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Site-specific PEGylation of recombinant immunotoxin αFAP-PE38 for half-life extension

(USC Thesis Other)

Site-specific PEGylation of recombinant immunotoxin αFAP-PE38 for half-life extension

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Content S i t e - S p e c i f i c P E G yl at i on of R e c om b i n an t I m m u n ot oxi n
α F A P - P E 38 f or H al f - L i f e E xt e n s i on
b y
Y i r u J i n
A T h e s i s P r e s e n t e d t o t h e
F A C U L T Y O F T H E U S C G R A D U A T E S C H O O L
U N I V E R S I T Y O F S O U T H E R N C A L I F O R N I A
I n P a r t i a l F u l f i l l m e n t o f t h e
R e q u i r e m e n t s f o r t h e D e g r e e
M A S T E R O F S C I E N C E
P H A R M A C E U T I C A L S C I E N C E S
A u g u s t 2 0 1 8
I
T a b l e o f C o n t e n t s
A c k n ow l e d ge m e n t s ...................................................................................................................... I V
L i s t of F i gu r e s ................................................................................................................................ V
L i s t of T a b l e s ................................................................................................................................ V I
L i s t of A b b r e vi at i on s .................................................................................................................. V I I
A b s t r ac t .......................................................................................................................................... X
1 I n t r od u c t i on ............................................................................................................................ 1
1.1 F i br obl a s t s i n T um or S t r om a ............................................................................................. 1
1.1.1 T um or - P r om ot i ng E f f e c t s of C A F s ............................................................................ 1
1.1.2 C A F s a s a N ove l T a r ge t f or C a nc e r T he r a py ............................................................. 3
1.2 F A P - T a r ge t i ng I m m unot oxi n α F A P - P E 38 ........................................................................ 4
1.2.1 S t r uc t ur e of α F A P - P E 38 ............................................................................................. 5
1.2.2 M e c ha ni s m of A c t i on of α F A P - P E 38 ......................................................................... 8
1.2.3 A nt i t um or A c t i vi t y of α F A P - P E 38 ............................................................................. 8
1.2.4 L i m i t a t i on of α F A P - P E 38 T he r a py ............................................................................ 9
1.3 P E G y l a t i on ......................................................................................................................... 9
1.3.1 R a ndom P E G y l a t i on ................................................................................................. 10
1.3.2 S i t e - S pe c i f i c P E G y l a t i on .......................................................................................... 10
1.4 G e ne t i c I n c or por a t i on of U A A ........................................................................................ 11
I I
1.5 R e s e a r c h O bj e c t i ve s ......................................................................................................... 16
2 M at e r i al s an d M e t h od s ........................................................................................................ 17
2.1 R e a ge nt s ........................................................................................................................... 17
2.2 C e l l l i ne s .......................................................................................................................... 17
2.3 B a c t e r i a l S t r a i ns a nd P l a s m i ds ......................................................................................... 17
2.4 S i t e - D i r e c t e d M ut a ge ne s i s ............................................................................................... 19
2.5 C ons t r uc t i on of E xpr e s s i on V e c t or s of a a R S G e ne s ....................................................... 22
2.6 E x pr e s s i on a nd P ur i f i c a t i on of R e c om bi na nt I m m unot oxi ns .......................................... 22
2.7 P r ot e i n R e f ol di ng ............................................................................................................. 24
2.8 P E G y l a t i on ....................................................................................................................... 25
2.8.1 P E G y l a t i on a t t he C y s t e i ne R e s i due ......................................................................... 25
2.8.2 P E G y l a t i on a t t he pA c F R e s i due .............................................................................. 25
2.8.3 P E G y l a t i on a t t he pA z F R e s i due .............................................................................. 26
2.9 S E C A na l y s i s ................................................................................................................... 26
2.10 F A C S - B a s e d C e l l S ur f a c e S t a i ni ng A s s a y ...................................................................... 27
2.11 C ol or i m e t r i c X T T C e l l V i a bi l i t y A s s a y .......................................................................... 27
3 P E G yl at i on of α F A P - P E 38 at t h e C ys t e i n e R e s i d u e .......................................................... 29
3.1 P r ot e i n E xpr e s s i on, R e f ol di ng a nd P ur i f i c a t i on .............................................................. 29
I I I
3.2 P E G y l a t i on of R e f ol de d α F A P - C 3( C y s ) - P E 38 ............................................................... 31
3.3 P ur i f i c a t i on of P E G y l a t e d α F A P - C 3( C y s ) - P E 38 by S E C ............................................... 33
3.4 B i ndi ng A c t i vi t y of P E G y l a t e d α F A P - C 3( C y s ) - P E 38 .................................................... 35
3.5 C y t ot ox i c A c t i vi t y of P E G y l a t e d α F A P - C 3( C y s ) - P E 38 ................................................. 37
4 S i t e - S p e c i f i c P E G yl at i on of α F A P - P E 38 at t h e U A A R e s i d u e .......................................... 39
4.1 E x pr e s s i on a nd P ur i f i c a t i on of U A A - I n c or por a t e d α F A P - P E 38 V a r i a nt s a s I n c l us i on
B odi e s ......................................................................................................................................... 39
4.2 P E G y l a t i on of α F A P - P E 38 V a r i a nt s ............................................................................... 41
4.2.1 R e f ol di ng a nd P E G y l a t i on of α F A P - C D 8h( C 29pA c F ) - P E 38 .................................. 41
4.2.2 P E G y l a t i on of R e f ol de d α F A P - C 3( pA z F ) - P E 38 ...................................................... 43
4.2.3 P E G y l a t i on of α F A P - C D 8h( C 29pA c F ) - P E 38 unde r D e na t ur i ng C ondi t i ons .......... 45
4.3 R e f ol di ng a nd P ur i f i c a t i on of P E G y l a t e d α F A P - C D 8h( C 29pA c F ) - P E 38 by S E C ......... 47
4.4 B i ndi ng A c t i vi t y of P E G y l a t e d α F A P - C D 8h( C 29pA c F ) - P E 38 ...................................... 49
4.5 C y t ot ox i c A c t i vi t y of P E G y l a t e d α F A P - C D 8h( C 29pA c F ) - P E 38 ................................... 51
5 D i s c u s s i on .............................................................................................................................. 53
R e f e r e n c e s ..................................................................................................................................... 57
I V
A c k n o w l e d g e m e n t s
I w oul d f i r s t l i ke t o e x pr e s s m y s i nc e r e g r a t i t ude t o m y a dvi s or , D r . J i a nm i ng X i e , f or
of f e r i ng m e t he oppor t uni t y t o w or k on t hi s pr oj e c t a nd f or hi s g ui da nc e dur i ng m y s t udy a t t he
U ni ve r s i t y of S out he r n C a l i f or ni a . H i s i m m e n s e k n o w l e d g e , p e r p e t u a l e n t h u s i a s m f o r s c i e n c e , a n d
t h o u g h t f u l i n s i g h t s h a v e h e l p e d m e o n t h e r i g h t t r a c k f o r m y r e s e a r c h a n d k e p t i n s p i r i n g m e .
I w oul d l i ke t o e x t e nd m y g r a t i t ude t o m y c om m i t t e e m e m be r s , D r . W e i - C hi a ng S he n a nd D r .
P i n W a ng , f or t he i r va l ua bl e c om m e nt s on m y t he s i s .
I a m g r a t e f ul t o D r . L i a ng R ong , Q i a nqi a n Z ha o a nd R e be c c a L i m , w ho w e r e a l w a y s
a c c e s s i bl e a nd w i l l i ng t o he l p m e t hr oughout t he pr oj e c t . I w oul d l i ke t o t ha nk F a n F e i f or hi s
ki nd he l p a nd a s s i s t a nc e w i t h c e l l c ul t ur e a nd i n v i t r o a s s a y s . I w oul d l i ke t o e x t e nd m y
a ppr e c i a t i on t o D r . X i a oy a ng Z ha ng f or hi s t e c hni c a l a dvi c e . M y a ppr e c i a t i on a l s o g oe s t o T a o
M a a nd M a ny u n C he n f or a l w a y s be i ng s uppor t i ve .
F i na l l y , I w oul d l i ke t o e x pr e s s m y g r a t i t ude t o m y f a m i l y m e m be r s a nd f r i e nds f or pr ovi di ng
m e w i t h unf a i l i ng s uppor t a nd c ont i nuous e nc our a ge m e nt t hr oughout m y y e a r s of s t udy . T hi s
a c c om pl i s hm e nt w oul d not ha ve be e n pos s i bl e w i t hout t he m .
V
L i s t o f F i g u r e s
F i gu r e 1. S c he m a t i c of t he i m m unot oxi n α F A P - P E 38 .
F i gu r e 2. P E a nd i t s t r unc a t e d f or m P E 38.
F i gu r e 3. G e ne t i c i nc or por a t i on of U A A s i nt o pr ot e i ns a l l ow s s e l e c t i ve m odi f i c a t i on .
F i gu r e 4. P l a s m i d m a ps of e x pr e s s i on ve c t or s .
F i gu r e 5. D N A a nd a m i no a c i d s e que nc e s of t he pe pt i de c onne c t or s .
F i gu r e 6. P ur i f i c a t i on of α F A P - C D 8h- P E 38 a nd α F A P - C 3( C y s ) - P E 38 .
F i gu r e 7. P E G y l a t i on of r e f ol de d α F A P - C 3( C y s ) - P E 38 .
F i gu r e 8. P ur i f i c a t i on of P E G y l a t e d α F A P - C 3( C y s ) - P E 38 .
F i gu r e 9. B i ndi ng a c t i vi t y of P E G y l a t e d α F A P - C 3( C y s ) - P E 38 .
F i gu r e 10. C y t ot ox i c a c t i vi t y of P E G y l a t e d α F A P - C 3( C y s ) - P E 38 .
F i gu r e 1 1 . P ur i f i c a t i on of U A A - i nc or por a t e d α F A P - P E 38 va r i a nt s .
F i gu r e 12. P E G y l a t i on of r e f ol de d α F A P - C D 8h ( C 29pA c F ) - P E 38 .
F i gu r e 13. P E G y l a t i on of r e f ol de d α F A P - C 3( pA z F ) - P E 38 .
F i gu r e 14. P E G y l a t i on of α F A P - C D 8h ( C 29pA c F ) - P E 38 unde r de na t ur i ng c ondi t i ons .
F i gu r e 15. P ur i f i c a t i on of P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 .
F i gu r e 16. B i ndi ng a c t i vi t y of P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 .
F i gu r e 17. C y t ot ox i c a c t i vi t y of P E G y l a t e d α F A P - C D 8h( C 29pA c F ) - P E 38 .
V I
L i s t o f T a b l e s
T a b l e 1. P r i m e r s f or s i t e - di r e c t e d m ut a g e ne s i s of t hr e e c hos e n s i t e s .
V I I
L i s t o f A b b r e v i a t i o n s
a a R S : a m i noa c y l - t R N A s y n t he t a s e
A D P : a de nos i ne di phos pha t e
α F A P : a nt i - F A P a nt i body
B T T A A : 2- [ 4- { ( bi s [ ( 1- t e r t - but y l - 1 H - 1,2,3- t r i a z ol - 4- y l ) m e t hy l ] a m i no) m e t hy l } - 1 H - 1,2,3- t r i a z ol -
1- y l ] a c e t i c a c i d
C A F s : c a nc e r - a s s oc i a t e d f i br obl a s t s
C D 8h: hum a n C D 8 hi ng e
C uA A C : c oppe r - c a t a l y z e d a z i de –a l ky n e c y c l oa ddi t i on
C X C L 12: c he m oki ne C - X - C m ot i f l i g a nd 12
C X C R 4: c he m oki ne C - X - C m ot i f r e c e pt or 4
C y 5 .5: C y a n i ne 5.5 N H S e s t e r
D T T : di t hi ot hr e i t ol
E . c ol i : Es c he r i c hi a C ol i
E C M : e x t r a c e l l ul a r m a t r i x
E D T A : e t hy l e ne di a m i ne t e t r a a c e t i c a c i d
E F 2: e l onga t i on f a c t or 2
E G F s : e pi de r m a l g r ow t h f a c t or s
E M T : e pi t he l i a l - m e s e nc hy m a l t r a ns i t i on
F A C S : f l uor e s c e nc e - a c t i va t e d c e l l s or t i ng
F A P : f i br obl a s t a c t i va t i on pr ot e i n
V I I I
F B S : f e t a l bovi ne s e r um
F G F s : f i br obl a s t g r ow t h f a c t or s
G S H : g l ut a t hi one
G S S G : g l ut a t hi one di s ul f i de
H G F : he pa t oc y t e g r ow t h f a c t or
I C 5 0 : ha l f m a x i m a l i nhi bi t or y c onc e nt r a t i on
I G F - 1: i ns ul i n- l i ke g r ow t h f a c t or 1
I P T G : i s opr o py l β - D - 1- t hi og a l a c t opy r a nos i de
L B : Lur i a br ot h
M . j annas c hi i : M e t hanoc oc c us j annas c hi i
m A b: m onoc l ona l a nt i body
M D S C s : m y e l oi d- de r i ve d s uppr e s s or c e l l s
M M P s : m a t r i x m e t a l l opr ot e i na s e s
m P D A : m e t a - phe ny l e ne di a m i ne
M W C O : m ol e c ul a r w e i g ht c ut - of f
N i - N T A : ni c ke l - ni t r i l ot r i a c e t i c a c i d
pA c F : pa r a - a c e t y l phe ny l a l a ni ne
pA z F : pa r a - a z i dophe ny l a l a ni ne
P B S : phos pha t e - buf f e r e d s a l i ne
P D : pha r m a c ody n a m i c
P D G F : pl a t e l e t - de r i ve d g r ow t h f a c t or
I X
P E : P s e udom onas e x ot oxi n A
P E G : pol y e t hy l e ne g l y c o l
P K : pha r m a c oki ne t i c
P M S F : phe ny l m e t hy l s ul f ony l f l uor i de
R F 1: r e l e a s e f a c t or 1
s c F v: s i ng l e - c ha i n va r i a bl e f r a gm e n t
S D F - 1: s t r om a l c e l l - de r i ve d f a c t or - 1
S D S - P A G E : s odi um dode c y l s ul f a t e - pol y a c r y l a m i de g e l e l e c t r ophor e s i s
S E C : s i z e e x c l us i on c hr om a t ogr a phy
S O D 1: s upe r ox i de di s m ut a s e 1
t 1 / 2 : ha l f - l i f e
T A M s : t um or - a s s oc i a t e d m a c r opha ge s
T C E P : t r i s ( 2- c a r box y e t hy l ) phos phi ne
T G F - β : t r a ns f or m i ng g r ow t h f a c t or - β
T r e gs : r e gul a t or y T c e l l s
T r i s : t r i s ( hy d r ox y m e t hy l ) a m i nom e t ha ne
U A A : unna t ur a l a m i no a c i d
X T T : 2,3- bi s ( 2- m e t hox y - 4- ni t r o- 5- s ul f ophe ny l ) - 2 H - t e t r a z ol i um - 5- c a r box a ni l i de
X
A b s t r a c t
F i br obl a s t a c t i va t i on pr ot e i n ( F A P ) i s a c e l l - s ur f a c e pr ot e a s e hi g hl y e x pr e s s e d i n
c a nc e r - a s s oc i a t e d f i br obl a s t s ( C A F s ) —a t y p e of c e l l s i n t he t um or m i c r oe nvi r onm e nt t ha t
pr om ot e t he pr ogr e s s i on of hum a n e pi t he l i a l c a nc e r s . T o de pl e t e F A P - e xpr e s s i ng C A F s f or
t r e a t i ng c a nc e r , a r e c om bi na nt i m m unot oxi n, α F A P - P E 38, w a s r e c e nt l y de ve l ope d by f us i ng a
s i ng l e - c ha i n va r i a bl e f r a gm e nt ( s c F v) a ga i ns t F A P w i t h a t r unc a t e d f or m of P s e udom onas
e x ot oxi n A ( P E 38) . H ow e ve r , t he s hor t pl a s m a ha l f - l i f e of α F A P - P E 38 ha s l i m i t e d i t s t he r a pe ut i c
e f f i c a c y . T he a i m of t hi s t he s i s i s t o m odi f y α F A P - P E 38 w i t h pol y e t hy l e ne g l y c o l ( P E G ) , a
pol y m e r t ha t ha s pr e vi ou s l y be e n us e d t o e x t e nd t he pl a s m a ha l f - l i f e of ot he r t he r a pe ut i c pr ot e i ns .
W e ha ve g e ne r a t e d P E G y l a t e d α F A P - P E 38 us i ng t w o s t r a t e gi e s : ( 1) P E G y l a t i on a t a c y s t e i ne
r e s i due , a nd ( 2) P E G y l a t i on a t a n unna t ur a l a m i no a c i d r e s i due . W e f i nd t ha t a P E G y l a t e d
α F A P - P E 38 pr ot e i n, i n w hi c h a 10 kD a P E G i s a t t a c he d t o a ke t one - c ont a i ni ng unna t ur a l a m i no
a c i d i n t he l i nke r , c a n s pe c i f i c a l l y bi nd t o hum a n F A P - e xpr e s s i ng 293T c e l l s a nd i nhi bi t t he i r
vi a bi l i t y i n a dos e - de pe nde nt m a nne r . H ow e ve r , t he α F A P - P E 38 pr ot e i ns m odi f i e d w i t h l a r g e r
P E G , i nc l udi ng t he 20 a nd 30 kD a va r i a nt s , ha ve l os t bi ndi ng a f f i ni t y a nd c y t ot oxi c e f f e c t t o
hum a n F A P - e x pr e s s i ng 293T c e l l s , l i ke l y due t o i nc r e a s e d s t e r i c hi ndr a nc e . F ur t he r s t udi e s a r e
ne e de d f or i n v i v o e va l ua t i on of t he pha r m a c oki ne t i c pr ope r t i e s a nd pha r m a c ol ogi c e f f e c t s of t he
10 kD a P E G y l a t e d α F A P - P E 38 pr ot e i n .
1
1 I n t r o d u c t i o n
1.1 F i b r ob l as t s i n T u m or S t r om a
I t i s now w e l l a c c e pt e d t ha t c a nc e r i ni t i a t i on a nd pr ogr e s s i on de pe nd not s ol e l y on
c e l l - a ut onom ous de f e c t s but a l s o on t he s ur r oundi ng s t r om a . T he t um or s t r om a c ons i s t s of
i m m une c e l l s , bl ood a nd l y m pha t i c ve s s e l s , f i br obl a s t s , e x t r a c e l l ul a r m a t r i x ( E C M ) , a s w e l l a s
s i g na l i ng m ol e c ul e s ( i nc l udi ng c y t oki ne s , c he m oki ne s , g r ow t h f a c t or s , a nd e x os om e s ) ( H a na ha n
a nd C ous s e ns , 2012; Q ua i l a nd J oy c e , 2013) . F i br obl a s t s r e pr e s e nt t he m os t pr om i ne nt c e l l t y p e
w i t hi n t he s t r om a of m a ny c a nc e r s , m os t not a bl y br e a s t a nd pa nc r e a t i c c a nc e r ( P i e t r a s a nd
O s t m a n, 2010 ) . T he s e c e l l s , of t e n r e f e r r e d t o a s c a nc e r - a s s oc i a t e d f i br obl a s t s ( C A F s ) , a r e
phe not y p i c a l l y a nd f unc t i ona l l y di s t i ng ui s ha bl e f r om t he i r c ount e r pa r t s i n nor m a l t i s s ue s ( K a l l ur i
a nd Z e i s be r g , 2006) . F i br obl a s t s i n nor m a l t i s s ue s , w hi c h a r e us ua l l y qui e s c e nt , be c om e
a c t i va t e d t o f a c i l i t a t e r e pa i r a nd r e ge ne r a t i on i n r e s pons e t o t i s s ue i nj ur y a nd a s s oc i a t e d s t i m ul i .
O nc e w ound he a l i ng i s a c c om pl i s he d, how e ve r , a c t i va t e d f i br obl a s t s r e ve r t t o a r e s t i ng
phe not y p e or unde r g o a popt os i s ( T o m a s e k e t a l ., 2002) . I n c ont r a s t , C A F s a r e pe r pe t ua l l y
a c t i va t e d, e x hi bi t i ng hi g he r pr ol i f e r a t i on r a t e , e nha nc e d m i g r a t or y c a pa c i t y , e nha nc e d a ut oc r i ne
g r ow t h f a c t or - i nduc e d s i g na l i ng, a nd i nc r e a s e d l e ve l s of s e c r e t or y m ol e c ul e s ( K a l l ur i , 2016) .
A c c um ul a t i ng e vi de nc e s ug ge s t s t ha t t he y a r e a ke y de t e r m i na nt i n t he m a l i g na nt pr ogr e s s i on of
c a nc e r ; t he r e by t he y ha ve e m e r g e d a s a n i m por t a nt t a r g e t f or c a nc e r t he r a p y ( F r a nc o e t a l ., 2010;
G onda e t a l ., 2010) .
1.1.1 T u m or - P r om ot i n g E f f e c t s of C A F s
C A F s s e c r e t e va r i ous g r ow t h f a c t or s , s uc h a s e pi de r m a l g r ow t h f a c t or s ( E G F s ) , he pa t oc y t e
2
g r ow t h f a c t or ( H G F ) , f i br obl a s t g r ow t h f a c t or s ( F G F s ) a nd i ns ul i n- l i ke g r ow t h f a c t or 1 ( I G F - 1) .
T he s e g r ow t h f a c t or s a r e m i t og e ni c f or m a l i g na nt c e l l s ( C i r r i a nd C hi a r ugi , 201 1 ; G r uga n e t a l .,
2010; S a t y a m oor t hy e t a l ., 2001) . A s c a nc e r pr ogr e s s e s , c a nc e r c e l l s c a n obt a i n t he a bi l i t y t o
br e a k dow n t he unde r l y i ng ba s e m e nt m e m br a ne a nd i nva de i nt o t he s ur r oundi ng t i s s ue . S om e
c a nc e r c e l l s a t t a i n t hi s i nva s i ve be ha vi or t hr ough a c qui s i t i on of a m e s e nc hy m a l phe not y p e ,
know n a s t he e pi t he l i a l - m e s e nc hy m a l t r a ns i t i on ( E M T ) ( K a l l ur i a nd W e i nbe r g , 2009) . C A F s m a y
i nduc e t he E M T of c a nc e r c e l l s t hr oug h pa r a c r i ne s i g na l i ng w i t h g r ow t h f a c t or s a nd
pr o- m i gr a t or y c y t oki ne s , i nc l udi ng t r a ns f or m i ng g r ow t h f a c t or - β ( T G F - β ) a nd s t r om a l
c e l l - de r i ve d f a c t or - 1 ( S D F - 1) / c he m oki ne C - X - C m ot i f l i g a nd 12 ( C X C L 12) ( O r i m o e t a l ., 2005;
Y u e t a l ., 2014) .
C A F s a r e t he m a j or c e l l t y p e r e s pons i bl e f or t he s y n t he s i s , de gr a da t i on, a nd m odi f i c a t i on of
t he E C M , a c om pl e x a s s e m bl y of m a c r om ol e c ul e s t ha t not onl y s e r ve s a s a phy s i c a l s c a f f ol d f or
c e l l a dhe s i on a nd m i g r a t i on but a l s o ha s a c r i t i c a l r ol e i n t he e vol ut i on a nd s pr e a d of c a nc e r s
( G a ggi ol i e t a l ., 2007) . C A F s c a n i nc r e a s e t he pr oduc t i on of E C M pr ot e i ns , s uc h a s c ol l a ge ns
( t y p e I , I I I , a nd V ) , f i br one c t i n, l a m i ni n a nd t e na s c i n C . T he y a l s o c ont r i but e t o de r e gul a t e d
e x pr e s s i on of va r i ous de gr a di ng e nz y m e s i nc l udi ng m a t r i x m e t a l l opr ot e i na s e s ( M M P s ) t ha t
m e di a t e E C M r e m ode l i ng ( E r dog a n a nd W e bb, 2017) . A bnor m a l E C M dy n a m i c s c a n di r e c t l y
pr om ot e c e l l ul a r t r a ns f or m a t i on a nd m e t a s t a s i s a nd a l s o he l p g e ne r a t e a t um or i g e ni c ni c he t ha t
f ur t he r f a c i l i t a t e s c a nc e r pr ogr e s s i on ( L u e t a l ., 2012) .
I n a ddi t i on, C A F s he l p t o s ha pe t he t um or m i c r oe nvi r onm e nt by m odul a t i ng t he a nt i t um or
i m m une r e s pons e s ( Z i a ni e t a l ., 2018) . T he y a r e c a pa bl e of pr ovi di ng m ul t i pl e r e gul a t or y s i g na l s
3
w i t h t he pot e nt i a l t o i nhi bi t i m m une e f f e c t or c e l l s . F or e x a m pl e , T G F - β 1 s e c r e t e d by C A F s i s a
pot e nt i m m unos uppr e s s or w hi c h a t t e nua t e s t he a c qui s i t i on a nd e x pr e s s i on of T - c e l l e f f e c t or
f unc t i ons ( A hm a dz a de h a nd R os e nbe r g , 2005) . I t a l s o i nhi bi t s t he f unc t i on of na t ur a l ki l l e r c e l l s
a nd C D 8
+
c y t ot ox i c T l y m phoc y t e s , t hus bl oc ki ng a nt i t um or c y t ot oxi c a c t i vi t i e s ( B a l s a m o e t a l .,
2009; Y a ng e t a l ., 2010b) . A ddi t i ona l l y , C A F s m i g ht i ndi r e c t l y i m pa i r a nt i t um or i m m un i t y by
pr om ot i ng t he r e c r ui t m e nt of i m m unos uppr e s s i ve c e l l s s uc h a s r e gul a t or y T c e l l s ( T r e g s ) , t y p e I I
t um or - a s s oc i a t e d m a c r opha ge s ( T A M s ) , a nd m y e l oi d- de r i ve d s uppr e s s or c e l l s ( M D S C s )
( A ugs t e n e t a l ., 2009; D e ng e t a l ., 2017; T a n e t a l ., 201 1 ) .
1.1.2 C A F s as a N ove l T a r ge t f or C an c e r T h e r ap y
M os t t he r a pe ut i c s t r a t e gi e s a ga i ns t c a nc e r ha ve f oc us e d on t a r g e t i ng va r i ous a s pe c t s of t um or
c e l l s di r e c t l y . U nl i ke t um or c e l l s , s t r om a l c e l l s w i t hi n t he t um or m i c r oe nvi r onm e nt a r e
g e ne t i c a l l y s t a bl e , a nd t hus r e pr e s e nt a n a t t r a c t i ve t he r a pe ut i c t a r g e t w i t h r e duc e d r i s k of dr ug
r e s i s t a nc e a nd t um or r e c ur r e nc e ( Q ua i l a nd J oy c e , 2013) . R e c e nt a t t e nt i on ha s be e n g i ve n t o
C A F s due t o t he i r t um or - pr om ot i ng c a pa c i t y . T he r e a r e t w o s t r a t e gi e s f or C A F - di r e c t e d t he r a py
( G a s c a r d a nd T l s t y , 2016; K a l l ur i a nd Z e i s be r g , 2006) . O ne i s t o r e ve r t t he C A F phe not y p e t o a
nor m a l f i br obl a s t phe not y p e . T hi s c a n be done by i nt e r f e r i ng w i t h C A F phe not y p e - di r e c t e d
s i g na l i ng pa t hw a y s , s uc h a s pl a t e l e t - de r i ve d g r ow t h f a c t or ( P D G F ) , T G F - β , he dge hog, N ot c h,
F G F , a nd S D F - 1 / c he m oki ne C - X - C m ot i f r e c e pt or 4 ( C X C R 4) pa t hw a y s . F or e x a m pl e ,
V i s m ode g i b , a he dge hog pa t hw a y i nhi bi t or , ha s a l r e a dy be e n a ppr ove d f or t he t r e a t m e nt
of ba s a l - c e l l c a r c i nom a ( S e kul i c e t a l ., 2012) . A ddi t i ona l l y , a nt i - f i br ot i c a ge nt s ( e .g., nons t e r oi da l
a nt i - i nf l a m m a t or y dr ugs ) ha ve be e n us e d t o m odul a t e E C M r e m ode l i ng ( L y ons e t a l ., 201 1 ) .
4
T he ot he r s t r a t e gy i s t o e l i m i na t e C A F s by t a r g e t i ng C A F - s pe c i f i c pr ot e i ns . F or e xa m pl e ,
f i br obl a s t a c t i va t i on pr ot e i n ( F A P ) i s a t y p e I I i nt e g r a l m e m br a ne g l y c o pr ot e i n of t he s e r i ne
pr ot e a s e f a m i l y s e l e c t i ve l y e x pr e s s e d by C A F s i n e pi t he l i a l c a r c i nom a s , w hi c h m a ke s i t a
pr om i s i ng c a ndi da t e f or s pe c i f i c t a r g e t i ng of C A F s ( B r e nne n e t a l ., 2012) . S m a l l - m ol e c ul e
i nhi bi t or s of F A P e nz y m a t i c a c t i vi t y ha ve be e n de ve l ope d; how e ve r , t he i r a nt i - t um or a c t i vi t y i s
s ubopt i m a l be c a us e F A P c oul d pr om ot e t um or g r ow t h a nd i nva s i on t hr ough non- e nz y m a t i c
f unc t i ons ( H ua ng e t a l ., 201 1 ) . T he a nt i - F A P m onoc l ona l a nt i body ( α F A P m A b) s i br ot uz um a b,
w hi c h bi nds s pe c i f i c a l l y t o t um or s i t e s a nd i s w e l l t ol e r a t e d w i t hout a ppa r e nt s i de e f f e c t s ( S c ot t
e t a l ., 2003) , f a i l e d a n e a r l y pha s e I I c l i ni c a l t r i a l f or m e t a s t a t i c c ol or e c t a l c a nc e r a s t he e x pe c t e d
e f f i c a c y w a s not m e t ( H of he i nz e t a l ., 2003) . T o e nha nc e t he a nt i t um or e f f i c a c y , a va r i e t y of
a nt i body - b a s e d t he r a pe ut i c a ge nt s ha ve be e n de ve l ope d us i ng r e c om bi na nt D N A t e c hnol ogy .
S om e a r e c ur r e nt l y be i ng i nve s t i g a t e d i n pha s e I c l i ni c a l t r i a l s , s uc h a s α F A P / i nt e r l e uki n- 2
i m m unoc y t oki ne R O 687428 1, α F A P / a nt i - d e a t h r e c e pt or 5 bi s pe c i f i c a nt i bod y R O 6874813 , a nd
F A P - s pe c i f i c r e - di r e c t e d T c e l l s ( S c hube r t h e t a l ., 2013) .
1.2 F A P - T a r ge t i n g I m m u n ot oxi n α F A P - P E 38
I m m unot oxi ns a r e a c l a s s of a nt i body - b a s e d t he r a pe ut i c a ge nt s i n w hi c h m A bs or a nt i body
f r a gm e nt s a r e e x pl oi t e d f or t a r g e t e d de l i ve r y of pot e nt c y t ot oxi c a ge nt s t o a c hi e ve s e l e c t i ve c e l l
ki l l i ng . M a ny i m m unot oxi ns us i ng P s e udom onas e x ot oxi n A ( P E ) or di pht he r i a t oxi n a s t he
pa y l oa d a r e de ve l ope d f or c a nc e r t r e a t m e nt ( W e i dl e e t a l ., 2014) . α F A P - P E 38 , a F A P - t a r g e t i ng
i m m unot oxi n de ve l ope d by our c ol l a bor a t or , ha s be e n de m ons t r a t e d t o ha ve a nt i t um or a c t i vi t i e s
i n a 4T 1 m ur i ne br e a s t c a nc e r m ode l ( F a ng e t a l ., 2016) .
5
1.2.1 S t r u c t u r e of α F A P - P E 38
T he c ons t r uc t of α F A P - P E 38 w a s pr oduc e d by f us i ng a n α F A P s i ng l e - c ha i n va r i a bl e
f r a gm e nt ( s c F v) t o a t r unc a t e d f or m of P E ( P E 38) w i t h a hum a n C D 8 hi ng e ( C D 8h) ( F i g. 1) . T he
α F A P s c F v i s a 250- a m i no a c i d f us i on pr ot e i n c ont a i ni ng t he va r i a bl e r e gi ons of t he he a vy a nd
l i g ht c ha i ns of t he α F A P m A b c onne c t e d w i t h a ( G G G G S ) 3 l i nk e r . I t ha s c r os s - r e a c t i vi t y a ga i ns t
hum a n F A P ( hF A P ) a nd m ur i ne F A P ( m F A P ) ( B r oc ks e t a l ., 2001) . T he s m a l l e r s i z e of t he s c F v
i n c om pa r i s on t o t ha t of t he m A b c a n i m pr ove t he t i s s ue pe ne t r a t i on i n s ol i d t um or s . T he C D 8h
i s a 48- a m i no a c i d pe pt i de c onne c t or w hi c h c oul d he l p i nc r e a s e t he s c F v e x pr e s s i on ( C l a s s on e t
a l ., 1992) . P E i s a pr ot e i n t oxi n w i t h t hr e e f unc t i ona l dom a i ns : a r e c e pt or - bi ndi ng dom a i n
( do m a i n I a ) , a t r a ns l oc a t i on dom a i n ( do m a i n I I ) , a nd a c a t a l yt i c dom a i n ( do m a i n s I b a nd I I I ) ( F i g.
2) . B e c a us e dom a i n I a m a y c a us e nons pe c i f i c c y t ot ox i c i t y , P E 38 , a t r unc a t e d f or m de voi d of
dom a i n I a , i s of t e n us e d f or c ons t r uc t i on of t he ne w g e ne r a t i on of i m m unot oxi ns ( W e l don a nd
P a s t a n, 201 1 ) .
6
F i gu r e 1. S c h e m at i c of t h e i m m u n ot oxi n α F A P - P E 38 . α F A P - P E 38 i s a f us i on pr ot e i n
c ons i s t i ng of a n α F A P s c F v , a C D 8h c onne c t or , a nd P E 38 ( a t r unc a t e d f or m of P E ) .
7
F i gu r e 2. P E an d i t s t r u n c at e d f or m P E 38. ( A ) S c he m a t i c of P E a nd P E 38. P E c ons i s t s of
t hr e e f unc t i ona l dom a i ns : a r e c e pt or - bi ndi ng dom a i n ( dom a i n I a ) , a t r a ns l oc a t i on dom a i n
( dom a i n I I ) , a nd a c a t a l y t i c dom a i n ( dom a i ns I b a nd I I I ) . P E 38 i s a t r unc a t e d f or m de voi d of
dom a i n I a . ( B ) C r y s t a l s t r u c t u r e of P E . D om a i n I a ( 1- 25 2) i s s how n i n gr e e n, dom a i n I I ( 25 3- 364)
i s s how n i n y e l l ow , dom a i n I b ( 365- 404) i s s how n i n bl ue , a nd dom a i n I I I ( 405- 613) i s s how n i n
r e d.
8
1.2.2 M e c h an i s m of A c t i on of α F A P - P E 38
The i nt oxi c a t i on pa t hw a y of P E - ba s e d i m m unot oxi ns i s w e l l unde r s t ood ( W e l don a nd P a s t a n,
201 1 ) . U pon bi ndi ng t o t he t a r g e t of i nt e r e s t ( F A P i n t hi s c a s e ) , t he i m m unot oxi n i s i nt e r na l i z e d
by r e c e pt or - m e di a t e d e ndoc y t os i s a nd t r a ns l oc a t e d t o a c i di c c om pa r t m e nt s i n t he c e l l . I t i s t he n
c l e a ve d be t w e e n r e s i due s R 279 a nd G 280 of dom a i n I I by t he e ndopr ot e a s e f ur i n ( O ga t a e t a l .,
1992) , g e ne r a t i ng a c a t a l y t i c a l l y a c t i ve C - t e r m i na l f r a gm e nt ( O ga t a e t a l ., 1990) . T he C - t e r m i na l
f r a gm e nt i s s ubs e que nt l y t r a ns por t e d t o t he t r a ns - G ol gi ne t w or k ( S m i t h e t a l ., 2006) a nd
unde r g o e s r e t r ogr a de t r a ns por t t o t he e ndopl a s m i c r e t i c ul um ( J a c ks on e t a l ., 1999) f ol l ow e d by
t r a ns l oc a t i on t o t he c y t os ol . O nc e i n t he c y t os ol , t he c a t a l y t i c dom a i n bl oc ks t he e uka r y o t i c
t r a ns l a t i on e l ong a t i on f a c t or 2 ( E F 2) vi a a n a de nos i ne di phos pha t e ( A D P ) - r i bos y l a t i on
m e c ha ni s m , c a us i ng t he a r r e s t of pr ot e i n s y n t he s i s ( H w a ng e t a l ., 1987) . T hi s e ve nt i ni t i a t e s t he
a popt ot i c c a s c a de by l ow e r i ng M c l - 1 l e ve l s a nd unl e a s hi ng B a k t o pr om ot e a popt ot i c de a t h ( D u
e t a l ., 2010) .
1.2.3 A n t i t u m or A c t i vi t y of α F A P - P E 38
α F A P - P E 38 e x hi bi t e d a nt i t um or a c t i vi t i e s i n a 4T 1 m ur i ne br e a s t c a nc e r m ode l ( F a ng e t a l .,
2016) . S pe c i f i c a l l y , m i c e r e c e i vi ng f our s e que nt i a l i nt r a ve nous i nj e c t i ons w i t h 0.5 m g / kg
α F A P - P E 38 s how e d s i g ni f i c a nt r e t a r da t i on of t um or g r ow t h w i t hout a ppa r e nt s i de - e f f e c t s
obs e r ve d f or t he dur a t i on of t he e x pe r i m e nt . F ur t he r e x a m i na t i on of c e l l s f r om e x c i s e d t um or
t i s s ue s i ndi c a t e d t ha t α F A P - P E 38 i nduc e d a popt os i s of F A P - pos i t i ve t um or s t r om a l c e l l s . I n
a ddi t i on, t he a na l y s i s of m R N A a nd pr ot e i n e x pr e s s i on i n 4T 1 t um or t i s s ue s de m ons t r a t e d t ha t
α F A P - P E 38 a l t e r e d t he t um or m i c r oe nvi r onm e nt by c ha ngi ng t he e x pr e s s i on of g r ow t h f a c t or s ,
9
c y t oki ne s , c he m oki ne s a nd M M P s , a nd by i nhi bi t i ng t he r e c r ui t m e nt of T A M s .
1.2.4 L i m i t at i on of α F A P - P E 38 T h e r ap y
D e s pi t e t he de m ons t r a t e d a nt i t um or a c t i vi t y of α F A P - P E 38 , i t s ha l f - l i f e i n bl ood c i r c ul a t i on
i s l e s s t ha n 30 m i n, r e s ul t i ng i n poor bi oa va i l a bi l i t y a nd l i m i t e d di f f us i on i n s ol i d t um or s . C l e a r l y
a s t r a t e gy t o e x t e nd t he pl a s m a ha l f - l i f e of α F A P - P E 38 i s ne e de d f or i t s a ppl i c a t i on i n t he c l i ni c .
1.3 P E G yl at i on
C he m i c a l m odi f i c a t i on w i t h pol y e t hy l e ne g l y c ol ( P E G ) , know n a s P E G y l a t i on , i s a
w e l l - e s t a bl i s he d t e c hnol ogy f or e x t e ndi ng t he ha l f - l i f e of t he r a pe ut i c pr ot e i ns ( J e vs e va r e t a l .,
2010) . T he a t t a c he d P E G c ha i ns c a n s hi e l d t he pr ot e i n s ur f a c e c ha r g e s , a nd t he y a l s o i nc r e a s e t he
pr ot e i n ’ s hy d r ody n a m i c r a di us due t o t he i r a bi l i t y t o c oor di na t e w i t h t w o t o t hr e e w a t e r
m ol e c ul e s pe r m onom e r uni t ( G oka r n e t a l ., 2012) . T he s e a l t e r e d phy s i c oc he m i c a l pr ope r t i e s
c ont r i but e t o r e duc e d r e na l c l e a r a nc e of t he P E G y l a t e d pr ot e i n. P E G c ha i ns a l s o pr ovi d e t he
pr ot e i n w i t h a pr ot e c t i ng m a s k w hi c h he l ps t o r e duc e i t s pha goc y t i c upt a ke by pa r e nc hy m a l c e l l s
( Y a ng e t a l ., 2010a ) a nd i t s ops oni z a t i on ( O w e ns a nd P e ppa s , 2006) , l e a di ng t o a n i nc r e a s e d
r e s i de nc e t i m e i n s y s t e m i c c i r c ul a t i on . I n a ddi t i on t o pr ol onge d ha l f - l i ve s , P E G y l a t e d t he r a pe ut i c
pr ot e i ns m a y i m pa r t s e ve r a l ot he r a dva nt a ge s ove r t he i r unm odi f i e d f or m , s uc h a s i nc r e a s e d
w a t e r s ol ubi l i t y , i m pr ove d s t a bi l i t y a nd de c r e a s e d i m m unog e ni c i t y . I t s houl d be not e d t ha t
P E G y l a t i on c a n of t e n l e a d t o a l os s of bi ndi ng a f f i ni t y of t he r a pe ut i c pr ot e i n s t o t he i r t a r g e t
r e c e pt or s due t o s t e r i c i nt e r f e r e nc e of P E G w i t h t he bi ndi ng i nt e r a c t i on . H ow e ve r , t he r e s ul t i ng
l os s i n pot e nc y c a n be of f s e t by a n i nc r e a s e d c i r c ul a t i on t i m e . S uc c e s s f ul P E G y l a t i on of
t he r a pe ut i c pr ot e i ns r e qui r e s t o f i nd a ba l a nc e be t w e e n t he i r pha r m a c oki ne t i c ( P K ) a nd
1 0
pha r m a c ody n a m i c ( P D ) pr ope r t i e s ( F i s hbur n, 2008) .
1.3.1 R an d om P E G yl at i on
E a r l y s t a ge de ve l opm e nt s of P E G y l a t i on m a i nl y t a r g e t e d pr i m a r y a m i no g r oups i n t he s i de
c ha i n of a l y s i ne r e s i due or a t t he N - t e r m i nus . T he s e pr i m a r y a m i no g r oups c a n c onj uga t e w i t h
P E G de r i va t i ve s c a r r y i ng a s uc c i ni m i de or a n a l de hy d e vi a r e duc t i ve a m i na t i on ( R obe r t s e t a l .,
2002) . B e c a us e e a c h pr ot e i n ha s m ul t i pl e nuc l e ophi l e s a nd e x c e s s P E G r e a ge nt s a r e us e d t o
a c hi e ve r e a s ona bl e c onve r s i ons , t he pr oduc t i s of t e n he t e r oge ne ous c ont a i ni ng pr ot e i n - P E G
c onj uga t e s w i t h va r i ous num be r s a nd s i t e s of P E G c ha i ns . T hi s c om pl i c a t e s t he pur i f i c a t i on,
i de nt i f i c a t i on a nd a na l y s i s of t he P E G y l a t e d pr oduc t s . M os t i m por t a nt l y , P E G y l a t i on ne a r or a t
t he a c t i ve s i t e of t he pr ot e i n c a n i na c t i va t e t he pr ot e i n .
1.3.2 S i t e - S p e c i f i c P E G yl at i on
Thi ol c onj uga t i on w i t h na t ur a l l y unpa i r e d c y s t e i ne r e s i due s c a n pr ovi de l e s s he t e r oge ne ous
pr oduc t s s i nc e t he num be r of f r e e c y s t e i ne r e s i due s on t he s ur f a c e of a pr ot e i n i s m uc h l e s s t ha n
t ha t of l y s i ne r e s i due s . V a r i ous t hi ol - r e a c t i ve P E G de r i va t i ve s , s uc h a s m a l e i m i de , vi ny l s ul f one ,
i odoa c e t a m i de a nd or t hopy r i dy l di s ul f i de , ha ve be e n us e d f or c y s t e i ne c onj uga t i on t hr oug h
f or m a t i on of e i t he r a t hi oe t he r or a di s ul f i de l i nka g e ( R obe r t s e t a l ., 2002) . H ow e ve r , t he
unpa i r e d c y s t e i ne r e s i due i n a pr ot e i n i s us ua l l y bur i e d i n hi dde n hy d r ophobi c poc ke t s of t he
t e r t i a r y s t r uc t ur e t ha t a r e not e a s i l y a c c e s s i bl e t o hy d r ophi l i c P E G f or c onj ug a t i on unde r na t i ve
c ondi t i ons . F or pr ot e i ns t ha t do not ha ve a ny unpa i r e d or a c c e s s i bl e c y s t e i ne r e s i due , t he c y s t e i ne
r e s i due c a n be i nt r oduc e d a t t he de s i r e d P E G y l a t i on s i t e by m e a ns of s i t e - di r e c t e d m ut a g e ne s i s .
F or e x a m pl e , t he i m m unot oxi n a nt i - T a c ( F v) - P E 38 w a s P E G y l a t e d a t a g e ne t i c a l l y e ngi ne e r e d
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c y s t e i ne r e s i due i n t he pe pt i de c onne c t or C 3 ( A S G C G P E ) be t w e e n t he s c F v a nd t he t oxi n, w hi c h
r e s ul t e d i n a f i ve - t o e i g ht - f ol d i nc r e a s e i n pl a s m a ha l f - l i ve s i n m i c e a nd a t hr e e - t o f our - f ol d
i nc r e a s e i n a nt i t um or a c t i vi t y ( T s ut s um i e t a l ., 2000) . H ow e ve r , t he s hor t c om i ng of t hi s m e t hod i s
t ha t t he a ddi t i on of f r e e c y s t e i ne r e s i due s by g e ne t i c e ngi ne e r i ng i nc r e a s e s t he pos s i bi l i t y of
i nc or r e c t di s ul f i de f or m a t i on a nd pr ot e i n f or m a t i on.
S i t e - s pe c i f i c i nc or por a t i on of unna t ur a l a m i no a c i ds ( U A A s ) i nt o pr ot e i ns c a n pr ovi de
bi o- or t hogona l f unc t i ona l i t y f or P E G y l a t i on. A n e vol ve d E s c he r i c hi a C ol i ( E . c ol i ) t y r o s y l
t R N A / t R N A s y n t he t a s e pa i r ha s be e n g e ne r a t e d t ha t s e l e c t i ve l y i nc or por a t e s a n U A A i nt o t he
pr ot e i n i n y e a s t i n r e s pons e t o t he a m be r nons e ns e c odon, U A G . T hi s m e t hod w a s us e d t o c r e a t e
a pa r a - a z i dophe ny l a l a ni ne ( pA z F ) - c ont a i ni ng hum a n s upe r ox i de di s m ut a s e 1 ( S O D 1) w hi c h w a s
s ubs e que nt l y c onj uga t e d t o a n a l ky n e - be a r i ng P E G r e a ge nt vi a “ c l i c k ” r e a c t i on ( D e i t e r s e t a l .,
2004) . T he i nc or por a t i on e f f i c i e nc y w a s f ur t he r i nc r e a s e d by r e l e a s e f a c t or 1 ( R F 1 ) knoc kout i n
l i ve c e l l s w hi c h a l l ow e d r i bo s om a l i nc o r po r a t i on of U A A s a t m ul t i pl e s i t e s ( J ohns on e t a l ., 201 1 ) .
S i t e - s pe c i f i c P E G y l a t i on c a n g e ne r a t e c ont r ol l e d, w e l l - de f i ne d c onj ug a t e d pr oduc t s w i t h
i m pr ove d pr oduc t pr of i l e s ove r t hos e obt a i ne d t hr ough non- s pe c i f i c r a ndom c onj uga t i ons .
1.4 G e n e t i c I n c or p or at i on of U A A
C onve nt i ona l m ut a g e ne s i s i s l i m i t e d t o 20 na t ur a l a m i no a c i ds a s bui l di ng bl oc ks . T he
i nt r oduc t i on of U A A m ut a g e ne s i s ha s e x pa nde d t he r e pe r t oi r e of a m i no a c i ds t ha t c a n be
i nc or por a t e d i nt o pr ot e i ns c ons i de r a bl y . S i t e - s pe c i f i c i nc or por a t i on of U A A s ha s w i de - r a ngi ng
a ppl i c a t i ons i n t he s t r uc t ur e - f unc t i on s t udi e s of pr ot e i ns , i nc l udi ng s t udi e s of pr ot e i n s t a bi l i t y a nd
f ol di ng , pr ot e i n - l i g a nd a nd pr ot e i n - pr ot e i n i nt e r a c t i ons , a nd a l s o e na bl e s s e l e c t i ve m odi f i c a t i on
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of t he r a pe ut i c pr ot e i ns ( N e um a nn- S t a ubi t z a nd N e um a nn, 2016) . O ne of t he a ppr oa c he s t o
i nc or por a t i ng U A A s i s us i ng i n v i t r o or i n v i v o s t op - c odon s uppr e s s or t R N A t e c hnol ogy .
T he r i bos om e t r a ns l a t e s m R N A i nt o a pol y p e pt i de by c om pl e m e nt i ng t r i pl e t - c odons w i t h
m a t c hi ng a m i noa c y l a t e d t R N A s . T hr e e of t he 64 na t ur a l l y - oc c ur r i ng t r i pl e t c odons do not c ode
f or a n a m i no a c i d, but i ns t e a d r e s ul t i n t e r m i na t i on of t he t r a ns l a t i on pr oc e s s vi a t he r e c r ui t m e nt
of r e l e a s e f a c t or s ( F i g. 3A ) . T he y a r e na m e d a s oc hr e ( U A A ) , opa l ( U G A ) , a nd a m be r ( U A G ) .
The a m be r c odon ( U A G ) i s t he l e a s t f r e que nt l y us e d s t op c odon i n E . c ol i ( ~ 7% ) a nd r a r e l y
t e r m i na t e s e s s e nt i a l g e ne s ( N a ka m ur a e t a l ., 2000) . T he r e f or e , i t i s a n i de a l c a ndi da t e f or
r e a s s i g nm e nt vi a t he de s i g n of a s ui t a bl e s uppr e s s or t R N A . T he r e a s s i gnm e nt l e a ds t o a n U A A
i nc or por a t i on s y s t e m t ha t i s i n c om pe t i t i on w i t h t r a ns l a t i ona l t e r m i na t i on by t he hos t m a c hi ne r y .
S c hul t z a n d c ol l e a gue s f i r s t us e d a c he m i c a l l y a c y l a t e d , U A G - s pe c i f i c s uppr e s s or t R N A t o
s i t e - s pe c i f i c a l l y i nc or por a t e U A A s i nt o t he e nz y m e β - l a c t a m a s e i n a c e l l f r e e s y s t e m ( N or e n e t
a l ., 1989) . T o i m pr ove t he e f f i c i e nc y , t he y de ve l ope d a n e vol ve d s uppr e s s or
t R N A / a m i noa c y l - t R N A s y n t he t a s e ( t R N A / a a R S ) pa i r de r i ve d f r om t he a r c ha e ba c t e r i a
M e t hanoc oc c us j annas c hi i ( M . j annas c hi i ) f or s i t e - s pe c i f i c i nc or por a t i on of U A A s i nt o pr ot e i ns
i n E . c ol i e x pr e s s i on s y s t e m s ( S a nt or o e t a l ., 2003) . T he M . j annas c hi i t R N A / a a R S pa i r nor m a l l y
e nc ode s f or a t y r os i ne a nd t hus i s or t hog ona l t o E . c ol i e ndoge nous t R N A / s y n t he t a s e pa i r s . T he
m i ni m a l a nt i c odon r e c ogni t i on of t he a a R S t o i t s t R N A m a ke s i t pos s i bl e t o m ut a t e t he a m i no
a c i d bi ndi ng poc ke t f r om t y r o s i ne t o a n U A A w i t h l i t t l e l os s of a f f i ni t y a nd a m i noa c y l a t i on
e f f i c i e nc y of t he a a R S f or t he t R N A ( W a l s a nd O va a , 2014) . I n r e s pons e t o a n a m be r c odon
dur i ng t r a ns l a t i on, t he U A A i s a m i noa c y l a t e d t o t he c om pl e m e nt a r y s uppr e s s or t R N A by a n
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or t hogona l a a R S , a nd t he a m i noa c y l - t R N A , a l ong w i t h s om e e l onga t i on f a c t or s , de l i ve r s t he
U A A t o t he r i bos om e f or i nc or por a t i on i nt o t he pol y p e pt i de c ha i n t ha t i s be i ng pr oduc e d ( F i g.
3B ) . T he t r a ns l a t i on m i g ht a l s o be t e r m i na t e d by t he r e c r ui t m e nt of R F 1, r e s ul t i ng i n t he
g e ne r a t i on of a t r unc a t e d pr ot e i n. T he f ul l - l e ng t h pr ot e i n c a n be s e pa r a t e d a nd s e l e c t i ve l y
m odi f i e d vi a c he m os e l e c t i ve m odi f i c a t i on .
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F i gu r e 3. G e n e t i c i n c or p or at i on of U A A s i n t o p r ot e i n s al l ow s s e l e c t i ve m od i f i c at i on ( W a l s
a nd O va a , 2014) . ( A ) N or m a l t r a ns l a t i on i s t e r m i na t e d by t he r e c r ui t m e nt of r e l e a s e f a c t or s . I n
t he c a s e of a n a m be r c odon, R F 1 i s r e c r ui t e d a nd t e r m i na t e s t he t r a ns l a t i on of t he pol y p e pt i de ,
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r e l e a s i ng t he ne w l y s y n t he s i z e d pr ot e i n. ( B ) A m be r c odon s uppr e s s i on t o i nc or por a t e U A A s
m a ke s us e of t he a m be r c odon a s a c odi ng c odon i n t r a ns l a t i on. T he c om pl e m e nt a r y a m be r t R N A
i s a m i noa c y l a t e d by a n or t hogona l a a R S t ha t i s s pe c i f i c a l l y de s i gne d t o a c c e pt onl y U A A s . T hi s
r e s ul t s i n a pr ot e i n t ha t c ont a i ns a n U A A a nd c a n be s e l e c t i ve l y m odi f i e d vi a c he m os e l e c t i ve
m odi f i c a t i on.
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1.5 R e s e ar c h O b j e c t i ve s
α F A P - P E 38 i s a pot e nt F A P - t a r g e t i ng i m m unot oxi n w i t h pot e nt i a l f or c a nc e r t he r a py .
H ow e ve r , i t s s hor t ha l f - l i f e i n v i v o c r i t i c a l l y l i m i t s t he t he r a pe ut i c e f f i c a c y . P E G y l a t i on a s a t ool
t o i m pr ove P K pr ope r t i e s of t he r a pe ut i c pr ot e i ns i s w e l l e s t a bl i s he d a nd ha s be e n w i de l y us e d.
T he ove r a l l g oa l of m y pr oj e c t i s t o c onj uga t e α F A P - P E 38 t o P E G f or ha l f - l i f e e x t e ns i on. T w o
s t r a t e gi e s a r e us e d f or pr ot e i n m odi f i c a t i on: ( 1) P E G y l a t i on a t a c y s t e i ne r e s i due i n t he C 3
c onne c t or , a nd ( 2) s i t e - s pe c i f i c P E G y l a t i on a t a n U A A r e s i due i n t he C D 8h c onne c t or .
The pr oj e c t ha s t he f ol l ow i ng obj e c t i v e s :
1. T o g e ne r a t e α F A P - P E 38 va r i a nt s c ont a i ni ng a f r e e c y s t e i ne r e s i due or a n U A A r e s i due i n
t he pe pt i de c onne c t or be t w e e n t he s c F v a nd t he t oxi n.
2. T o c onj ug a t e t he α F A P - P E 38 va r i a nt s t o P E G .
3. T o opt i m i z e t he P E G y l a t i on r e a c t i ons f or i nc r e a s i ng t he y i e l d of t he P E G y l a t e d pr oduc t s .
4. T o i nve s t i g a t e t he e f f e c t s of P E G y l a t i on on t he bi ndi ng a nd c y t ot ox i c a c t i vi t i e s of t he
i m m unot oxi ns .
1 7
2 M a t e r i a l s a n d M e t h o d s
2.1 R e age n t s
C he m i c a l s w e r e pur c ha s e d f r om S i g m a - A l dr i c h a nd V W R unl e s s ot he r w i s e s t a t e d . E nz y m e s
w e r e pur c ha s e d f r om N e w E ngl a nd B i ol a bs a nd L i f e T e c hnol og i e s .
2.2 C e l l l i n e s
T he 293T a nd 293T - hF A P c e l l s us e d i n t hi s s t udy w e r e obt a i ne d f r om our c ol l a bor a t or , D r .
P i n W a ng . T he 293T - hF A P c e l l l i ne w a s pr e vi ous l y g e ne r a t e d by D r . W a ng a nd c ol l e a gue s us i ng
s t a bl e t r a ns duc t i on of 293T c e l l s w i t h l e nt i vi r us ps e udot y p e d w i t h ve s i c ul a r s t om a t i t i s vi r us
g l y c o pr ot e i n ( Y a ng e t a l ., 2008) . C e l l s w e r e c ul t ur e d i n D ul be c c o ’ s M odi f i e d E a gl e M e di um
( D M E M ) s uppl e m e nt e d w i t h 10% f e t a l bovi ne s e r um ( F B S ) a nd 1x pe ni c i l l i n/ s t r e pt om y c i n/
gl ut a m i ne .
2.3 B ac t e r i al S t r ai n s an d P l as m i d s
E. c ol i D H 5 α c om pe t e nt c e l l s f r om L i f e T e c hnol og i e s w e r e us e d f or pr opa ga t i on of pl a s m i ds .
E . c ol i B L 2 1( D E 3) c om pe t e nt c e l l s f r om N e w E ngl a nd B i ol a bs w e r e us e d a s a hos t f or
e x pr e s s i on of r e c om bi na nt i m m unot oxi ns .
T he pl a s m i d pE T 28a - α F A P - C D 8h- P E 38 - H i s 6 ( F i g . 4A ) e nc odi ng α F A P - C D 8h- P E 38 w a s
obt a i ne d f r om D r . W a ng . T he pl a s m i d pE T 28a - α F A P - C 3- P E 38 - H i s 6 w a s c ons t r uc t e d by
r e pl a c i ng t he C D 8h s e que nc e i n pE T 28a - α F A P - C D 8h- P E 38 - H i s 6 w i t h t he s e que nc e e nc odi ng a
pe pt i de c onne c t or , A S G G P E . P l a s m i ds pE V O L ( F i g. 4B ) , pB K - A c F R S a nd pB K - A z F R S w e r e
s t or e d i n ou r l a b o r a t o r y .
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F i gu r e 4. P l as m i d m ap s of e xp r e s s i on ve c t or s . ( A ) pE T 28a - α F A P - C D 8h- P E 38 - H i s 6 m a p. ( B )
pE V O L - a a R S m a p ( Y o ung e t a l ., 2010) .
1 9
2.4 S i t e - D i r e c t e d M u t age n e s i s
I n or de r t o s i t e - s pe c i f i c a l l y i nc or por a t e a pa r a - a c e t y l phe ny l a l a ni ne ( pA c F ) i nt o α F A P - P E 38 ,
t he c odon of C 29 ( U G C ) i n C D 8h ( F i g. 5A ) w a s m ut a t e d i nt o a n a m be r c odon ( U A G ) . T he
pl a s m i d pE T 28a - α F A P - C D 8h- P E 38 - H i s 6 w a s s ubj e c t e d t o s i t e - di r e c t e d m ut a g e ne s i s us i ng a s e t
of m ut a g e ni c pr i m e r s l i s t e d i n T a bl e 1 a nd t he Q ui kc ha nge s i t e - di r e c t e d m ut a g e ne s i s ki t ( A gi l e nt
T e c hnol og i e s ) , a c c or di ng t o m a nuf a c t ur e r ’ s i ns t r uc t i ons . T he P C R pr oduc t s w e r e t r e a t e d
w i t h D pn I r e s t r i c t i on e nz y m e a t 37 ° C f or 1 h t o di g e s t t he pa r e nt a l t e m pl a t e s . T he r e m a i ni ng
m ut a t e d pl a s m i ds w e r e t r a ns f or m e d i nt o β - m e r c a pt oe t ha nol - t r e a t e d X L 1 - B l u e s upe r c om pe t e nt
c e l l s ( A gi l e nt T e c hnol og i e s ) . T he t r a ns f or m a t i on r e a c t i on w a s i nc uba t e d on i c e f or 30 m i n, he a t
s hoc k e d a t 42 ° C f or 45 s a nd t he n i nc uba t e d on i c e f or 2 m i n . A f t e r t he a ddi t i on of 900 μ L S O C
m e di um , t he t r a ns f or m e d c e l l s w e r e i nc uba t e d a t 37 ° C f or 1 h w i t h s ha ki n g a t 230 r pm . T he
t r a ns f or m a t i on m i xt ur e w a s pl a t e d on a L ur i a br ot h ( L B ) a ga r pl a t e c ont a i ni ng ka na m y c i n f or
s e l e c t i on a nd i nc uba t e d a t 37 ° C ove r ni ght . S i ng l e c ol oni e s w e r e s e l e c t e d , f ol l ow e d by D N A
s e que nc i ng a na l y s i s ( G E N E W I Z ) t o ve r i f y t h a t t he s e l e c t e d c l one s c ont a i ne d t he de s i r e d
m ut a t i on.
T he i ns e r t i on of a c y s t e i ne or a pA z F i nt o t he C 3 c onne c t or ( F i g. 5B ) f ol l ow e d t he s a m e
s i t e - di r e c t e d m ut a g e ne s i s pr ot oc ol a nd w a s ve r i f i e d by D N A s e que nc i ng a na l y s i s .
2 0
F i gu r e 5. D N A an d am i n o ac i d s e q u e n c e s of t h e p e p t i d e c on n e c t or s . ( A ) T he U G C c odon of
C 29 i n C D 8h w a s m ut a t e d t o a n a m be r c odon, U A G . ( B ) T he i ns e r t i on s i t e s f or i nc or por a t i ng a
c y s t e i ne or a pA z F i nt o t he C 3 c onne c t or .
2 1
T a b l e 1. P r i m e r s f or s i t e - d i r e c t e d m u t age n e s i s of t h r e e c h os e n s i t e s
F : f or w a r d pr i m e r ; R : r e ve r s e pr i m e r . U nde r l i ne d l e t t e r s a r e m ut a t e d ba s e s i n s i t e - di r e c t e d
m ut a g e ne s i s pr i m e r s .
2 2
2.5 C on s t r u c t i on of E xp r e s s i on V e c t or s of aaR S G e n e s
T he pA c F R S g e ne c a r r y i ng B gl I I a nd Sal I r e s t r i c t i on s i t e s w a s a m pl i f i e d f r om t he pB K
pl a s m i d by P C R us i ng P f x D N A pol y m e r a s e . T he P C R pr oduc t s w e r e pur i f i e d us i ng t he
Z y m oc l e a n ™ G e l D N A R e c ove r y K i t ( G e ne s e e S c i e nt i f i c ) , f ol l ow e d by doubl e di g e s t i on of t he
P C R pr oduc t s a nd t he pE V O L ve c t or w i t h B gl I I a nd Sal I - H F r e s t r i c t i on e nz y m e s a t 37 ° C f or a t
l e a s t 4 h. T he di g e s t e d P C R pr oduc t s w e r e pur i f i e d us i ng a P C R P ur i f i c a t i on ki t ( L i f e
T e c hnol og i e s ) , a nd t he ve c t or ba c kbone s w e r e pur i f i e d us i ng t he Z y m oc l e a n ™ G e l D N A
R e c ove r y K i t . A f t e r w a r ds , t he pur i f i e d P C R pr oduc t s a nd ve c t or ba c kbone s w e r e l i g a t e d w i t h T 4
l i g a s e a t 16 ° C f or 2 h. E . c ol i D H 5 α c e l l s w e r e t r a ns f or m e d w i t h t he l i g a t i on pr oduc t s by he a t
s hoc k a t 42 ° C f or 22 s , a nd t he n g r ow n on a L B a ga r pl a t e w i t h 30 μ g / m L c hl or a m phe ni c ol a t
37 ° C ove r ni ght . P l a s m i d D N A e x t r a c t e d f r om pos i t i ve c l one s w a s s ubj e c t e d t o D N A s e que nc i ng
a na l y s i s t o c onf i r m t ha t t he pA c F R S g e ne w a s i n s e r t e d ups t r e a m of t he c ons t i t ut i ve g l nS ′
pr om ot e r i n t o t he pE V O L ve c t or . T he s e c ond c opy of t he pA c F R S g e ne w a s i ns e r t e d ups t r e a m of
t he a r a bi nos e - i nduc i bl e a r a B A D pr om ot e r f ol l ow i ng t he s a m e pr oc e dur e s . I n s e r t i on of t w o
c opi e s of t hi s g e ne c a n he l p t o i nc r e a s e t he e x pr e s s i on y i e l d of pA c F R S . The c ons t r uc t e d
pE V O L - pA c F R S I - pA c F R S I I - t R N A ve c t or w a s ve r i f i e d by D N A s e que nc i ng a na l y s i s .
S i m i l a r l y , t he pE V O L - pA z F R S I - pA z F R S I I - t R N A ve c t or w a s g e ne r a t e d by s ubc l oni ng t w o
c opi e s of t he pA z F R S g e ne i nt o t he pE V O L ve c t or a nd ve r i f i e d by D N A s e que nc i ng a na l y s i s .
2.6 E xp r e s s i on an d P u r i f i c at i on of R e c om b i n an t I m m u n ot oxi n s
T o e x pr e s s r e c om bi na nt i m m unot oxi ns α F A P - C D 8h- P E 38 a nd α F A P - C 3( C y s ) - P E 38 , E . c ol i
B L 2 1( D E 3) c e l l s t r a ns f or m e d w i t h pE T 28a - α F A P - C D 8h- P E 38 - H i s 6 a nd pE T 28a - α F A P -
2 3
C 3( C y s ) - P E 38 - H i s 6 w e r e g r ow n a t 37 ° C i n L B m e di um c ont a i ni ng 0.05 m g / m L ka na m y c i n,
r e s pe c t i ve l y . W he n t he a bs or ba nc e of ba c t e r i a l c ul t ur e a t 600 nm ( O D 6 0 0 ) r e a c he d 0.6, i s opr opy l
β - D - 1- t hi o g a l a c t opy r a nos i de ( I P T G ) w a s a dde d t o a f i na l c onc e nt r a t i on of 1 m M t o i nduc e
pr ot e i n e x pr e s s i on. A f t e r e x pr e s s i on f or 4 h, c e l l s w e r e ha r ve s t e d by c e nt r i f uga t i on a t 4500 r pm
f or 30 m i n .
T o e x pr e s s U A A - i nc or por a t e d i m m unot oxi ns , pE T 28a - α F A P - C D 8h ( C 29pA c F ) - P E 38 - H i s 6 or
pE T 28a - α F A P - C 3( pA z F ) - P E 38 - H i s 6 w a s c ot r a ns f or m e d i nt o B L 2 1( D E 3) c om pe t e nt c e l l s w i t h
t he pE V O L pl a s m i d ha r bor i ng t he t R N A / a a R S pa i r s pe c i f i c f or t he de s i r e d U A A . T he
t r a ns f or m e d E . c ol i B L 2 1( D E 3) c e l l s w e r e g r ow n a t 37 ° C i n L B m e di um c ont a i ni ng 0.05
m g / m L ka na m y c i n, 0.03 m g / m L c hl or a m phe ni c ol a nd 1m M c or r e s pondi ng U A A . W he n
O D 6 0 0 r e a c he d 0.6, L - ( + ) - a r a bi nos e ( 0.2% ) a nd I P T G ( 1 m M ) w e r e a dde d t o i nduc e pr ot e i n
e x pr e s s i on . A f t e r e x pr e s s i on f or 4 h, c e l l s w e r e ha r ve s t e d by c e nt r i f uga t i on a t 4500 r pm f or 30
m i n . S i nc e t he a z i do g r oup i s l i g ht - s e ns i t i ve , l i g ht e xpos ur e s houl d be a voi de d w he n e x pr e s s i ng
α F A P - C 3( pA z F ) - P E 38 , a s w e l l a s dur i ng t he f ol l ow i ng ope r a t i ons .
C e l l pe l l e t f r om e a c h 1 L c ul t ur e w a s r e s us pe nde d i n 10 m L l y s i s buf f e r c ont a i ni ng 1 m M
phe ny l m e t hy l s ul f ony l f l uor i de ( P M S F ) , 1 m M di t hi ot hr e i t ol ( D T T ) , 10 m M M g C l 2 , 1 uni t
D N a s e I , 1 m g / m L l y s oz y m e , 0.001 m g / m L pe ps t a t i n A , a nd 0.001 m g / m L l e upe pt i n . C e l l
s us pe ns i on w a s i nc uba t e d on i c e f or 1 hour , f ol l ow e d by s oni c a t i on t o di s r upt t he c e l l s
c om pl e t e l y . A f t e r s oni c a t i on, t he i nc l us i on body pe l l e t w a s c ol l e c t e d by c e nt r i f uga t i on a t 13,000
r pm , 4 ° C f or 30 m i n. I n c l us i on bodi e s w e r e w a s he d t w i c e w i t h 25 m L of de t e r g e nt buf f e r ( 1%
s odi um de ox y c hol a t e , 1% N P - 40, 50 m M T r i s [ pH 8.0] , 200 m M N a C l , 1 m M E D T A , 1 m M
2 4
D T T ) , t hr e e t i m e s w i t h 25 m L of w a s h buf f e r 1 ( 0.5% T r i t on X - 100, 50 m M T r i s [ pH 8.0] , 100
m M N a C l , 0.1 % N a N 3 , 1 m M E D T A , 1 m M D T T ) , t w i c e w i t h 25 m L of w a s h buf f e r 2 ( 50 m M
T r i s [ pH 8.0] , 100 m M N a C l , 0.1% N a N 3 , 1 m M E D T A , 1 m M D T T ) , a nd onc e w i t h 20 m L of 2
M ur e a buf f e r ( 2 M ur e a , 10 m M T r i s , 100 m M N a H 2 P O 4 , pH 8.0) . A l l t he buf f e r s f or l y s i s a nd
w a s hi ng of t he α F A P - C 3( pA z F ) - P E 38 i nc l us i on bodi e s di d not c ont a i n D T T . A f t e r e a c h w a s h,
t he i nc l us i on body pe l l e t w a s c ol l e c t e d by c e nt r i f uga t i on a t 13,000 r pm , 4 ° C f or 10 m i n . T he
r e c om bi na nt pr ot e i n f r om t he w a s he d i nf us i on bodi e s w a s s ol ubi l i z e d w i t h 8 M ur e a buf f e r ( 8 M
ur e a , 10 m M T r i s , 100 m M N a H 2 P O 4 , pH 8.0) , f ol l ow e d by c e nt r i f uga t i on t o r e m ove i ns ol ubl e
m a t e r i a l s .
I n or de r t o r e m ove t he t r unc a t e d pr ot e i n, U A A - i nc or por a t e d r e c om bi na nt pr ot e i ns w e r e
f ur t he r pur i f i e d unde r de na t ur i ng c ondi t i ons by ni c ke l - ni t r i l ot r i a c e t i c a c i d ( N i - N T A ) a f f i ni t y
c hr om a t ogr a phy us i ng a 5 m L H i s T r a p ™ H P c ol um n ( G E H e a l t hc a r e ) . T he pr ot e i n s a m pl e w a s
l oa de d ont o t he c ol um n pr e - e qui l i br a t e d w i t h 5 c ol um n vol um e s of bi ndi ng buf f e r ( 8 M ur e a
buf f e r , pH 8.0) . F ol l ow i ng w a s hi ng w i t h 10 c ol um n vol um e s of w a s h buf f e r ( 8 M ur e a buf f e r , pH
6.3) , t he pr ot e i n w a s e l ut e d w i t h 5 c ol um n vol um e s of e l ut i on buf f e r 1 ( 8 M ur e a buf f e r , pH 5.9)
a nd 5 c ol um n vol um e s of e l ut i on buf f e r 2 ( 8 M ur e a buf f e r , pH 4.3) . E l ua t e s w e r e a na l y z e d by
S D S - P A G E us i ng t he B i s - T r i s g e l ( G e nS c r i pt ) , f ol l ow e d by s t a i ni ng w i t h G e l C ode B l ue S t a i n
R e a ge nt ( T he r m o F i s he r S c i e nt i f i c ) .
2.7 P r ot e i n R e f ol d i n g
T he pr ot e i n s ol ut i on ( 0.1 –1 m g / m L ) w a s t r a ns f e r r e d i nt o a 10 kD a m ol e c ul a r w e i ght c ut - of f
( M W C O ) S na ke S ki n di a l y s i s t ubi ng ( T he r m o F i s he r S c i e nt i f i c ) a nd di a l y z e d s e que nt i a l l y a ga i ns t
2 5
a s e r i e s of r e f ol di ng buf f e r ( 10 m M T r i s , 100 m M N a H 2 P O 4 , 2 m M g l ut a t hi one ( G S H ) , 0.2 m M
g l ut a t hi one di s ul f i de ( G S S G ) , pH 8.0) c ont a i ni ng de c r e a s i ng c onc e nt r a t i on s of ur e a ( 4, 2, 1 a nd 0
M ) a t 4 ° C . F or α F A P - C 3( pA z F ) - P E 38 r e f ol di ng, G S H a nd G S S G w e r e not a dde d e x c e pt i n t he
r e f ol di ng buf f e r c ont a i ni ng 1 M ur e a .
2.8 P E G yl at i on
P E G y l a t i on w a s c onduc t e d by c onj ug a t i on of P E G t o t he e ngi ne e r e d r e s i due of α F A P - P E 38
va r i a nt s . P E G y l a t i on r e a c t i ons w e r e a na l y z e d by S D S - P A G E a nd t he r e a c t i on m i xt ur e s w e r e
s ubj e c t e d t o s i z e e x c l us i on c hr om a t ogr a phy ( S E C ) t o s e pa r a t e t he P E G y l a t e d s pe c i e s .
2.8.1 P E G yl at i on at t h e C ys t e i n e R e s i d u e
A f t e r r e f ol di ng of α F A P - C 3( C y s ) - P E 38 , t he pr ot e i n s ol ut i on w a s c onc e nt r a t e d us i ng a 10
kD a M W C O A m i c on U l t r a c onc e nt r a t or ( M i l l i por e ) .T he r e f ol de d α F A P - C 3( C y s ) - P E 38 ( 50 μ M )
w a s i nc uba t e d w i t h 1 m M t r i s ( 2- c a r box y e t hy l ) phos phi ne ( T C E P ) i n P E G y l a t i on buf f e r ( 20 m M
s odi um phos pha t e , 50 m M N a C l , 10 m M E D T A , pH 7.0) f or 90 m i n a t r oom t e m pe r a t ur e w i t h
g e nt l e s ha ki ng . A f t e r r e m ovi ng T C E P , t he r e duc e d α F A P - C 3( C y s ) - P E 38 ( 50 μ M ) w a s i nc uba t e d
w i t h a 20- f ol d m ol a r e x c e s s of 10 a nd 20 kD a m P E G - m a l e i m i de ( B i oc he m pe g ) , r e s pe c t i ve l y , i n
P E G y l a t i on buf f e r ove r ni ght a t r oom t e m pe r a t ur e w i t h g e nt l e s ha ki ng .
2.8.2 P E G yl at i on at t h e p A c F R e s i d u e
A f t e r r e f ol di ng of α F A P - C D 8h ( C 29pA c F ) - P E 38 , t he pr ot e i n s ol ut i on w a s c onc e nt r a t e d us i ng
a 10 kD a M W C O A m i c on U l t r a c onc e nt r a t or . T he r e f ol de d α F A P - C D 8h ( C 29pA c F ) - P E 38 ( 50
μ M ) w a s i nc uba t e d w i t h a 20- f ol d m ol a r e x c e s s of 30 kD a P E G - a m i noox y ( S unbr i g ht ) a nd 10
m M m e t a - phe ny l e ne di a m i ne ( m P D A ) i n 0.1 M s odi um a c e t a t e ( pH 4.2) a t r oom t e m pe r a t ur e
2 6
w i t h g e nt l e s ha ki ng f or 24 h.
I n a n a t t e m pt t o i nc r e a s e t he P E G y l a t i on y i e l d, P E G y l a t i on of α F A P - C D 8h ( C 29pA c F ) - P E 38
w a s a l s o c a r r i e d out unde r de na t ur i ng c ondi t i ons i n 8 M ur e a buf f e r a t pH 2.5. A f t e r i nc uba t i on a t
37 ° C w i t h g e nt l e s ha ki ng f or 40 h, t he r e a c t i on m i xt ur e w a s di l ut e d a nd t he n s ubj e c t e d t o
N i - N T A a f f i ni t y c hr om a t ogr a phy t o r e m ove e x c e s s P E G - a m i nooxy . T he di l ut e d m i xt ur e w a s
l oa de d ont o a H i s T r a p ™ H P c ol um n pr e - e qui l i br a t e d w i t h 5 c ol um n vol um e s of 8 M ur e a buf f e r
( pH 8.0) , a nd t he n w a s w a s he d w i t h 10 c ol um n vol um e s of t he s a m e buf f e r , f ol l ow e d by e l ut i on
w i t h 5 c ol um n vol um e s of 8 M ur e a buf f e r c ont a i ni ng 300 m M i m i da z ol e ( pH 8.0) . T he pr ot e i n
w a s r e f ol de d us i ng t he pr ot oc ol de s c r i be d a bove .
2.8.3 P E G yl at i on at t h e p A z F R e s i d u e
A f t e r r e f ol di ng of α F A P - C 3( pA z F ) - P E 38 , t he pr ot e i n s ol ut i on w a s c onc e nt r a t e d us i ng a 10
kD a M W C O A m i c on U l t r a c onc e nt r a t or . T he r e f ol de d α F A P - C 3( pA z F ) - P E 38 ( 50 μ M ) w a s
i nc uba t e d w i t h a f i ve - f ol d m ol a r e xc e s s of 10 kD a m P E G - a l ky n e ( A dva nc e d B i oc he m i c a l s ) , 500
μ M C uS O 4, 2.5 m M 2- [ 4- { ( bi s [ ( 1 - t e r t - but y l - 1H - 1,2,3- t r i a z ol - 4- y l ) m e t hy l ] a m i no) m e t hy l } - 1H -
1,2,3- t r i a z ol - 1- y l ] a c e t i c a c i d ( B T T A A ) a nd 5 m M s odi um a s c or ba t e i n 0.1 M s odi um phos pha t e
buf f e r ( pH 7.0) ove r ni ght a t r oom t e m pe r a t ur e w i t h g e nt l e s ha ki ng .
2.9 S E C A n al ys i s
S E C w a s pe r f or m e d us i ng a S upe r de x 200 I n c r e a s e 10/ 300 G L c ol um n ( G E H e a l t hc a r e )
f i t t e d t o t he Ä K T A P r ot e i n P ur i f i c a t i on S y s t e m ( G E H e a l t hc a r e ) . T he c ol um n w a s e qui l i br a t e d
w i t h P B S a t 4 ° C . T he pr ot e i n s a m pl e w a s a ppl i e d a nd t he n e l ut e d f r om t he c ol um n w i t h P B S a t
a f l ow r a t e of 0.5 m L / m i n. F r a c t i ons s how i ng U V a bs or pt i on a t 280 nm w e r e c ol l e c t e d a nd
2 7
a na l y z e d by S D S - P A G E .
2.10 F A C S - B as e d C e l l S u r f ac e S t ai n i n g A s s ay
C y a n i ne 5.5 N H S e s t e r ( C y 5 .5, L u m i pr obe ) w a s us e d f or t he l a be l i ng of pr ot e i ns . T he
pur i f i e d pr ot e i n w a s i nc uba t e d w i t h a n e i ght - f ol d m ol a r e x c e s s of C y 5 .5 i n 0.1 M N a H C O 3
s ol ut i on ( pH 8.3) a t 4 ° C i n t he da r k. A f t e r ove r ni ght i nc uba t i on, 1 μ L of 1 M T r i s ( pH 8.0) w a s
a dde d t o que nc h t he r e a c t i on. T he C y 5 .5- c onj uga t e d pr ot e i n w a s pur i f i e d a nd e x c ha ng e d i nt o
P B S us i ng Z e ba S pi n D e s a l t i ng C ol um ns ( T he r m o F i s he r S c i e nt i f i c ) . C e l l s w e r e ha r ve s t e d,
w a s he d t hr e e t i m e s w i t h i c e - c ol d F A C S buf f e r ( P B S c ont a i ni ng 2% F B S , 0.05% N a N 3 a nd 2 m M
E D T A ) , a nd t he n i nc uba t e d w i t h 80 nM C y 5 .5- c onj ug a t e d pr ot e i n i n F A C S buf f e r f or 30 m i n on
i c e . A f t e r s t a i ni ng , c e l l s w e r e w a s he d t hr e e t i m e s w i t h i c e - c ol d F A C S buf f e r t o r e m ove unbound
pr ot e i n . S a m pl e s w e r e a na l y z e d on B D L S R I I f l ow c y t om e t e r us i ng F A C S D i va s of t w a r e ( B D
B i os c i e nc e s ) . F l ow c y t om e t r y da t a w e r e a na l y z e d w i t h F l ow J o s of t w a r e ( T r e e S t a r ) .
2.1 1 C ol or i m e t r i c X T T C e l l V i ab i l i t y A s s ay
2,3- bi s ( 2- m e t hox y - 4- ni t r o- 5- s ul f ophe ny l ) - 2 H - t e t r a z ol i um - 5- c a r box a ni l i de ( X T T ) i s a
t e t r a z ol i um de r i va t i ve t ha t c a n be r e duc e d t o a w a t e r - s ol ubl e , or a nge c ol or e d f or m a z a n pr oduc t
by m i t oc hondr i a l de hy d r oge na s e i n l i vi ng c e l l s . T he c y t ot ox i c a c t i vi t y of unP E G y l a t e d a nd
P E G y l a t e d i m m unot oxi ns a ga i ns t 293T - hF A P c e l l s w a s m e a s ur e d us i ng a X T T c e l l vi a bi l i t y
a s s a y ki t ( B i ot i um ) a c c or di ng t o m a nuf a c t ur e r ’ s i ns t r uc t i ons . B r i e f l y , c e l l s w e r e pl a t e d i n
t r i pl i c a t e s ont o a 96- w e l l t i s s ue c ul t ur e pl a t e a t a de ns i t y of 1  10
5
c e l l s pe r w e l l . O n t he ne x t
da y , s e r i a l di l ut i ons of t he pr ot e i n w e r e a dde d t o c e l l s a t va r i ous c onc e nt r a t i ons r a ngi ng f r om 1 
10
- 6
t o 2  10
- 3
m g / m L i n a f i na l vol um e of 200 μ L . C e l l s w e r e i nc uba t e d unde r s t a nda r d
2 8
c ondi t i ons f or 48 h. A f t e r t r e a t m e nt , 50 μ L of t he a c t i va t e d X T T s ol ut i on ( a c t i va t i on
r e a ge nt / X T T s ol ut i on = 1: 200, v/ v) w a s a dde d t o e a c h w e l l a nd t he pl a t e w a s i nc uba t e d f or a n
a ddi t i ona l 4 h. C e l l vi a bi l i t y w a s de t e r m i ne d by c ol or i m e t r i c a na l y s i s of t he r e duc t i on of X T T t o
f or m a z a n a t 450 nm a bs or ba nc e w a ve l e ngt h a nd 630 nm r e f e r e nc e w a ve l e ngt h us i ng t he E poc h 2
m i c r opl a t e s pe c t r ophot om e t e r ( B i oT e k) . T he r e qui r e d pr ot e i n c onc e nt r a t i on t o a c hi e ve a 50%
r e duc t i on of c e l l vi a bi l i t y r e l a t i ve t o unt r e a t e d c e l l s ( I C 5 0 ) w a s c a l c ul a t e d us i ng G r a phP a d P r i s m
( G r a phP a d S of t w a r e ) .
2 9
3 P E G y l a t i o n o f α F A P - P E 3 8 a t t h e C y s t e i n e R e s i d u e
3.1 P r ot e i n E xp r e s s i on , R e f ol d i n g an d P u r i f i c at i on
B ot h α F A P - C D 8h- P E 38 a nd α F A P - C 3( C y s ) - P E 38 w e r e e x pr e s s e d a s i nc l us i on bodi e s i n E .
c ol i B L 2 1( D E 3) c e l l s . T he i nc l us i on bodi e s w e r e i s ol a t e d, de na t ur e d, a nd i n v i t r o r e f ol de d. T he
pr ot e i ns w e r e f i r s t pur i f i e d by N i –N T A a f f i ni t y c hr om a t ogr a phy , a nd t he e l ut e d f r a c t i on w a s
a na l y z e d by S D S - P A G E . T he r e s ul t s how e d a m a j or ba nd a t 70 kD a , c ons i s t e nt w i t h t he e x pe c t e d
m ol e c ul a r w e i ght of t he e x pr e s s e d pr ot e i n ( F i g. 6A a nd 6B ) . H ow e ve r , t he r e w e r e a l s o a ddi t i ona l
ba nds w i t h hi g he r m ol e c ul a r w e i ght , l i ke l y due t o t he f or m a t i on of di m e r s a nd a ggr e ga t e s . T he
pr ot e i ns w e r e t he n f ur t he r pur i f i e d by S E C ( F i g. 6C a nd 6D ) . S D S - P A G E a na l y s i s s how e d t ha t
t he e l ut e d f r a c t i ons of P e a k 3 w e r e t he de s i r e d m onom e r i c pr ot e i ns ( F i g. 6E a nd 6F ) . A f t e r
c onc e nt r a t i on, a ppr ox i m a t e l y 3 m g pr ot e i n w a s obt a i ne d pe r l i t e r of E . c ol i i n L B br ot h.
3 0
F i gu r e 6. P u r i f i c at i on of α F A P - C D 8h - P E 38 an d α F A P - C 3( C ys ) - P E 38 . α F A P - C D 8h- P E 38
a nd α F A P - C 3( C y s ) - P E 38 w e r e pur i f i e d by N i - N T A a f f i ni t y c hr om a t ogr a phy ( A a nd B ) a nd S E C
( C a nd D ) . S D S - P A G E a na l y s i s ( E a nd F ) s how e d t ha t t he e l ut e d f r a c t i ons of P e a k 3 w e r e t he
de s i r e d m onom e r i c pr ot e i ns . B C : be f or e c ol um n; F T : f l ow - t hr ough; W : w a s h; E : e l ut i on.
3 1
3.2 P E G yl at i on of R e f ol d e d α F A P - C 3( C ys ) - P E 38
The f r e e t hi ol g r oup of t he c y s t e i ne r e s i due i n t he l i nke r r e gi on of t he pr ot e i n i s us ua l l y
c y s t e i ny l a t e d dur i ng c e l l di s r upt i on ( J e vs e va r e t a l ., 2012) . I n or de r t o r e a c t i va t e t he t hi ol g r oup,
t he r e f ol de d α F A P - C 3( C y s ) - P E 38 w a s r e duc e d by T C E P a nd t he n r e a c t e d w i t h m P E G - m a l e i m i de
r e a ge nt s a s de s c r i be d i n S e c t i on 2.8.1. T he m ono- P E G y l a t e d pr ot e i n w a s g e ne r a t e d w i t h a y i e l d
of a bout 27% . H ow e ve r , m ul t i - P E G y l a t e d pr oduc t s w e r e a l s o de t e c t e d ( F i g . 7) .
3 2
F i gu r e 7. P E G yl at i on of r e f ol d e d α F A P - C 3( C ys ) - P E 38 . T he r e f ol de d α F A P - C 3( C y s ) - P E 38
w a s r e duc e d by T C E P a nd t he n r e a c t e d w i t h m P E G - m a l e i m i de r e a ge nt s . T he m ono- P E G y l a t e d
α F A P - C 3( C y s ) - P E 38 w a s g e ne r a t e d, w hi l e m ul t i - P E G y l a t e d pr oduc t s w e r e a l s o de t e c t e d.
3 3
3.3 P u r i f i c at i on of P E G yl at e d α F A P - C 3( C ys ) - P E 38 b y S E C
A f t e r P E G y l a t i on, t he m ono- P E G y l a t e d α F A P - C 3( C y s ) - P E 38 w a s s e pa r a t e d f r om t he
r e a c t i on m i xt ur e by S E C . P E G c onj ug a t i on i nc r e a s e d t he m ol e c ul a r s i z e , t he r e by t he P E G y l a t e d
pr ot e i n w a s e l ut e d e a r l i e r t ha n t he unP E G y l a t e d pr ot e i n ( F i g. 8) . F or t he 10 a nd 20 kD a
P E G y l a t e d α F A P - C 3( C y s ) - P E 38 , f r a c t i ons 24- 25 a nd 21- 23 w e r e pool e d a nd c onc e nt r a t e d,
r e s pe c t i ve l y .
3 4
F i gu r e 8. P u r i f i c at i on of P E G yl at e d α F A P - C 3( C ys ) - P E 38 . A f t e r P E G y l a t i on, t he 10 a nd 20
kD a P E G y l a t e d α F A P - C 3( C y s ) - P E 38 w e r e s e pa r a t e d f r om t he r e a c t i on m i xt ur e s by S E C ( A a nd
B ) . S D S - P A G E a na l y s i s ( C a nd D ) i ndi c a t e d t ha t t he f r a c t i ons of P e a k 1 c ont a i ne d t he
m ono- P E G y l a t e d pr ot e i ns . F or t he 10 a nd 20 kD a P E G y l a t e d α F A P - C 3( C y s ) - P E 38 , f r a c t i ons
24- 25 a nd 21- 23 w e r e pool e d a nd c onc e nt r a t e d, r e s pe c t i ve l y .
3 5
3.4 B i n d i n g A c t i vi t y of P E G yl at e d α F A P - C 3( C ys ) - P E 38
P E G pol y m e r s m i g ht c a us e s t e r i c hi ndr a nc e w hi c h w i l l bl oc k t he bi ndi ng of i m m unot oxi ns t o
t he F A P m ol e c ul e s e x pr e s s e d on t he c e l l s ur f a c e . U s i ng F A C S - ba s e d c e l l s ur f a c e s t a i ni ng a s s a y ,
i n v i t r o bi ndi ng a na l y s i s of α F A P - C D 8h- P E 38 a nd α F A P - C 3( C y s ) - P E 38 s how e d t ha t t he y ha d
s pe c i f i c bi ndi ng a c t i vi t y t o 293T - hF A P c e l l s ( F i g. 9A a nd 9B ) . H o w e ve r , t he c onj uga t i on of
α F A P - C 3( C y s ) - P E 38 t o 10 a nd 20 kD a P E G r e s ul t e d i n a c om pl e t e l os s of F A P - s pe c i f i c bi ndi ng
a c t i vi t y ( F i g. 9C a nd 9D ) .
3 6
F i gu r e 9. B i n d i n g ac t i vi t y of P E G yl at e d α F A P - C 3( C ys ) - P E 38 . α F A P - C D 8h- P E 38 a nd
α F A P - C 3( C y s ) - P E 38 s how e d s pe c i f i c bi ndi ng a c t i vi t y t o 293T - hF A P c e l l s ( A a nd B ) . H o w e ve r ,
t he c onj ug a t i on of α F A P - C 3( C y s ) - P E 38 t o 10 a nd 20 kD a P E G r e s ul t e d i n a c om pl e t e l os s of
F A P - s pe c i f i c bi ndi ng a c t i vi t y ( C a nd D ) . W i de - t y p e : α F A P - C D 8h- P E 38 ; C y s : α F A P - C 3( C y s ) -
P E 38 ; 10 k- C y s : 10 kD a P E G y l a t e d α F A P - C 3( C y s ) - P E 38 ; 20 k- C y s : 20 kD a P E G y l a t e d α F A P -
C 3( C y s ) - P E 38 .
3 7
3.5 C yt ot oxi c A c t i vi t y of P E G yl at e d α F A P - C 3( C ys ) - P E 38
T he c y t ot ox i c a c t i vi t y of t he P E G y l a t e d i m m unot oxi n s a ga i ns t 293T a nd 293T - hF A P c e l l s
w a s m e a s ur e d by c ol or i m e t r i c X T T c e l l vi a bi l i t y a s s a y f or m e t a bol i c a c t i vi t y . T he unP E G y l a t e d
α F A P - C D 8h- P E 38 w a s a bl e t o i nhi bi t t he vi a bi l i t y of 293T - hF A P c e l l s i n a dos e - de pe nde nt
m a nne r w i t h t he I C 5 0 va l ue of 50 ng / m L . F or c om pa r i s on, no c y t ot oxi c e f f e c t w a s obs e r ve d i n
t he c ont r ol 293T c e l l s ( F i g. 10A ) . H ow e ve r , t he 10 a nd 20 kD a P E G y l a t e d α F A P - C 3( C y s ) - P E 38
ha d no e f f e c t on c e l l vi a bi l i t y , l i ke l y due t o t he l os s of bi ndi ng a f f i ni t y ( F i g. 10B a nd 10C ) .
3 8
F i gu r e 10. C yt ot oxi c ac t i vi t y of P E G yl at e d α F A P - C 3( C ys ) - P E 38 . T he unP E G y l a t e d
α F A P - C D 8h- P E 38 i nhi bi t e d t he vi a bi l i t y of 293T - hF A P c e l l s i n a dos e - de pe nde nt m a nne r w i t h
t he I C 5 0 va l ue of 50 ng / m L . F or c om pa r i s on, no c y t ot ox i c e f f e c t w a s obs e r ve d i n t he c ont r ol
293T c e l l s ( A ) . H ow e ve r , t he 10 a nd 20 kD a P E G y l a t e d α F A P - C 3( C y s ) - P E 38 ha d no e f f e c t on
c e l l vi a bi l i t y ( B a nd C ) .
3 9
4 S i t e - S p e c i f i c P E G y l a t i o n o f α F A P - P E 3 8 a t t h e U A A R e s i d u e
S i nc e α F A P - P E 38 c ont a i ns m ul t i pl e c y s t e i ne r e s i due s , i t i s pos s i bl e t ha t P E G y l a t i on ha s
oc c ur r e d a t va r i ous s i t e s . T o a ddr e s s t hi s i s s ue , w e a t t e m pt e d t o us e t he U A A i nc or por a t i on
t e c hni que t o i ns e r t a pA c F ( a ke t one - c ont a i ni ng a m i no a c i d) i nt o α F A P - P E 38 i n a s i t e - s pe c i f i c
m a nne r . T hi s w oul d a l l ow s ubs e que nt P E G y l a t i on vi a S c hi f f ba s e f or m a t i on be t w e e n pA c F a nd
a n a m i nooxy - d e r i ve d P E G r e a ge nt .
4.1 E xp r e s s i on an d P u r i f i c at i on of U A A - I n c o r p or at e d α F A P - P E 38 V a r i an t s as I n c l u s i on
B od i e s
B a s e d on t he c r y s t a l s t r uc t ur e of α F A P - C D 8h- P E 38 a nd pr e l i m i na r y s c r e e ni ng r e s ul t s , t he
C 29 r e s i due i n t he C D 8h c onne c t or i s l oc a t e d a t t he pr ot e i n s ur f a c e di s t a l t o t he a c t i ve s i t e , a nd
t he r e f or e w a s s e l e c t e d a s t he P E G y l a t i on s i t e . T he U G C c odon f or C 29 i n C D 8h w a s m ut a t e d t o
a n a m be r c odon, U A G . I n r e s pons e t o t he a m be r c odon dur i ng t r a ns l a t i on, a pA c F c a n be
i nc or por a t e d i n t o t he pr ot e i n a t t he s e l e c t e d s i t e us i ng t he t R N A / a a R S pa i r s pe c i f i c f or pA c F .
A not he r α F A P - P E 38 va r i a nt c ont a i ns a pA z F r e s i due a t t he s a m e pos i t i on i n t he C 3 c onne c t or
w he r e t he c y s t e i ne w a s i ns e r t e d. U A A - i nc or por a t e d α F A P - P E 38 va r i a nt s be a r i ng a C - t e r m i na l
H i s 6 t a g w e r e e x pr e s s e d a s i nc l us i on bodi e s i n E . c ol i B L 2 1( D E 3) c e l l s a nd t he n pur i f i e d unde r
de na t ur i ng c ondi t i ons by N i - N T A a f f i ni t y c hr om a t ogr a phy ( F i g. 1 1 ) . S D S - P A G E a na l y s i s
c onf i r m e d t ha t t he t r unc a t e d pr ot e i n ( 29 kD a ) w a s r e m ove d.
4 0
F i gu r e 1 1 . P u r i f i c at i on of U A A - i n c o r p or at e d α F A P - P E 38 var i an t s . U A A - i nc or por a t e d
α F A P - P E 38 va r i a nt s w e r e pur i f i e d unde r de na t ur i ng c ondi t i ons by N i - N T A a f f i ni t y
c hr om a t ogr a phy . S D S - P A G E a na l y s i s c onf i r m e d t ha t t he t r unc a t e d pr ot e i n ( 29 kD a ) w a s
r e m ove d.
4 1
4.2 P E G yl at i on of α F A P - P E 38 V a r i an t s
4.2.1 R e f ol d i n g an d P E G yl at i on of α F A P - C D 8h ( C 29p A c F ) - P E 38
T he pur i f i e d i nc l us i on bodi e s of α F A P - C D 8h ( C 29pA c F ) - P E 38 w e r e di s s ol ve d i n 8 M ur e a
buf f e r , a nd t he n di a l y z e d a ga i ns t a s e r i e s of r e f ol di ng buf f e r c ont a i ni ng de c r e a s i ng c onc e nt r a t i ons
of ur e a , a s de s c r i be d i n S e c t i on 2.7. N o pr e c i pi t a t e w a s obs e r ve d dur i ng t he r e f ol di ng pr oc e s s .
T he r e f ol de d pr ot e i n w a s c onc e nt r a t e d a nd t he n e x c ha ng e d i nt o 0.1 M s odi um a c e t a t e ( pH 4.2) .
P E G y l a t i on r e a c t i on c ondi t i ons w e r e e x t e ns i ve l y s t udi e d, s uc h a s pr ot e i n c onc e nt r a t i on, m ol a r
r a t i o of pr ot e i n t o P E G r e a ge nt , pH , t e m pe r a t ur e , a nd r e a c t i on t i m e . T he opt i m i z e d P E G y l a t i on
r e a c t i on w a s pe r f or m e d by i nc uba t i ng 50 μ M α F A P - C D 8h ( C 29pA c F ) - P E 38 w i t h a 20- f ol d m ol a r
e x c e s s of 30 kD a P E G - a m i noox y i n 0.1 M s odi um a c e t a t e ( pH 4.2) a t r oom t e m pe r a t ur e w i t h
g e nt l e s ha ki ng f or 24 h, w i t h t he a ddi t i on of 10 m M m P D A a s t he c a t a l y s t . D ur i ng t he
i nc uba t i on, how e ve r , a c ons i de r a bl e a m ount of pr ot e i n w a s pr e c i pi t a t e d. A ddi t i ona l l y ,
S D S - P A G E a na l y s i s of t he r e a c t i on m i xt ur e i ndi c a t e d t ha t a l a r g e por t i on of
α F A P - C D 8h ( C 29pA c F ) - P E 38 r e m a i ne d unr e a c t e d ( F i g. 12) . T he y i e l d of t he P E G y l a t e d pr oduc t
w a s onl y 1% ba s e d on t he s t a r t i ng a m ount of t he r e f ol de d α F A P - C D 8h ( C 29pA c F ) - P E 38. F ur t he r
opt i m i z a t i on w a s r e qui r e d by r e duc i ng pr ot e i n pr e c i pi t a t i on a nd/ or i nc r e a s i ng t he e f f i c i e nc y of
P E G y l a t i on .
4 2
F i gu r e 12. P E G y l at i on of r e f ol d e d α F A P - C D 8h ( C 29p A c F ) - P E 38 . U s i ng m P D A a s t he c a t a l y s t ,
t he r e f ol de d α F A P - C D 8h ( C 29pA c F ) - P E 38 w a s c onj uga t e d t o P E G - a m i noox y a t pH 4.2 vi a
oxi m e l i g a t i on. I n c r e a s i ng t he P E G c onc e nt r a t i on f r om a 10- f ol d m ol a r e x c e s s t o a 20- f ol d m ol a r
e x c e s s c a n i m pr ove t he y i e l d of t he P E G y l a t e d pr oduc t . H ow e ve r , a l a r g e por t i on of
α F A P - C D 8h ( C 29pA c F ) - P E 38 r e m a i ne d unr e a c t e d.
4 3
4.2.2 P E G yl at i on of R e f ol d e d α F A P - C 3( p A z F ) - P E 38
O xi m e l i g a t i on be t w e e n t he ke t one - c ont a i ni ng α F A P - C D 8h( C 29pA c F ) - P E 38 a nd t he
a m i nooxy f unc t i ona l i z e d P E G r e a ge nt r e qui r e d a c i di c pH , w hi c h m i g ht a c c ount f or t he s e ve r e
pr ot e i n pr e c i pi t a t i on obs e r ve d dur i ng t he i nc uba t i on pr oc e s s . T o a ddr e s s t hi s i s s ue , a pA z F ( a n
a z i de - c ont a i ni ng U A A ) w a s i nc or por a t e d i nt o t he s a m e s i t e i n t he C 3 c onne c t or w he r e t he
c y s t e i ne w a s i n s e r t e d, w hi c h w oul d a l l ow P E G y l a t i on a t ne ut r a l pH vi a “ c l i c k c he m i s t r y ” a nd
t he r e by he l p t o r e duc e pr ot e i n pr e c i pi t a t i on . U s i ng t hi s s t r a t e gy , a P E G y l a t i on r e a c t i on w a s
c a r r i e d out by i nc uba t i ng 50 μ M r e f ol de d α F A P - C 3( pA z F ) - P E 38 w i t h a f i ve - f ol d m ol a r e x c e s s of
10 kD a m P E G - a l ky n e , 500 μ M C uS O 4 , 2.5 m M B T T A A a nd 5 m M s odi um a s c or ba t e i n 0.1 M
s odi um phos pha t e buf f e r ( pH 7.0) ove r ni ght a t r oom t e m pe r a t ur e w i t h g e nt l e s ha ki ng . C u( I ) ,
g e ne r a t e d by t he r e duc t i on of C u( I I ) w i t h s odi um a s c or ba t e , a c t s a s t he c a t a l y s t , a nd B T T A A a s a
l i g a nd c a n a c c e l e r a t e t he c oppe r - c a t a l y z e d a z i de - a l ky n e c y c l oa ddi t i on ( C uA A C )
( B e s a nc e ne y - W e bl e r e t a l ., 201 1 ) . N o pr e c i pi t a t e s w e r e obs e r ve d dur i ng t he i nc uba t i on ; how e ve r ,
P E G y l a t i on vi a t he C uA A C r e a c t i on w a s uns uc c e s s f ul . A s s how n by S D S - P A G E a na l y s i s , t he
s t a r t i ng m a t e r i a l α F A P - C 3( pA z F ) - P E 38 w a s pa r t i a l l y de g r a de d , a nd no P E G y l a t e d pr ot e i n w a s
de t e c t e d ( F i g. 13) . A pos s i bl e r e a s on f or t he f a i l e d c onj uga t i on c oul d be t ha t pA z F w a s
hy d r ophobi c a nd t hus i t w a s not e x pos e d t o t he s ol ve nt .
4 4
F i gu r e 13. P E G yl at i on of r e f ol d e d α F A P - C 3( p A z F ) - P E 38 . C onj ug a t i on of t he r e f ol de d
α F A P - C 3( pA z F ) - P E 38 t o m P E G - a l ky n e vi a t he C uA A C r e a c t i on w a s uns uc c e s s f ul . A s s how n by
S D S - P A G E a na l y s i s , t he s t a r t i ng m a t e r i a l α F A P - C 3( pA z F ) - P E 38 w a s pa r t i a l l y de gr a de d , a nd no
P E G y l a t e d pr ot e i n w a s de t e c t e d .
4 5
4.2.3 P E G yl at i on of α F A P - C D 8h ( C 29p A c F ) - P E 38 u n d e r D e n at u r i n g C on d i t i on s
W e hy p ot he s i z e d t ha t P E G y l a t i on w oul d be m or e e f f i c i e nt unde r de na t ur i ng c ondi t i ons
be c a us e t he i nc or por a t e d U A A r e s i due ( pA c F a nd pA z F ) i n t he r e c om bi na nt pr ot e i n w oul d be
m or e e x pos e d t o t he s ol ve nt . T o t e s t t hi s , α F A P - C D 8h ( C 29pA c F ) - P E 38 ( 50 μ M ) w a s i nc uba t e d
w i t h a 20- f ol d m ol a r e x c e s s of 10 or 20 kD a P E G - a m i nooxy a nd 10 m M m P D A ( a s t he c a t a l y s t )
i n 8 M ur e a buf f e r a t pH 2.5, 37 ° C w i t h g e nt l e s ha ki ng f or 40 h. S D S - P A G E a na l y s i s of t he
r e a c t i on m i xt ur e s s how e d t ha t t he m a j or i t y of t he pr ot e i n w a s c onj uga t e d t o 10 or 20 kD a
P E G - a m i noox y ( F i g. 14) . A f t e r r e m ova l of e x c e s s P E G - a m i noox y by N i - N T A a f f i ni t y
c hr om a t ogr a phy , t he pr ot e i ns w e r e r e f ol de d by di a l y s i s , dur i ng w hi c h no pr e c i pi t a t e s w e r e
obs e r ve d. S i m i l a r l y , t he 30 kD a P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 w a s obt a i ne d a nd
r e f ol de d ( da t a not s how n) .
4 6
F i gu r e 14. P E G yl at i on of α F A P - C D 8h ( C 29p A c F ) - P E 38 u n d e r d e n at u r i n g c on d i t i on s . U s i ng
m P D A a s t he c a t a l y s t , α F A P - C D 8h ( C 29pA c F ) - P E 38 w a s c onj uga t e d t o P E G - a m i nooxy i n 8 M
ur e a buf f e r a t pH 2.5. S D S - P A G E a na l y s i s of t he 10 a nd 20 kD a P E G y l a t i on r e a c t i ons s how e d
t ha t t he P E G y l a t i on e f f i c i e nc y w a s s i g ni f i c a nt l y hi g he r unde r de na t ur i ng c ondi t i ons .
4 7
4.3 R e f ol d i n g an d P u r i f i c at i on of P E G yl at e d α F A P - C D 8h ( C 29p A c F ) - P E 38 b y S E C
T he 10, 20 a nd 30 kD a P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 w e r e a l l r e f ol de d us i ng t he
s a m e pr ot oc ol de s c r i be d pr e vi ous l y . T he pr ot e i ns w e r e t he n di a l y z e d a ga i ns t P B S , c onc e nt r a t e d,
a nd pur i f i e d by S E C . P e a k f r a c t i ons s how i ng U V a bs or pt i on a t 280 nm w e r e c ol l e c t e d a nd
a na l y z e d by S D S - P A G E .
F or t he 10 kD a P E G y l a t i on, t hr e e pe a ks w e r e c ol l e c t e d ( F i g. 15A ) . S A S - P A G E a na l y s i s
s how e d t ha t P e a k 1 ( t he m a j or pe a k) w a s m os t l y a g gr e ga t e s w i t h hi g h m ol e c ul a r w e i ght s , P e a k 2
w a s t he P E G y l a t e d pr oduc t , a nd P e a k 3 w a s t he unP E G y l a t e d s ubs t r a t e ( F i g. 15B ) . T he y i e l d of
P E G y l a t e d pr oduc t ( P e a k 2) w a s 10–12% , w hi c h w a s s i g ni f i c a nt l y e nha nc e d c om pa r e d t o t he
y i e l d obt a i ne d by P E G y l a t i on unde r na t i ve c ondi t i on s ( s e e S e c t i on 4.2.1) .
F or t he 20 a nd 30 kD a P E G y l a t i on, s i m i l a r r e s ul t s w e r e obs e r ve d; how e ve r , t he r e w a s
i nc r e a s i ng ove r l a p be t w e e n t he t w o pe a ks c or r e s pondi ng t o a ggr e ga t e s a nd t he de s i r e d
P E G y l a t e d pr ot e i n ( F i g. 15C –F ) . T he P e a k 2 f r a c t i ons , e s pe c i a l l y t hos e f r om t he 30 kD a
P E G y l a t i on r e a c t i on m i xt ur e , c oul d s t i l l c ont a i n a ggr e ga t e s a nd t hus r e qui r e f ur t he r pur i f i c a t i on .
4 8
F i gu r e 15. P u r i f i c at i on of P E G yl at e d α F A P - C D 8h ( C 29p A c F ) - P E 38 . A f t e r r e f ol di ng, t he
r e a c t i on m i xt ur e s w e r e s ubj e c t e d t o S E C ( A , C a nd E ) . S A S - P A G E a na l y s i s ( B , D a nd F )
s how e d t ha t P e a k 1 ( t he m a j or pe a k) w a s m os t l y a g gr e ga t e s w i t h hi g h m ol e c ul a r w e i ght s , P e a k 2
w a s t he P E G y l a t e d pr oduc t , a nd P e a k 3 w a s t he unP E G y l a t e d s ubs t r a t e . F or t he 10, 20 a nd 30
kD a P E G y l a t e d pr ot e i ns , t he P e a k 2 f r a c t i ons 25- 26, 23- 24 a nd 22- 24 w e r e pool e d a nd
c onc e nt r a t e d r e s pe c t i ve l y , w i t h y i e l ds of 10–12% .
4 9
4.4 B i n d i n g A c t i vi t y of P E G yl at e d α F A P - C D 8h ( C 29p A c F ) - P E 38
T he 10 kD a P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 ( F i g. 16B ) s how e d s pe c i f i c bi ndi ng
a c t i vi t y t o 293T - hF A P c e l l s , w hi c h w a s s i m i l a r t o t ha t of α F A P - C D 8h- P E 38 w i t hout P E G y l a t i on
( F i g. 16A ) . H ow e ve r , t he 20 or 30 kD a P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 di d not ha ve
F A P - s pe c i f i c bi ndi ng a c t i vi t y ( F i g. 16C a nd 16D ) . T he l os s of bi ndi ng a c t i vi t y c oul d be
a t t r i but e d t o t he g r e a t e r s t e r i c s hi e l di ng e f f e c t of l ong e r P E G c ha i ns .
5 0
F i gu r e 16. B i n d i n g ac t i vi t y of P E G yl at e d α F A P - C D 8h ( C 29p A c F ) - P E 38 . T he 10 kD a
P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 ( B ) s how e d s pe c i f i c bi ndi ng a c t i vi t y t o 293T - hF A P
c e l l s , w hi c h w a s s i m i l a r t o t ha t of α F A P - C D 8h- P E 38 w i t hout P E G y l a t i on ( A ) . H ow e ve r , t he 20
or 30 kD a P E G y l a t e d α F A P - C D 8 h( C 29pA c F ) - P E 38 di d not ha ve F A P - s pe c i f i c bi ndi ng a c t i vi t y
( C a nd D ) . 10 k- pA c F : 10 kD a P E G y l a t e d α F A P - C D 8h( C 29pA c F ) - P E 38 ; 20 k- pA c F : 20 kD a
P E G y l a t e d α F A P - C D 8h( C 29pA c F ) - P E 38 ; 30 k- pA c F : 30 kD a P E G y l a t e d
α F A P - C D 8h( C 29pA c F ) - P E 38 .
5 1
4.5 C yt ot oxi c A c t i vi t y of P E G yl at e d α F A P - C D 8h ( C 29p A c F ) - P E 38
B o t h α F A P - C D 8h- P E 38 ( F i g. 17A ) a nd t he 10 kD a P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) -
P E 38 ( F i g. 17B ) i nhi bi t e d c e l l vi a bi l i t y i n a dos e - de pe nde nt m a nne r i n 293T - hF A P c e l l s , w he r e a s
no c y t ot oxi c e f f e c t w a s obs e r ve d i n 293T c e l l s . T he I C 5 0 va l ue of 10 kD a P E G y l a t e d α F A P -
C D 8h ( C 29pA c F ) - P E 38 w a s 100 ng / m L , w hi c h w a s t w o- f ol d hi g he r t ha n t ha t of
α F A P - C D 8h- P E 38 ( 50 ng / m L ) . H ow e ve r , t he 20 a nd 30 kD a P E G y l a t e d
α F A P - C D 8h ( C 29pA c F ) - P E 38 ha d no c y t ot oxi c e f f e c t on e i t he r c e l l l i ne , c ons i s t e nt w i t h t he l os s
of bi ndi ng a f f i ni t i e s a s r e ve a l e d pr e vi ous l y ( F i g. 17C a nd 17D ) .
5 2
F i gu r e 17. C yt ot oxi c ac t i vi t y of P E G yl at e d α F A P - C D 8h ( C 29p A c F ) - P E 38 . B ot h α F A P - C D 8h-
P E 38 ( A ) a nd t he 10 kD a P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 ( B ) i nhi bi t e d t he vi a bi l i t y of
293T - hF A P c e l l s i n a dos e - de pe nde nt m a nne r , w he r e a s no c y t ot oxi c e f f e c t w a s obs e r ve d i n 293T
c e l l s . T he I C 5 0 va l ue of 10 kD a P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 w a s 100 ng / m L , w hi c h
w a s t w o- f ol d hi g he r t ha n t ha t of α F A P - C D 8h- P E 38 ( 50 ng / m L ) . H ow e ve r , t he 20 a nd 30 kD a
P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 ha d no c y t ot ox i c e f f e c t on e i t he r c e l l l i ne ( C a nd D ) .
5 3
5 D i s c u s s i o n
T he i nt r oduc t i on of t he “ m a gi c bul l e t ” c onc e pt by P a ul E hr l i c h ha s l e d t o t he s e a r c h f or
t he r a pe ut i c a ge nt s t ha t a l l ow s e l e c t i ve ki l l i ng of c a nc e r s w hi l e s pa r i ng nor m a l c e l l s ( S t r e bha r dt
a nd U l l r i c h, 2008) . A l a r g e va r i e t y of m A b s ha ve be e n i de nt i f i e d t ha t bi nd t o a nt i g e ns on c a nc e r
c e l l s or t he i r s uppor t i ng s t r om a , a nd t he r e a r e m a ny m e c ha ni s m s by w hi c h a nt i bodi e s c a n
m e di a t e c e l l ki l l i ng ( A da m s a nd W e i ne r , 2005) . D e s pi t e t he c l i ni c a l s uc c e s s of t he r a pe ut i c m A bs ,
na ke d a nt i bodi e s a r e r a r e l y pot e nt e nough by t he m s e l ve s , a nd t hus a r e us ua l l y a dm i ni s t e r e d i n
c om bi na t i on w i t h c he m ot he r a py ( D os i o e t a l ., 201 1 ) . I m m unot oxi ns r e pr e s e nt a c l a s s of
m A b- ba s e d a ge nt s t ha t e x pl oi t t he bi ndi ng s pe c i f i c i t y of m A b f or t a r g e t e d de l i ve r y of a ba c t e r i a l
or pl a nt t oxi n pa y l oa d t o e nha nc e a nt i t um or e f f e c t s . T he m a j or i t y of t he m e x e r t t he f unc t i on of
c e l l ki l l i ng by i nhi bi t i ng pr ot e i n s y n t he s i s , w hi c h i s t oxi c t o bot h di vi di ng a nd non- di vi di ng c e l l s
( A nt i g na ni a nd F i t z g e r a l d, 2013) .
A l t houg h i m m unot oxi ns ha ve de m ons t r a t e d e f f i c a c y i n s e ve r a l t y p e s of he m a t ol og i c
m a l i g na nc i e s ( F i t z G e r a l d e t a l ., 201 1 ) , t he s uc c e s s of t hi s t y p e of t r e a t m e nt ha s not y e t be e n
a c hi e ve d i n s ol i d t um or s . O ne of t he m a j or hur dl e s i s t he i r poor pe ne t r a t i on i nt o bul ky t um or
m a s s e s be c a us e of t he l a r g e m ol e c ul a r s i z e (  190 kD a ) . T hi s c a n be a ddr e s s e d by us i ng s c F v
i ns t e a d of f ul l - s i z e m A b t o c ons t r uc t t a r g e t i ng m oi e t i e s ( S ha n e t a l ., 2013) . C om pa r e d w i t h
m A b- ba s e d i m m unot oxi ns , t h i s ne w g e ne r a t i on of i m m unot oxi ns e x hi bi t m a r ke dl y i m pr ove d
pe ne t r a t i on c a pa bi l i t y t o t um or m a s s e s , but t he y a r e c l e a r e d r a pi dl y f r om t he bl oods t r e a m . T he i r
ha l f - l i ve s i n v i v o ( t 1 / 2 = ~ 20 m i n) a r e t oo l i m i t e d f or di f f us i on t o oc c ur i n s ol i d t um or s , l e a di ng t o
a na r r ow t i m e w i ndow of a c t i vi t y obt a i ne d w i t h a bol us i nj e c t i on ( L i e t a l ., 2017) . T o ove r c om e
5 4
t he pr obl e m s a s s oc i a t e d w i t h s hor t ha l f - l i ve s , a num be r of di f f e r e nt a ppr oa c he s ha ve be e n
de ve l ope d. P E G y l a t i on i s t he m os t e s t a bl i s he d ha l f - l i f e e x t e ns i on t e c hnol og y i n t he c l i ni c w i t h
pr ove n s a f e t y i n hum a ns f or ove r t w o de c a de s . C ova l e nt a t t a c hm e nt of P E G t o i m m unot oxi ns
c a n not onl y pr ol ong t he i r ha l f - l i ve s i n bl ood c i r c ul a t i on , but a l s o of f e r ot he r a dva nt a g e s s uc h a s
i m pr ove d s ol ubi l i t y a nd s t a bi l i t y a nd r e duc e d i m m unog e ni c i t y ( S w i e r c z e w s ka e t a l ., 2015) .
P E G y l a t i on s t r a t e gy ha s de ve l ope d f r om non- s pe c i f i c r a ndom c onj uga t i ons t o t he m or e r e c e nt
s i t e - s pe c i f i c c onj uga t i on m e t hods w hi c h g e ne r a t e c ont r ol l e d, w e l l - de f i ne d c onj uga t e d pr oduc t s
w i t h i m pr ove d pr oduc t pr of i l e s ( N i s c ha n a nd H a c ke nbe r g e r , 2014) .
I n t hi s s t udy , P E G y l a t i on of t he i m m unot oxi n α F A P - P E 38 va r i a nt a t a c y s t e i ne r e s i due
g e ne r a t e d bot h m ono- a nd m ul t i - P E G y l a t e d pr oduc t s . T o m odi f y t he pr ot e i n a t a s i ng l e
pr e - de t e r m i ne d s i t e , a n U A A w a s s i t e - s pe c i f i c a l l y i nc or por a t e d i nt o α F A P - C D 8h- P E 38 us i ng a n
or t hogon a l t R N A / a a R S pa i r . α F A P - C D 8h ( C 29pA c F ) - P E 38 w a s c oupl e d by oxi m e l i g a t i on t o
P E G - a m i noox y t hr ough t he ke t one g r oup of pA c F . T hi s m P D A - c a t a l y z e d oxi m e f or m a t i on w a s
c a r r i e d out a t pH 4.2 s i nc e t he r e a c t i on r a t e c a n be g r e a t l y a c c e l e r a t e d a t l ow e r pH va l ue s
( M a hm oodi e t a l ., 2015) . H ow e ve r , t he y i e l d of t he P E G y l a t e d pr oduc t w a s onl y a ppr ox i m a t e l y
1% , w hi c h w a s pa r t i a l l y ow i ng t o s e r i ous pr ot e i n pr e c i pi t a t i on a t l ow pH . T o e na bl e P E G y l a t i on
a t ne ut r a l pH , a di f f e r e nt U A A w a s e x a m i ne d. S pe c i f i c a l l y , t he a z i de - c ont a i ni ng a m i no a c i d
pA z F w a s i nc or por a t e d t o pr oduc e α F A P - C 3( pA z F ) - P E 38, w hi c h w a s t he n c onj ug a t e d w i t h
m P E G - a l ky n e vi a t he C uA A C r e a c t i on a t ne ut r a l pH . W hi l e pr ot e i ns r e m a i ne d s ol ubl e , t he
C uA A C r e a c t i on w a s uns uc c e s s f ul . A pos s i bl e r e a s on f or t he f a i l e d c onj uga t i on c oul d be t ha t
pA z F w a s hy d r ophobi c a nd t hus i t w a s not e x pos e d t o t he s ol ve nt .
5 5
T o e nha nc e t he a c c e s s i bi l i t y of U A A a nd a l s o r e duc e pr ot e i n pr e c i pi t a t i on a t l ow pH , t he
c onj uga t i on r e a c t i on be t w e e n α F A P - C D 8h ( C 29pA c F ) - P E 38 a nd P E G - a m i nooxy w a s c a r r i e d out
unde r de na t ur i ng c ondi t i ons . M uc h hi g he r c onj ug a t i on e f f i c i e nc y w a s obs e r ve d, s ug ge s t i ng t ha t
t he i nc or por a t e d pA c F w a s hi g hl y a c c e s s i bl e . A f t e r i n v i t r o r e f ol di ng, t he 10 kD a P E G y l a t e d
α F A P - C D 8h ( C 29pA c F ) - P E 38 w a s obt a i ne d w i t h a y i e l d of 10–12% . T hi s w a s m uc h e nha nc e d
f r om 1% , t he y i e l d of P E G y l a t i on unde r na t i ve c ondi t i on s .
W hi l e P E G y l a t i on hol ds s e ve r a l a dva nt a ge s , i t of t e n c om e s a t t he e x pe ns e of r e duc e d bi ndi ng
a f f i ni t y f or t he t a r g e t r e c e pt or . C om pa r e d w i t h unP E G y l a t e d i m m unot oxi ns , t he 10 kD a
P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 w a s s how n t o ha ve s i m i l a r bi ndi ng a f f i ni t y f or F A P . I t
w a s s t i l l c y t ot oxi c a ga i ns t hF A P - e x pr e s s i ng 293T c e l l s , a l t houg h i t s I C 5 0 va l ue w a s t w o- f ol d
hi g he r t ha n t ha t of t he unP E G y l a t e d α F A P - C D 8h- P E 38. I n c ont r a s t , t he 20 a nd 30 kD a
P E G y l a t e d α F A P - C D 8h ( C 29pA c F ) - P E 38 l os t t he i r a bi l i t y t o bi nd t o F A P , l i ke l y due t o a n
i nc r e a s e d s t e r i c hi ndr a nc e br ought by t he l a r g e P E G c ha i n s . N o c y t ot ox i c e f f e c t s a ga i ns t
hF A P - e xpr e s s i ng 293T c e l l s w e r e obt a i ne d w i t h t he 20 a nd 30 kD a P E G y l a t e d i m m unot oxi ns
e i t he r . I t ha s be e n know n t ha t s t e r i c hi ndr a nc e f r om t he P E G c ha i n c a n c a us e l os s of pr ot e i n
a c t i vi t y a f t e r P E G c onj ug a t i on. T hi s , how e ve r , c a n be of f s e t by t he l ong e r c i r c ul a t i ng ha l f - l i f e of
P E G y l a t e d m ol e c ul e s . T he r e f or e , i t i s c r i t i c a l t o m odi f y t he pa r e nt m ol e c ul e w i t h a ppr opr i a t e
P E G t o a l t e r t he ba l a nc e of P D a nd P K i n a c oor di na t e d m a nne r f or a n ove r a l l i m pr ove d
pha r m a c ol ogi c a l pr of i l e ( F i s hbur n, 2008) . I n our s t udy , t he 10 kD a P E G y l a t e d
α F A P - C D 8h ( C 29pA c F ) - P E 38 i s t he m os t pr om i s i ng c a ndi da t e f or f ur t he r i n v i v o e va l ua t i on. I t
s houl d be not e d t ha t a not he r 10 kD a P E G y l a t e d pr oduc t , i n w hi c h P E G w a s a t t a c he d t o t he
5 6
c y s t e i ne r e s i due i n t he C 3 c onne c t or , di d not s how c om pa r a bl e bi ndi ng or c y t ot oxi c a c t i vi t y .
S i nc e t he c y s t e i ne r e s i due i s c l os e r t o t he t a r g e t i ng m oi e t y of t he i m m unot oxi n, i t i s l i ke l y t ha t
t he bi ndi ng of 10 kD a P E G y l a t e d α F A P - C 3( C y s ) - P E 38 t o F A P i s di s r upt e d by t he e nha nc e d
s t e r i c hi ndr a nc e .
I n s um m a r y , our s t udy ha s s uc c e s s f ul l y i nc or por a t e d pA c F , a ke t one - c ont a i ni ng U A A , i nt o
t he i m m unot oxi n α F A P - P E 38 a t t he de s i r e d s i t e by us i ng a n or t hog on a l t R N A / a a R S pa i r . T h i s
ha s a l l ow e d s i t e - s pe c i f i c P E G y l a t i on of t he i m m unot oxi n, pr oduc i ng a c he m i c a l l y - de f i ne d,
hom og e ne ous pr oduc t w hi c h i s ha r d t o a c hi e ve by l y s i ne - or c y s t e i ne - P E G y l a t i on. T o e nha nc e
t he P E G y l a t i on y i e l d , w e de ve l ope d a nove l m e t hod by c a r r y i ng out t he c onj uga t i on r e a c t i on
unde r de na t ur i ng c ondi t i ons f ol l ow e d by i n v i t r o r e f ol di ng . T he 10 kD a P E G y l a t e d
α F A P - C D 8h ( C 29pA c F ) - P E 38 obt a i ne d by t hi s m e t hod w a s s how n t o r e t a i n s pe c i f i c bi ndi ng a nd
c y t ot oxi c a c t i vi t i e s t o 293T - hF A P c e l l s . F ut ur e i n v i v o s t udi e s a r e t hus w a r r a nt e d t o de t e r m i ne t he
P K pr ope r t i e s , bi odi s t r i but i on, t he r a pe ut i c a c t i vi t y , t oxi c i t y a nd i m m unog e ni c i t y of t he
P E G y l a t e d i m m unot oxi n.
5 7
R e f e r e n c e s
A da m s , G .P . , a nd W e i ne r , L . M . ( 2005) . M onoc l ona l a nt i body t he r a py of c a nc e r . N a t B i ot e c hnol
23, 1 1 47- 1 1 57.
A hm a dz a de h, M ., a nd R os e nbe r g , S .A . ( 2005) . T G F - be t a 1 a t t e nua t e s t he a c qui s i t i on a nd
e x pr e s s i on of e f f e c t or f unc t i on by t um or a nt i g e n- s pe c i f i c hum a n m e m or y C D 8 T c e l l s . J
I m m unol 174, 5215- 5223.
A nt i g na ni , A ., a nd F i t z g e r a l d, D . ( 2013) . I m m unot oxi ns : t he r ol e of t he t oxi n. T o xi ns ( B a s e l ) 5,
1486- 1502.
A ugs t e n, M ., H a ggl of , C ., O l s s on, E ., S t ol z , C ., T s a g oz i s , P . , L e vc he nko, T . , F r e de r i c k, M .J .,
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f i br obl a s t s a nd a c t s a s a m ul t i - m oda l s t i m ul a t or of pr os t a t e t um or g r ow t h. P r oc N a t l A c a d S c i U
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B a l s a m o, M ., S c or da m a gl i a , F . , P i e t r a , G ., M a nz i ni , C ., C a nt oni , C ., B oi t a no, M ., Q ue i r ol o, P . ,
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5 8
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Abstract (if available)

Abstract Fibroblast activation protein (FAP) is a cell-surface protease highly expressed in cancer-associated fibroblasts (CAFs)—a type of cells in the tumor microenvironment that promote the progression of human epithelial cancers. To deplete FAP-expressing CAFs for treating cancer, a recombinant immunotoxin, αFAP-PE38, was recently developed by fusing a single-chain variable fragment (scFv) against FAP with a truncated form of Pseudomonas exotoxin A (PE38). However, the short plasma half-life of αFAP-PE38 has limited its therapeutic efficacy. The aim of this thesis is to modify αFAP-PE38 with polyethylene glycol (PEG), a polymer that has previously been used to extend the plasma half-life of other therapeutic proteins. We have generated PEGylated αFAP-PE38 using two strategies: (1) PEGylation at a cysteine residue, and (2) PEGylation at an unnatural amino acid residue. We find that a PEGylated αFAP-PE38 protein, in which a 10 kDa PEG is attached to a ketone-containing unnatural amino acid in the linker, can specifically bind to human FAP-expressing 293T cells and inhibit their viability in a dose-dependent manner. However, the αFAP-PE38 proteins modified with larger PEG, including the 20 and 30 kDa variants, have lost binding affinity and cytotoxic effect to human FAP-expressing 293T cells, likely due to increased steric hindrance. Further studies are needed for in vivo evaluation of the pharmacokinetic properties and pharmacologic effects of the 10 kDa PEGylated αFAP-PE38 protein.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Jin, Yiru (author)

Core Title Site-specific PEGylation of recombinant immunotoxin αFAP-PE38 for half-life extension

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 08/06/2018

Defense Date 06/20/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag fibroblast activation protein,immunotoxin,OAI-PMH Harvest,polyethylene glycol,site-specific PEGylation,unnatural amino acid

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Xie, Jianming (committee chair), Shen, Wei-Chiang (committee member), Wang, Pin (committee member)

Creator Email yirujin@usc.edu,yirujin74@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-55205

Unique identifier UC11671843

Identifier etd-JinYiru-6662.pdf (filename),usctheses-c89-55205 (legacy record id)

Legacy Identifier etd-JinYiru-6662.pdf

Dmrecord 55205

Document Type Thesis

Format application/pdf (imt)

Rights Jin, Yiru

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA