Z h ig u a n g E r icZ h a n g 1 7 , 2 0 0 6Ap r il

6 9 C D a n d Te r lip r e s s in c lo n in g t a r g e t s

6 9 C D c lo n in g t a r g e t s( )G a r y a n d H a k ju

6 9 C D b a c k g r o u n d in f o

6 9 - 3 F ix in g m u C D p C R m is s e n s e m u t a t io n

6 9 F u n c t io n s o f C D e x p r e s s io n v e c t o r s

CRD: Caborhydrate recognition domain

N (Neck) region

TM: Transmembrane

Cy (Cytoplasmic) region

- Type II integral membrane protein, C-type lectin

6 9 – C D E a r ly Ac t iv a t io n M a r k e r

6 9 – C D E a r ly A c t iv a t io nM a r k e r

6 9 ( )F u n c t io n o f C D in v it r o - C o s t im u la t o r y r e c e p t o r

P r o in f la m m a t o r y r e c e p t o r

In h ib it o r y r e c e p t o r

6 9 ( )F u n c t io n o f C D in v iv o

- N o p o s it iv e c o s t im u la t o r y e f f e c t

6 9 N o in f la m m a t o r y c o n d it io n s in C D t r a n s g e n ic m ic e

N e g a t iv e r e g u la t o r o f im m u n e r e s p o n s e s

6 9 E x a c e r b a t e d f o rm o f C IA in C D KOm ic e

- R e d u c e d le v e l o f TG F b in in f la m e d jo in t s

- - An t i TG F b a n t ib o d y in c r e a s e s C IAs e v e r it y

6 9 - C D c o n c lu s io n s

6 9 ( F r o m a n t ib o d ie s t o m u C D S p a in’ )c o lla b o r a t o r s w o r k

◆ 2 .2 a n t ib o d y e x a c e r b a t e s t y p e II ( )c o lla g e n in d u c e d a r t h r it is C IA

◆ 2 .3 a n t ib o d y a m e lio r a t e s d is e a s e

6 9 C D is a n e a r ly s t a g e t a r g e t

◆ S u p p o r t in g e v id e n c e o f e f f ic a c y in m ic e

◆ M o A u n c le a r b u t lik e ly in v o lv e s TG F b

◆ – N a t u r e o f p o t e n t ia l d r u g n o t c le a r h o w ?t o s c r e e n

3 9 6 9 4 N EF h u C D n a t iv e IC D e x p r e s s io n v e c t o r

Tr a n s f e c t e d in t o J u r k a t c e lls f o r u s e

in f u n c t io n a l a s s a y s

Tr a n s f e c t e d in t o2 9 3 S c e l ls f o r

h y b r id o m a s c r e e n in g

Tr a n s f e c t e d in t o3 1 2 T c e l ls f o r

c e l lu la r im m u n iz a t io n

6 9 m u C D m u t a t io n s

◆ S ile n t a n d m is s e n s e m u t a t io n s in6 9 3 m u C D c D N A in p C R v e c t o r r e c e iv e d

f r o m S p a in

◆ D e c id e d t o f ix m is s e n s e m u t a t io n t o u s in g o v e r la p P C R

NP_001028294.1 20 KDHGTSIHFEKHHEGSIQVS 40Spain 20 KDHGTSIHFEKHHEGSIQVS 40 ********************

NP_001028294.1 100 TTTKSWALAQRSCSEDAATL 120Spain 100 TTTKSWALAQRSCSEDAAIL 120 ****************** *

AK153224.1 300 ACCACAACCAAGAGTTGGGCCTTGGCCCAACGCTCTTGTTCTGAAGATGCTGCTACTCTT 360AK151076.1 300 ACCACAACCAAGAGTTGGGCCTTGGCCCAACGCTCTTGTTCTGAAGATGCTGCTACTCTT 360L23638 300 ACCACAACCAAGAGTTGGGCCTTGGCCCAACGCTCTTGTTCTGAAGATGCTGCTACTCTT 360NM_001033122 300 ACCACAACCAAGAGTTGGGCCTTGGCCCAACGCTCTTGTTCTGAAGATGCTGCTACTCTT 360Spain 300 ACCACAACCAAGAGTTGGGCCTTGGCCCAACGCTCTTGTTCTGAAGATGCTGCTATTCTT 360 ******************************************************* ****

AK153224.1 60 AAGGACCATGGCACCAGTATACATTTTGAGAAGCATCATGAAGGATCCATTCAAGTTTCT 120AK151076.1 60 AAGGACCATGGCACCAGTATACATTTTGAGAAGCATCATGAAGGATCCATTCAAGTTTCT 120L23638 60 AAGGACCATGGCACCAGTATACATTTTGAGAAGCATCATGAAGGATCCATTCAAGTTTCT 120NM_001033122 60 AAGGACCATGGCACCAGTATACATTTTGAGAAGCATCATGAAGGATCCATTCAAGTTTCT 120Spain 60 AAGGACCATGGCACCAGTATACATTTTGAGAAACATCATGAAGGATCCATTCAAGTTTCT 120 ******************************** ***************************

6 9 F ix in g m u C D c D N A m is s e n s em u t a t io n

6 9 C D F c f u s io n e x p r e s s io n v e c t o r s f o r- c r o s s r e a c t iv it y s c r e e n

, Wa n t o n ly e x t r a c e llu la r r e g io n y e t t h e r e is a n e c k r e g io n w it h a d is u lf id e b o n d t h a t f o r m s d im e r

N o t s u r e w h a t w ill h a p p e n t o F c f u s io n p r o t e in w h e n f r e e d f r o m – , ?c e ll s u r f a c e d im e r t e t r a m e r

6 9 . 1 . 2 , C r e a t e C D M a n d M e x t r a c e llu la r m u t a n t s w it h a n d w it h o u t / n e c k r e g io n d is u lf id e b o n d

6 9 . 1 H u m a n a n d m o u s e C D M a n d. 2 M m u t a n t s

•huCD69 MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIA 60 •muCD69 MDSENCSITENSSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIA 60 • *.**** ::**** * * **::..** **..:****:**.: **: **:** ******* • . 1 -> . 2 ->M M•huCD69 LSV~GQYNCPGQYTFSMPSDSH~VSSCSEDWVGYQRKCYFISTVKRSWTSAQNACSEHGATL 120 •muCD69 LNV~GKYNCPGLYEKLESSDHH~VATCKNEWISYKRTCYFFSTTTKSWALAQRSCSEDAATL 120 • *.* *:***** * .** * *::*.::*:.*:*.***:**..:**: **.:***..*** • •huCD69 AVIDSEKDMNFLKRYAGREEHWVGLKKEPGHPWKWSNGKEFNNWFNVTGSDKCVFLKNTE 180 •muCD69 AVIDSEKDMTFLKRYSGELEHWIGLKNEANQTWKWANGKEFNSWFNLTGSGRCVSVNHKN 180 • *********.*****:*. ***:***:*..:.***:******.***:***.:** :::.: • •huCD69 VSSMECEKNLYWICNKPYK 199 •muCD69 VTAVDCEANFHWVCSKPSR 199

6 9 s N IF C D e x p r e s s io n v e c t o r s f o rim m u n iz a t io n s

3 9 U n f o r t u n a t e ly t h e c e lls t r a n s f e c t e d w it h N e f F c f u s io n v e c t o r s 6 9a r e e x p r e s s in g b u t n o t s e c r e t in g C D

6 9 -C r e a t e s N IF v e r s io n s o f C D w it h s ig n a l s e q u e n c e a t N t e rm ( 3 ’ ) - a n d p r e c is io n p r o t e a s e s it e in p r im e r a t C t e rm t o a id in s e c r e t io n

N e w Te r lip r e s s in C o n s t r u c t s

F o r u s e in f u n c t io n a l a s s a y s t o w a r d s f u r t h e r r e s e a r c h a n d d e v e lo p m e n t

p e n d in g Te r lip r e s s in FD A a p p r o v a l

Gly

Gly

GlyCys

TyrPhe

GlnAsn

Cys

ProLys

Gly

H

H2NArg in human vasopressin

1 - -8 - -t r ig ly c y l ly s in e v a s o p r e s s in

( 1 2 S in c e Te r l ip r e s s in is s o s m a ll o n ly a m in o) a c id s a n d it w il l b e jo in e d t o a c o m p a r a t iv e ly , 0 , 3 , 1 0 la r g e F c a lin k e r r e g io n o f a n d g ly c in e

r e s id u e s is t o b e a d d e d t o h o p e f u lly m in im iz e a n y in t e r f e r e n c e t o TP a c t iv it y w h e n b o u n d t o F c

Ad d it io n o f G ly c in e s p a c e r s

- - (0 ,3 ,1 0 )-S S Te r lip r e s s in G ly1 (1 ) F c G m c lo n in g s t r a t e g y

, S in c e b o t h S S a n d Te r lip r e s s in a r e s o s h o r t t h e y w ill n o t b e e a s i ly s u b c lo n e d b y P C R a n d

g e l e x t r a c t io n

Ad d it io n o f g ly c in e s p a c e r s p o s e s a d d it io n a lc h a lle n g e

B e s t s t r a t e g y a p p e a r s t o b e p r im e r w a lk in g 5 ’ 1 (1 ) b y P C R e x t e n d in g t h e e n d o f F c G m f r o m

3 9 - 1 (1 ) p r e v io u s c o n s t r u c t N e f s s u r o F c G m4IC D

- - (0 ,3 ,1 0 )-S S Te r lip r e s s in G ly1 (1 ) F c G m p r im e r w a lk in g

- - P a c K o z S S(5 0 )m e r

-1 (4 0 )S S m e r

- (5 0 )S S TP m e r

- 1 (1 ) TP F c G m(3 0 )m e r

. 1 - 1 (1 ) TP M F c G m(3 9 )m e r

. 2 - 1 (1 ) TP M F c G m(6 0 )m e r

1 (1 )F c G m

- F c R e v(3 2 )m e r

S u c c e s s iv e r o u n d s o f P C R r e q u ir e d d u e t o h ig h e r c h a n c e o f o l ig o s y n t h e s is e r r o r s w it h in c r e a s in g le n g t h

- - If t h e s e p r im e r s c o n t a in s y n t h e s is e r r o r s r e o r d e r @ h ig h e r p u r it y

- - (0 ,3 ,1 0 )-S S Te r lip r e s s in G ly1 (1 ) F c G m P r im e r s

5 ’ - – 3 ’AAC TG C C C G AAAG G C G AC AAAAC TC AC AC A-TP F c

S e q u e n c eP r im e r

5 ’ - ATTG C G G C C G C TC ATTTAC C C G G AG AC AG G G A – 3 ’-F c R e v

5 ’ - AAC TG C C C G AAAG G C G G AG G C G G TG G AG G TG G AG G C G G TG G AG G TG AC AAAAC

– 3 ’TC AC AC A

. 2 -TP M F c

5 ’ - AAC TG C C C G AAAG G C G G AG G C G G T – 3 ’G AC AAAAC TC AC AC A. 1 -TP M F c

5 ’ - TC TC C TC C G C G TG C G G TG G C G G ATG C TAC TTC C AG AAC TG C C C G AAAG G C– 3 ’

-S S TP

5 ’ - – 3 ’C G C C TG C TTC C TC G G C C TAC TG G C C TTC TC C TC C G C G TG C-1S S

5 ’ - –G C C TTAATTAAC AC C ATG C C TG AC AC C ATG C TG C C C G C C TG C TTC C TC G G3 ’

- -P a c K o z S S

- - (0 ,3 ,1 0 )-S S Te r lip r e s s in G ly1 (1 ) 2 % F c G m a g a r o s e g e l

, . 1 , . 2 TP TP M TP M g r a d ie n t P C R p r o d u c t s a f t e r 4 th and

final round o f p r im e r w a lk in g P C R

TO P O c lo n e d t o b e s e q u e n c e d a n d d ig e s t

s c r e e n e d f o r t a r g e t e x p r e s s io n v e c t o r

c r e a t io n u L v o l p e rla n e

P C Rc o m p o n e n t

2 0To t a l

11 /1 0 0 p r e v io u s P C R p r o d u c t g e lp u r if ie d

5 - P r im e r P a c K o z‘ 8 0 8 7 ’

5 _ P r im e r FC R EV‘ 7 7 7 4 ’

4 .42d H O

0 .2 iP r o o fp o ly m e r a s e

45 x iP r o o f H Fb u f f e r

0 .41 0 m M d N TP

8 5 0 b p

- - 7 8 0S S TP F c b p - . 1 - 7 8 9S S TP M F c b p

- . 2 - 8 10S S TP M F c b p

| 5 0 .5 | 5 3 .2 | 5 8 .4 | C C C6 4 .6 |C

An n e a lin g Te m p e r a t u r eG r a d ie n t s

1 2 , 2 0 0 6 , 2 % Ap r a g a r o s e g e l