 779 i n h e a l t h y v o l u n t e e r s . C i r c u l a t i o n . 2 0 0 7 ; 1 1 6 ( 2 3 ) : 2 6 7 8 ‑ 2 6 8 6 .
2 9 . Nimjee SM,
¥.弓1 i t e RR, B e c k e r RC
ヲS u l l e n g e rBA. A p t a m e r s a s
百1 e r a p e u t i c s . Annu Rev P h a n n a c o l T o x i c o l . 2017 ; 6 ( 5 7 ) : 6 1
・7 9 .
3 0 . Chen Y , R o b e r t s J R , 0 り e J N , e t a l . I d e n t i f i c a t i o n o f a VWFA l M u t a t i o n A t t e n u a t i n g Thrombus Growth b u t Not P l a t e l e t A d h e s i o n . Blood . 2 0 1 7 ; 1 3 0 ( S u p p l e m a n t 1 ) : 5 4 7 .
3 1 . Mazepa MA, M a s i a s C , C h a t u r v e d i S . How t a r g e t e d t h e r a p y d i s r u p t s t h e
t r e a t m e n t p a r a d i g m f o r a c q u i r e d T τ ?ー t h er i s k s , b e n e f i t s a n d u n k n o w n s . B l o o d . 2 0 1 9 ;
1 3 4 ( 5 ) : 4 1 5 ・ 4 2 0 .
ト
l a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
F i g u r e l e g e n d s
F i g u r e 1 The i n h i b i t o r y e f f e c t s o f TAGX ・ 0 0 0 4 ,AR C l 779
ヲand c a p l a c
包umabon p l a t e l e t a g g r e g a t i o n under s t a t i c c o n d i t i o n s
As s h o wn i n t h
巴u p p e r l e f t p a n e l , TAGX‑0004 c o m p l e t e l y i n h i b i t e d r i s t o c e t i n ‑ i n d u c e d p l a t e l e t a g g r e g a t i o n ( R I P A ) a t a c o n c e n t r a t i o n o f 5 0
品1 1 . I n c o n t r a s t , t h e c o n c e n t r a t i o n r e q u i r e d f o r ARCl 7 7 9 was 5 0 0 nM, and t h a t f o r c a p l a c i z u m b was 50 nM. As s hown i n l o w e r l e f t p a n e l , TAGX‑0004 i n h i b i t e d b o t r o c e t i n ‑ i n d u c e d p l a t e l e t a g g r e g a t i o n ( B I P A ) a t a c o n c e n
仕a t i o no f 50 nM. The c o n c e n t r a t i o n r e q u i r e d f o r ARCl 779 was 5 0 0 nM , a n d t h a t f o r c a p l a c i z u m a b was 5 0 nM.
F i g u r e 2 The i n h i b i t o r y e f f e c t s o f TAGX‑0004, ARC1779, and c a p l a c i z u m a b on p l a t e l e t thrombus f o r m a t i o n under h i g h s h e a r s t r e s s
P r e s s u r e c u r v e s a t v a r i o u s c o n c e n t r a t i o n s o f e a c h a g e n t a r e s h o w n ; c u r v e s e n d a t t h e
p o i n t o f c o m p l e t e m i c r o c a p i l l a i y o c c l u s i o n . As shown i n t h e u p p e r p a n e l , TAGX‑0004
i n h i b i t e d t h r o m b u s f o r m a t i o n u n d e r h i g h s h e a r c o n d i t i o n s a t a f i n a l c o n c e n t r a t i o n o f 5 0
nM. The m i d d l e p a n e l s h o w s t h a t ARC 1 7 7 9 d i d n o t a c h i e v e c o m p l e t e i n h i b i t i o n e v e n a t
a c o n c e n
仕 組ono f 1 0 0 0 nM. Th e low e r p a n e l s h o w s t h a t c a p l a c i z u m a b c o m p l e t e l y
i n h i b i t e d t h r o m b u s f o m 1 a t i o n a t a c o n c e n t : t a t i o n o f 50 nM.
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
F i g u r e 3 Bioph y s i c a l i n t e r a c t i o n a n a l y s i s o f TAGX‑0004 and ARC1779 w i t h e l e c
仕o p h o r e s i s m o b i l i t y s h i f t a s s a y (EMSA)
The w h i t e a I T o w h e a d i n d i c a t e s t h e compl e x o f w i l d ‑ t y p e r e c o m b i n a n t VWF Al w i t h a n a p t a m e r (TAGX‑0004 o r ARCl 7 7 9 ) . The b l a c k anowhead i n d i c a t e s unbound a p t a m e r . I n c r e a s i n g t h e c o n c e n t r a t i o n o f w i l d ‑ t y p e VWF Al i n c r e a s e d t h e d e n s i t y of c o m p l e x b a n d s a n d d e c r e a s e d t h a t o f unbound a p t a m e r b a n d s . T h e s e e x p e r i m e n t s were p e r f o r m e d f o u r t i m e s f o r e a c h a p t a m e r .
百i e d i s s o c i a t i o n r a t e s ( K u ) o f TAG X ‑ 0 0 0 4 a n d ARCl 7 7 9 were 2 . 2
土0 . 9nM and 3 5 . 5 ± 1 . 5 nM, r e s p e c t i v e l y .
F i g u r e 4 E l e c t r o p h o r e s i s mob
脳t ys h i f t a s s a y (EMSA) u s i n g a l a n i n e ‑ s c a n n i n g m u t a n t s f o r a n a l y z i n g VWF Al b i n d i n g t o TAGX
・0004and AR Cl 779
The w h i t e a r τ o w h e a d i n d i c a t e s t h e complex ofVWF Al w i t h
如a p t a m e r (TAGX‑0004
o r ARCI 7 7 9 ) . The b l a c k a r r o w h e a d i n d i c a t e s unbound a p t a m e r . The l e
負most l a n e i n
e a c h g e l ( ‑ ) r e p r e s e n t s a p t a m e r o n l y , w i t h o u t a VWF Al m u t a n t . Using b o t h a p t a m e r s ,
1 0 VWF Al m u t a n t s (Kl312A , Rl334A , R1336A, Kl348A, K1371A , E1376A,
Kl406A, K l 4 2 3 A , R l 4 2 6 , and Kl430A) f o n n e d c o m p l e x e s w i t h a p t a m e r s , a s d i d
wi l d
咽t y p e (WT) VWF A l . With TAGX‑0004, a s shown i n t h e t o p p a n e l s , t h r e e m u t a n t s
σ1366A, Rl395A , a n d R1399A, i n d i c a t e d i n r e d l e t t e r s ) d e m o n s t r a t e d d e c r e a s e d
d e n s i t i e s o f c o m p l e x b a n d s a nd i n c r e a s e d d e n s i t i e s o f unbound ba n d s , i n d i c a t i n g t h a t
t h e s e amino a c i d s w e r e im po r t a n t f o r t h e b i n d i n g o f V
京T F Al t o TAGX
町0004
目With
ARC l 7 7 9 , a s shown i n t h e l o w e r p a n e l s , f i v e m u t a n t s ( R l 2 8 7 A , Kl362A , RI 392A ,
H a e m a t o l o g i c a HAEMATOL/2019/235549 V e r s i o n 3
Rl395A, a n d Rl399A) d e m o n s t r a t e d d e c r e a s e d d e n s i t i e s o f c o m p l e x b a n d s and m c r e a s e d d e n s i t i e s o f unbound b a n d s .
F i g u r e 5 A n a l y s i s o f c a p l a c i z u m a b b i n d i n g s i t e s t o t h e VWF Al domain u s i n g a l a n i n e ‑ s c a n n i n g m u t a n t s w i t h s u r f a c e p i a s
田onr e s o n a n c e (SPR)
We p e r f o m 1 e d SPR a n a l y s i s t o i n v e s t i g a t e c a p l a c i z u m a b b i n d i n g s i t e s t o t h e VWF Al d o m a i n . A f t e r i m m o b i l i z i n g c a p l a c i z u m a b on t h e s e n s o r c h i p CMS, 1 6 a l a n i n e ‑ s u b s t i t u t e d VWF Al m u t a n t s w e r e a n a l y z e d . The r e l a t i v e b i n d i n g volume of w i l d ‑ t y p e VWF Al was d e f i n e d a s 1 0 0 % .
百i e b i n d i n g amount o f e a c h m u t a n t was e x p r e s s e d a s a r a t i o r e l a t i v e t o w i l d t y p e . F i v e m u t a n t s w e r e c o n s i d e r e d t o b i n d t o c a p l a c i z u m a b b a s e d on h a v i n g a r e l a t i v e r a t i o a b o v e t h e c u t o f f o f 80%. T h e s e r e s u l t s i n d i c a t e t h a t t h e s e f i v e amino a c i d s 匹 1 3 6 2
ラR l 3 9 2 ,R l 3 9 5 , R l 3 9 9 , a n d Kl406) i n t h e VWF Al domain were imp o r t a n t i n c a p l a c i z u m a b b i n d i n g .
町
g u r e 6 V
明T F Al domain b i n d i n g s i t e s t o TAGX ・ 0 0 0 4 , ARC1779, and c a p l a c i z u
盟ab
Amin o a c i d s c o l o r e d y e l l o w d i d n o t c o n t r i b u t e t o VWF Al b i n d i n g t o a p t a m e r s o r c a p l a c i z u m a b . Amino a c i d s c o l o r e d r e d w e r e n e c e s s m y f o r b i n d i n g t o a p t a mer s o r
C叩