Induction and analysis of antigen-specific T cell responses in melanoma patients and animal model

Induction and analysis of antigen-specific T cell responses in

melanoma patients and animal model

Bins, A.D.

Citation

Bins, A. D. (2007, March 15). Induction and analysis of antigen-specific T cell responses in

melanoma patients and animal model. Retrieved from https://hdl.handle.net/1887/11457

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the

Institutional Repository of the University of Leiden

Downloaded from: https://hdl.handle.net/1887/11457

Note: To cite this publication please use the final published version (if applicable).



$GULDDQ %LQV^a %DEV 9HUVWUHSHQ ^a &KULVWLQH 5ROOLHU  3HWUD 0RRLM  *HUULW

.RRSPDQ  &\QWKLD 5REVRQ^# 1HLO 6KHSSDUG^# 5DOI :DJQHU^ +DQV :ROI^

7RQ6FKXPDFKHU^-RQDWKDQ+HHQH\ DQG-RKQ+DDQHQ^

6XSHULRULPPXQRJHQLFLW\RI'1$WDWWRRLQJDV

FRPSDUHGWRLQWUDPXVFXODU'1$YDFFLQDWLRQ

LQQRQKXPDQSULPDWHV

'HSWRI,PPXQRORJ\7KH1HWKHUODQGV&DQFHU,QVWLWXWH3OHVPDQODDQ

&;$PVWHUGDP7KH1HWKHUODQGV

'HSW RI 9LURORJ\ %LRPHGLFDO 3ULPDWH 5HVHDUFK &HQWUH 5LMVZLMN 7KH

1HWKHUODQGV

#6LU:LOOLDP'XQQ6FKRRORI3DWKRORJ\8QLYHUVLW\RI2[IRUG2[IRUG8.

,QVWLWXWHRI0HGLFDO0LFURELRORJ\DQG+\JLHQH5HJHQVEXUJ*HUPDQ\

a%RWKDXWKRUVFRQWULEXWHGHTXDOO\

&RUUHVSRQGHQFHVKRXOGEHDGGUHVVHGWR-%$*MKDDQHQ#QNLQO

& + $ 3 7 ( 5  



,QWURGXFWLRQ

7KH GHYHORSPHQW RI KHWHURORJRXV SULPHERRVW YDFFLQDWLRQ UHJLPHQV KDV

DFFHQWXDWHGWKHQHHGIRUSRWHQWSULPLQJPHWKRGVWRLQFUHDVHLPPXQHUHVSRQVHV

XSRQ ERRVWLQJ ZLWK DGHQR RU SR[EDVHG LPPXQRJHQV '1$ YDFFLQHV DUH DQ

DWWUDFWLYHFRPSRQHQWIRUWKHSULPLQJSKDVHRIVXFKUHJLPHQVDV7FHOOLQGXFWLRQLV

XQKLQGHUHGE\YHFWRUVSHFLILFLPPXQLW\DQGFDQRFFXULQDIOH[LEOHVDIHDQGVLPSOH

PDQQHU^8QIRUWXQDWHO\WKHKLJKHIILFDF\RI'1$YDFFLQHVVHHQLQHDUO\DQGPRUH

UHFHQWPRXVHVWXGLHVKDVQRWEHHQPLUURUHGLQKXPDQVDQGLQQRQKXPDQSULPDWH

'1$YDFFLQDWLRQWULDOV7KHSRRUWUDFNUHFRUGRI'1$YDFFLQHVLQSULPDWHVKDV

EHHQDPDMRULPSHGLPHQWIRUWKHGHYHORSPHQWRIFOLQLFDO'1$YDFFLQHV

7KH ORZ LPPXQRJHQLFLW\ RI LQWUDGHUPDO '1$ YDFFLQHV LQ SULPDWHV KDV EHHQ

VXJJHVWHGWREHDFRQVHTXHQFHRIWKHORZ7/5H[SUHVVLRQRQVNLQUHVLGHQW$3&V^ RISULPDWHV)XUWKHUPRUHLQUHODWLRQWRERG\PDVVWKHW\SLFDO'1$GRVHVXVHGLQ

ODUJHDQLPDOVDUHJHQHUDOO\IROGORZHUWKDQWKRVHXVHGLQURGHQWV5HIOHFWLQJ

DQLQFRPSOHWHXQGHUVWDQGLQJRIWKHIDFWRUVLQYROYHGWKHLVVXHKDVEHHQWHUPHGµWKH

VLPLDQEDUULHU¶^

5HFHQWO\DQHZLQWUDGHUPDO'1$YDFFLQDWLRQPHWKRGZDVGHYHORSHGDQGRSWLPL]HG

E\ XV LQ PRXVH PRGHOV ,Q WKLV VWUDWHJ\ WHUPHG '1$ WDWWRRLQJ YDFFLQDWLRQ LV

SHUIRUPHG E\ DSSO\LQJ D GURSOHW RI '1$ WR WKH VNLQ IROORZHG E\ H[SRVXUH RI

WKH VNLQ WR WKH QHHGOH KHDG RI D UDSLGO\ RVFLOODWLQJ WDWWRR GHYLFH UHVXOWLQJ LQ

WKH FUHDWLRQ RI WKRXVDQGV RI SXQFWXUHV LQ WKH '1$FRDWHG DUHD ,Q PLFH '1$

WDWWRRLQJOHDGVWRDVLJQLILFDQWO\KLJKHUOHYHORIDQWLJHQSUHVHQWDWLRQLQWKHGUDLQLQJ

O\PSKQRGHDVFRPSDUHGWRLQWUDPXVFXODULQMHFWLRQDQGWRWKHLQGXFWLRQRIVWURQJ

7FHOOUHVSRQVHVZLWKLQDWLPHVSDQRIZHHNV^)XUWKHUPRUHWKLVQRYHOPHWKRGRI

LPPXQL]DWLRQPDNHVLWSRVVLEOHWRVFDOH'1$GRVHGLUHFWO\OLQHDUWRWKHWRWDOERG\

VXUIDFHDFURVVGLIIHUHQWVSHFLHV%DVHGRQWKHSRRUWUDFNUHFRUGRI'1$YDFFLQDWLRQ

DSSURDFKHVLQSULPDWHVLWVHHPHGRILPSRUWDQFHWRDVVHVVWKHHIIHFWLYHQHVVRIWKH

QHZO\GHYHORSHGDSSURDFKLQDQRQKXPDQSULPDWHPRGHO+HUHZHUHSRUWRQWKH

HIILFDF\RILQWUDGHUPDO'1$WDWWRRYDFFLQDWLRQLQ0DFDFDPXODWWD

([SHULPHQWDOVHWXS

:HYDFFLQDWHGRXWEUHGPDFDTXHVZLWKDPJPO'1$YDFFLQHFRQWDLQLQJWKH

SODVPLGV S257*31 DQG S257JS DW HTXLPRODU FRQFHQWUDWLRQ S257*31

HQFRGHV D ELRORJLFDOO\ LQDFWLYH JDJSROQHI IXVLRQ SURWHLQ RI WKH FODGH & +,9

VWUDLQ FQ S257JS HQFRGHV D VHFUHWHG YHUVLRQ RI WKH (QY SURWHLQ IURP

WKHVDPH+,9VWUDLQIRUDGHWDLOHGGHVFULSWLRQRIWKHSODVPLGVVHHVXSSOHPHQWDU\

LQIRUPDWLRQ7KHYDFFLQDWLRQUHJLPHQFRQVLVWHGRIWDWWRRDSSOLFDWLRQVDWGD\

LQWHUYDOV7KLVVKRUWLQWHUYDOYDFFLQDWLRQUHJLPHQZDVLGHQWLFDOWRWKDWSUHYLRXVO\

XVHGLQPLFHZKHUHLWLQGXFHVUREXVWF\WRWR[LF7FHOOUHVSRQVHVZLWKLQZHHNV^

,QDGGLWLRQWRDOORZDFRPSDULVRQZLWKUHWURVSHFWLYHGDWDVHWVVHHEHORZDQLPDOV

UHFHLYHGDQLGHQWLFDOVHWRIERRVWHUYDFFLQDWLRQVDIWHUZHHNV

'XULQJ HDFK WDWWRR DQ DUHD RI DSSUR[LPDWHO\ [ FP  FP RQ HDFK OHJ ZDV

WUHDWHG2QWZROHJVWRJHWKHUHDFKWUHDWPHQWFRYHUVDIUDFWLRQRIWKHERG\VXUIDFH





,175$'(50$/'1$7$7722,1*35,0(652%8677&(//5(63216(6,10$&&$&$08/$77$

WKDWLVVLPLODUWRWKHIUDFWLRQWUHDWHGLQSUHYLRXVURGHQWH[SHULPHQWVRIWKH

ERG\VXUIDFHIRUHDFKDSSOLFDWLRQ7KHILUVWRIWKHWKUHHWDWWRRVZDVSODFHGRQWKH

LQQHUWKLJKVWKHVHFRQGRQWKHXSSHUWKLJKVDQGWKHWKLUGRQWKHRXWHUWKLJKV:LWK

HDFKWDWWRRPORIDPJPO'1$VROXWLRQZDVDSSOLHGRQHDFKOHJDQGWKH

DUHDZDVWUHDWHGXVLQJDGLVSRVDEOHQHHGOHEDUHDFKFUHDWLQJVNLQSHUIRUDWLRQV

ZLWK D GLDPHWHU RI DSSUR[LPDWHO\  PP PRXQWHG RQ D URWDU\ WDWWRR PDFKLQH

RVFLOODWLQJDW+]8VLQJWKLVVHWXSDSSUR[LPDWHO\VNLQSHUIRUDWLRQV

DUHLQIOLFWHGGXULQJWKHPLQXWHWUHDWPHQWRIHDFKOHJ$WRWDORIDSSUR[LPDWHO\

PJ'1$ZDVDSSOLHGRYHUWKHFRXUVHRIWKHZKROHSULPHERRVWUHJLPHQ

.LGQH\ DQG OLYHU IXQFWLRQ SDUDPHWHUV ZKLWH DQG UHG EORRG FHOO FRXQWV DQG

LQIODPPDWRU\ SDUDPHWHUV ZHUH GHWHUPLQHG RQFH HYHU\ ZHHN DQG  GD\V DIWHU

HDFK WDWWRR %ORRG ZDV GUDZQ IRU HQXPHUDWLRQ RI ,)1Ȗ DQG ,/ SURGXFLQJ

SHULSKHUDO EORRG PRQRQXFOHDU FHOOV 3%0& E\ HQ]\PHOLQNHG LPPXQRVSRW

(/,VSRWDVVD\VDWZHHNDQGSRVWYDFFLQDWLRQDQGE\LQWUDFHOOXODUF\WRNLQH

VWDLQLQJ ,&6 DW ZHHN  SRVW YDFFLQDWLRQ$GGLWLRQDOO\ DW ZHHN  DQG  EORRG

ZDVGUDZQIRUPHDVXUHPHQWRI(QYHORSHVSHFLILF,J*DQWLERG\WLWHUVE\HQ]\PH

OLQNHGLPPXQRVRUEHQWDVVD\V(/,6$(/,VSRWDQG(/,6$GDWDJHQHUDWHGLQWKH

H[SHULPHQW ZHUH FRPSDUHG WR WKRVH RI  PDFDTXHV WKDW SUHYLRXVO\ UHFHLYHG D

SULPHERRVWYDFFLQDWLRQE\EROXVLQWUDPXVFXODULPLQMHFWLRQRIPORIWKHVDPH

'1$YDFFLQHEDWFKDWZHHNDQGZHHNPJRI'1$LQWRWDO$VDVHFRQG

FRPSDULVRQUHVSRQVHVZHUHFRPSDUHGWRWKRVHLQPDFDTXHVYDFFLQDWHGZLWKD

1<9$&1HZ<RUN9DFFLQLDPRGLILHGSR[YHFWRUH[SUHVVLQJWKHVDPH+,9JHQH

VHTXHQFHVPOLPLQMHFWLRQLQHDFKDUPDWDGRVHRI[^SIXPO7KHVWXG\

GHVLJQLVVFKHPDWLFDOO\UHSUHVHQWHGLQ)LJXUH$QDO\VHVRILPPXQHUHVSRQVHVLQ

ERWKFRQWUROJURXSVKDGEHHQSHUIRUPHGDWWKHVDPHWLPHSRLQWVSRVWYDFFLQDWLRQ

DQG XVLQJ H[DFWO\ WKH VDPH PHWKRGV DQG UHDJHQWV DV LQ WKH  DQLPDOV WKDW ZHUH

YDFFLQDWHGE\'1$WDWWRR7KHVWXG\SURWRFRODQGH[SHULPHQWDOSURFHGXUHVZHUH

DSSURYHGE\WKHLQVWLWXWLRQDOµDQLPDOHWKLFDOFDUHDQGXVH¶FRPPLWWHH

5HVXOWV

1RQHRIWKHWDWWRRHGDQLPDOVGHYHORSHGDGYHUVHVLGHHIIHFWVXSRQ'1$WDWWRRLQJ

DQGDOODQLPDOVJDLQHGZHLJKWQRUPDOO\GXULQJWKHFRXUVHRIWKHH[SHULPHQW7R

D YDULDEOH H[WHQW LQ DOO DQLPDOV D WKLQ \HOORZLVK FUXVW RI FRDJXODWHG WUDQVXGDWH

DSSHDUHG RQ WKH WUHDWHG VLWHV WKH GD\ DIWHU WDWWRRLQJ )LJ  +RZHYHU WKHVH

FUXVWVGLVDSSHDUHGZLWKLQGD\V7KHDQLPDOVGLGQRWVFUDWFKQRURWKHUZLVH

PDQLSXODWHWKHWUHDWHGVNLQDUHDV

6WULNLQJO\DWZHHNVDIWHUWKHVWDUWRIYDFFLQDWLRQ&',)1ȖVHFUHWLQJ3%0&

GLUHFWHGDJDLQVW3RORU1HIFRXOGEHGHWHFWHGLQRIWKHWDWWRRHGDQLPDOVE\,&6

VXSSO)LJ,QDFFRUGDQFHZLWKWKLVILQGLQJWKH(/,VSRWDQDO\VLVDWZHHNV

DIWHUWKHVWDUWRIYDFFLQDWLRQVKRZHGWKDWRIWKHWDWWRRHGDQLPDOVKDGGHYHORSHG

D UREXVW UHVSRQVH RI DQWLJHQVSHFLILF ,)1Ȗ VHFUHWLQJ 3%0& 7KHVH UHVSRQVHV

ZHUH SUHGRPLQDQWO\ GLUHFWHG DJDLQVW 3RO DQG FRQVLVWHG RI RQ DYHUDJH 

6)&6SRW)RUPLQJ&HOOVSHUPLOOLRQ3%0&UDQJLQJIURPWR6)&

7KH FRQVLVWHQF\ RI WKHVH YDFFLQHLQGXFHG F\WRWR[LF UHVSRQVHV LQ DOO  DQLPDOV

FRQWUDVWHGVKDUSO\ZLWKLPRU1<9$&YDFFLQDWLRQS DQGS ERWK





RIZKLFKLQGXFHGORZOHYHO+,9VSHFLILF7FHOOUHVSRQVHVLQDIUDFWLRQRIDQLPDOV

DW WKLV WLPH SRLQW DYHUDJH  6)& UDQJH  ILJ D )RXU ZHHNV DIWHU WKH

VHFRQGYDFFLQDWLRQZHHNVDIWHUWKHILUVWYDFFLQDWLRQWKHDQWLJHQVSHFLILF,)1Ȗ

UHVSRQVHVLQWKHWDWWRRHGJURXSZHUH6)&RQDYHUDJHFRPSDUHGWR6)&

DIWHU LP S  DQG  6)& DIWHU 1<9$& S  )LJ D 1RWDEO\ RQO\

IRUWKHJURXSWUHDWHGE\'1$WDWWRRWKHVHYDOXHVDUHVLJQLILFDQWO\KLJKHUWKDQWKH

EDFNJURXQGUHDFWLYLW\DJDLQVWWKHXVHG+,9DQWLJHQVWKDWZDVSUHVHQWEHIRUHWKH

RQVHWRIYDFFLQDWLRQ0DQQ:KLWQH\WHVWS IRUWDWWRRS IRULPDQG

S IRU1<9$&

,QWHUHVWLQJO\DWZHHNVDIWHUYDFFLQDWLRQLQDGGLWLRQWR,)1ȖSURGXFLQJ3%0&

D SRSXODWLRQ RI ,/ VHFUHWLQJ 3%0& ZDV DOVR GHWHFWHG LQ  RXW RI  WDWWRRHG

DQLPDOV )LJ E 7KHVH FHOOV ZHUH PDLQO\ GLUHFWHG DJDLQVW HSLWRSHV LQ 3RO DQG

1HIDQGFRQVLVWHGRI6)&RQDYHUDJHVHHVXSSO)LJIRUGHWDLOHGJUDSKV

,Q FRQWUDVW QR VLJQLILFDQW LQFUHDVH LQ ,/ VHFUHWLQJ 3%0& SRSXODWLRQV ZDV

REVHUYHGXSRQLP'1$RU1<9$&YDFFLQDWLRQ6)&IRULP'1$DQG

6)&IRU1<9$&VXSSO)LJ,QVSLWHRIWKHSUHVHQFHRI,/VHFUHWLQJ3%0&

QR VLJQLILFDQW LQFUHDVH LQ WLWHUV RI JS VSHFLILF ,J* RU ,J0 DQWLERGLHV ZHUH

REVHUYHGXSRQ'1$WDWWRRGDWDQRWVKRZQ 'LVFXVVLRQ

,QWKHFXUUHQWVWXG\ZHKDYHDQDO\]HGWKHLPPXQRJHQLFLW\RI'1$WDWWRRLQJLQ

QRQKXPDQSULPDWHV$VWURQJLQGXFWLRQRI,)1Ȗ7FHOOUHVSRQVHVZDVREVHUYHG

ZLWKLQZHHNVDIWHUSULPLQJDQGWKHREVHUYHGLQGXFWLRQRIF\WRWR[LFUHVSRQVHVE\

'1$ YDFFLQDWLRQ LQ D SULPDWH PRGHO RQO\ XSRQ SULPLQJ KDV WR RXU NQRZOHGJH

QRW EHHQ REVHUYHG SUHYLRXVO\ XVLQJ RWKHU PHWKRGRORJLHV $OVR DIWHU ERRVWHU

LPPXQL]DWLRQWKHIUHTXHQFLHVRIYDFFLQHVSHFLILF,)1ȖVHFUHWLQJFHOOVUHPDLQHG

IROGKLJKHUWKDQWKRVHREVHUYHGXSRQLP'1$YDFFLQDWLRQRUYDFFLQDWLRQ

ZLWKD1<9$&YHFWRUH[SUHVVLQJWKHVDPHVHTXHQFHV3RVVLEO\WKHIDFWWKDWWKH

WDWWRRHGDUHDFDQEHVFDOHGXSOLQHDUWRDQLQFUHDVHLQERG\VXUIDFHOHDGLQJWRWKH

DYDLODELOLW\RIWKHHQFRGHGDQWLJHQVWRDSRWHQWLDOO\JUHDWHUSRSXODWLRQRIGHUPDO

$3&FRQWULEXWHVWRWKHHIILFDF\RI'1$WDWWRRLQJLQODUJHUDQLPDOV7KHVHGDWD

LQGLFDWHWKDWIXUWKHUH[SORUDWLRQRILQWUDGHUPDO'1$YDFFLQDWLRQE\WDWWRRGHYLFH

RU HTXLYDOHQW PHDQV LQ FOLQLFDO WULDOV LV PDQGDWHG 6XFK WULDOV VKRXOG IRFXV RQ

WKH XVH RI WKLV WHFKQRORJ\ HLWKHU LQ WKHUDSHXWLF VHWWLQJV RU RQ WKH XVH RI '1$

WDWWRRWRLQGXFHKLJKOHYHO7FHOOUHVSRQVHVDJDLQVWPDMRUSDWKRJHQVLQKLJKULVN

SRSXODWLRQV

0DWHULDOVDQG0HWKRGV '1$YDFFLQH

:LWKLQWKH(&(XURYDFFYDFFLQHGHYHORSPHQWSURJUDP4/.&7 JHQHRSWLPLVHG *DJ3RO1HI DQG (QY JHQH YHFWRU LQVHUWV ZHUH GHVLJQHG E\

*(1($57 *PE+ XVLQJ WKH *HQH2SWLPL]HU70 VRIWZDUH SDFNDJH &ODGH &%

*DJ3RO1HI*31DQG(QYJSVHTXHQFHVZHUHGHVLJQHGEDVHGRQVHTXHQFH

LQIRUPDWLRQ GHULYHG IURP D FQ SURYLUXV FORQH VHTXHQFH VXEPLWWHG WR

*HQ%DQN7KHJHQHRSWLPL]HGFQ(QYFRQVWUXFWFRPSULVHVQXFOHRWLGHV



,175$'(50$/'1$7$7722,1*35,0(652%8677&(//5(63216(6,10$&&$&$08/$77$

HQFRGLQJDQDUWLILFLDOVLJQDOSHSWLGHIROORZHGE\JSFQ7KH¶SDUWRIWKH

&1 *31 SRO\JHQH FRQVWUXFW HQFRGHV WKH PRGLILHG P\ULVW\ODWLRQ GHILFLHQW JURXSVSHFLILFDQWLJHQ7KH*DJFRGLQJVHTXHQFHLVIROORZHGE\DESIUDJPHQW

HQFRGLQJWKH¶SDUWRISROLQFOXGLQJDPXWDWLRQOHDGLQJWRLQDFWLYDWLRQRIWKHYLUDO

SURWHDVH$ESIUDJPHQWHQFRGLQJDVFUDPEOHG1HIYDULDQWZDVIXVHGWRWKH¶

HQGRISROFRGLQJVHTXHQFHUHSODFLQJWKHDFWLYHVLWHRIWKHUHYHUVHWUDQVFULSWDVH

7KH¶SROUHDGLQJIUDPHODFNLQJWKHLQWHJUDVHJHQHZDVH[WHQGHGE\WKH¶HQG

RIWKHVFUDPEOHG1HIJHQH7KHVHTXHQFHVWUHWFKHQFRGLQJWKHDFWLYHVLWHRIWKH

UHYHUVH WUDQVFULSWDVH ZDV WUDQVORFDWHG WR WKH ¶ HQG RI WKH SRO\JHQH FRQVWUXFW

UHVXOWLQJLQDQRSHQUHDGLQJIUDPHHQFRGLQJWKHN'DQRQJO\FRV\ODWHGDUWLILFLDO

*31 SRO\SURWHLQ 7KH &FODGHV\Q*DJ3RO1HI JHQH ZDV FORQHG LQWR WKH S&5

6FULSWDPSFORQLQJYHFWRU6WUDWDJHQH/D-ROOD86$UHVXOWLQJLQWKHYHFWRU

S&56FULSW&V\Q*DJ3RO1HI 7KH FODGH&V\QJS JHQH ZDV FORQHG LQWR WKH

S&56FULSWDPSFORQLQJYHFWRUUHVXOWLQJLQWKHYHFWRUS&56FULSW&V\QJS

7KHS&56FULSWEDVHGYHFWRUVZHUHXVHGWRJHQHUDWHWKH'1$YDFFLQHFDQGLGDWH

SODVPLGVS257D*31DQGS257DJSS257D*31DQGS257DJSZHUH

VHOHFWHG DQG SURGXFHG LQ DFFRUGDQFH WR *03 VWDQGDUGV XVLQJ WKH ( FROL VWUDLQ

'+ODFGDS'&REUD7KHUDSHXWLFV,QF8SVFDOLQJDQGSURGXFWLRQRIF*03JUDGH

PDWHULDOZDVSHUIRUPHGE\&REUD%LRPDQXIDFWXULQJ3OF.HHOH6WDIIRUGVKLUH8.

7KHUDSHXWLFV,QF

1<9$&YHFWRU

7KH 1<9$& YHFWRU H[SUHVVLQJ *DJ3RO1HI DQG (QY RI FODGH & +,9 FQ

ZDV SURYLGHG E\ 'U 0- )UDFKHWWH 6DQRIL 3DVWHXU /\RQ )UDQFH )XQFWLRQDO

H[SUHVVLRQRIWKHGRQRUJHQHVZDVGHPRQVWUDWHGE\:HVWHUQEORWDQDO\VLVRIWKH

SURWHLQV SURGXFHG E\ WKH SUHPDVWHU VHHG ORW 7KH FOLQLFDO EDWFK RI WKH YDFFLQH

ZDVPDQXIDFWXUHGE\6DQRIL3DVWHXUXVLQJDSURFHVVSUHYLRXVO\GHYHORSHGIRUWKH

PDQXIDFWXUHRIRWKHU$/9$&DQG1<9$&EDVHGYDFFLQHV

,)1ȖDQG,/(/,6327DVVD\V

4XDQWLILFDWLRQ RI DQWLJHQ VSHFLILF F\WRNLQH VHFUHWLQJ FHOOV ZDV SHUIRUPHG RQ

IUHVKO\LVRODWHG3%0&E\,)1ȖDQG,/HQ]\PHOLQNHGLPPXQRVSRW(/,VSRW DVVD\V DFFRUGLQJ WR WKH PDQXIDFWXUHU¶V LQVWUXFWLRQV 8&\WHFK 8WUHFKW 7KH

1HWKHUODQGV 3RVLWLYH FRQWURO ZDV 6(% —JPO QHJDWLYH FRQWURO ZDV PHGLXP

DORQH$QWLJHQ VSHFLILF UHVSRQVHV ZHUH PHDVXUHG DJDLQVW —JPO SHSWLGH SRROV

PHUVZLWKDDRYHUODSVSDQQLQJWKHHQWLUH*DJ3RO1HISRO\JHQHDQGWKH(QY

FODGH&RI+,9FQ6\QSHS&RUSRUDWLRQ'XEOLQ&$86$(LJKWSHSWLGH

SRROVZHUHPDGHDVIROORZV*DJSHSWLGHV&J_*DJSHSWLGHV&J_

3ROSHSWLGHV&JS_3ROSHSWLGHV&S_&S_3RO

SHSWLGHV&S_1HISHSWLGHV&Q_(QYSHSWLGHV&1_(QY

SHSWLGHV&1_5HVXOWVDUHH[SUHVVHGDVWKHPHDQQXPEHURIVSRWIRUPLQJ

FHOOV6)&SHU3%0&IURPWULSOLFDWHVDPSOHVPLQXVEDFNJURXQGYDOXHV

PHDQQXPEHURI6)&SOXV[6'RIWULSOLFDWHVDPSOHVZLWKPHGLXPDORQH0HDQ

6)&RIHDFKDQLPDOGRWVDUHH[SUHVVHGSHUDQWLJHQUHVSRQVHVWR*DJSRROV

3ROSRROVDQG(QYSRROVZHUHFRPELQHG





,QWUDFHOOXODUF\WRNLQHDQDO\VLV

7KH SKHQRW\SH RI UHVSRQGLQJ7FHOOV ZDV DQDO\]HG E\ ,&6 DQG IORZ F\WRPHWULF

DQDO\VLV DV GHVFULEHG HOVHZKHUH^ ZLWK PLQRU PRGLILFDWLRQV 3%0& ZHUH 21

LQFXEDWHGLQWKHSUHVHQFHRIVWLPXODWRU\DQWLERGLHVVSHFLILFIRU&'DQG&'G

—JPO HDFK ZLWK PHGLXP DORQH QHJDWLYH FRQWURO —JPO 6(% SRVLWLYH

FRQWURO RU VWLPXODWHG ZLWK —JPO RI WKH VDPH  SHSWLGH SRROV DV XVHG LQ WKH

(/,6327DVVD\DVGHVFULEHGDERYH&HOOVZHUHVWDLQHGZLWKGLUHFWO\FRQMXJDWHG

DQWLERGLHV %HFWRQ 'LFNLQVRQ 3KDUPLQJHQ &$ 86$ WR &' SHULGLQLQ

FKORURSK\OOSURWHLQ FRPSOH[ 3HU&3ODEHOHG &' SK\FRHU\WKULQF\DQLQ 

3(&<ODEHOHG&'DOORSK\FRF\DQLQ$3&&<ODEHOHGDQGWR&'DQG

&'(QHUJ\&RXSOHG'\H(&'ODEHOHG%HFNPDQ&RXOWHU0DUVHLOOH)UDQFH IRUH[FOXVLRQRIPRQRF\WHVDQG%FHOOV7KHFHOOVZHUHIL[HGDQGSHUPHDELOLVHG

ZLWK&\WRIL[&\WRSHUPEXIIHU%HFWRQ'LFNLQVRQDQGVWDLQHGLQWUDFHOOXODUO\ZLWK

DQWLERGLHVVSHFLILFIRU,)1Ȗ$3&ODEHOHG,/3(ODEHOHG71)ĮIOXRUHVFHLQ

LVRWKLRF\DQDWH),7&ODEHOHGLQ3HUPZDVKVROXWLRQ%HFWRQ'LFNLQVRQ

±HYHQWVZHUHDFTXLUHGRQD)$&6$ULD%HFWRQ'LFNLQVRQ&$86$

)$&6 'LYD %HFNWRQ 'LFNLQVRQ DQG 6XPPLW 'DNR &RORUDGR 86$ VRIWZDUH

ZDVXVHGIRUDQDO\VLV

(/,6$

$QWLERGLHV WR +,9 (QY JS FQ LQ VHUXP ZHUH PHDVXUHG E\ (/,6$

3ODWHVZHUHFRDWHGRYHUQLJKWZLWK(QYLQP01D+&2_DQGZHUHEORFNHGIRU

KRXUZLWKQRQIDWPLONEHIRUHDSSOLFDWLRQRIVHUXPVHULDOO\GLOXWHGLQ%6$

3%6EXIIHU$IWHUKRXU—JPODQWLKXPDQ,J*RU,J0+53FRQMXJDWHZDVDGGHG

IRUKRXUEHIRUHDGGLWLRQRIXOWUD70%(/,6$GHYHORSPHQWUHDJHQW7KHUHDFWLRQ

ZDVVWRSSHGE\DGGLWLRQRI0+_62_5HVXOWVZHUHH[SUHVVHGDV,J*DQG,J0

HQGSRLQWGLOXWLRQWLWHUV

,P'1$GD\  %RRVWGD\  UHWURVSHFWLYH

JURXS

%RRVWGD\  QHZ GDWD JURXS

   (/,VSRW

ZHHNV

,P1<9$&GD\  %RRVWGD\  UHWURVSHFWLYH JURXS

(/,VSRW ,&6

7DWWRR '1$GD\ 

(/,6$

(/,6$

)LJ6FKHPDWLFUHSUHVHQWDWLRQRIWKHH[SHULPHQWDOVHWXS$UURZVLQGLFDWHWLPH

SRLQWV RI YDFFLQDWLRQ HLWKHU E\ '1$ WDWWRRLQJ LP '1$ LQMHFWLRQ RU 1<9$&

LQMHFWLRQ



,175$'(50$/'1$7$7722,1*35,0(652%8677&(//5(63216(6,10$&&$&$08/$77$

)LJ3KRWRJUDSKVRIWKHVNLQDUHDVWUHDWHGE\'1$WDWWRRLQJRIUHSUHVHQWDWLYH

DQLPDOVWDNHQHLWKHUGLUHFWO\DIWHUXSSHUURZRUWKUHHGD\VDIWHUORZHUURZWKH

WUHDWPHQW7KHORZHUDQGXSSHUURZVGLVSOD\WKHVDPHDQLPDOV











  

)LJ  4XDQWLILFDWLRQ RI DQWLJHQ VSHFLILF F\WRNLQH VHFUHWLQJ FHOOV E\ (/,VSRW DW

ZHHNDQGZHHNSRVWYDFFLQDWLRQLQWKH'1$WDWWRRYDFFLQDWHGQ JURXS

LP '1$ YDFFLQDWHG Q  JURXS DQG 1<9$& YDFFLQDWHG JURXS Q  7KH

DVVD\V ZHUH SHUIRUPHG RQ IUHVKO\ LVRODWHG 3%0& E\ $ ,)1Ȗ DQG % ,/

(/,VSRWDVVD\VDFFRUGLQJWRWKHPDQXIDFWXUHU¶VLQVWUXFWLRQV8&\WHFK8WUHFKW

7KH 1HWKHUODQGV &\WRNLQH UHVSRQVHV RI LQGLYLGXDO DQLPDOV DUH SORWWHG DV

GLDPRQGV WDWWRR JURXS FLUFOHV ,0 JURXS DQG WULDQJOHV 1<9$& JURXS7KH

JURXSDYHUDJHVDUHLQGLFDWHGDVWKLFNFORVHGOLQHVWDWWRRJURXSGRWWHGOLQHV,0

JURXSDQGWKLQFORVHGOLQHV1<9$&JURXS5HVXOWVDUHH[SUHVVHGDVWKHPHDQ

QXPEHURIVSRWIRUPLQJFHOOV6)&SHU^3%0&IURPWULSOLFDWHVDPSOHVPLQXV

EDFNJURXQG YDOXHV PHDQ QXPEHU RI 6)& SOXV [6' RI WULSOLFDWH VDPSOHV ZLWK

PHGLXPDORQH7KHUHSUHVHQWHGVSRWQXPEHULVFDOFXODWHGE\VXPPDWLRQRIWKH

UHDGRXWLQVHSDUDWHDVVD\VHDFKGRQHXSRQVWLPXODWLRQZLWKDGLVWLQFWIUDFWLRQRI

WKHFRPSOHWHSHSWLGHSRRO)RUDUHSUHVHQWDWLRQRIVSRWQXPEHUVXSRQVWLPXODWLRQ

ZLWKWKHVHSDUDWHIUDFWLRQVUHSUHVHQWLQJSROJDJQHIDQGHQYVHH6XSSO)LJ

GLUHFWO\DIWHUWDWWRRLQJ

,)1Ȗ ,/











  

ZHHNVDIWHUVWDUWRIWKHYDFFLQDWLRQ

6)&PLOOLRQ3%0&

GD\VDIWHUWDWWRRLQJ

$ %





6XSSO)LJ)ORZF\WRPHWULFDQDO\VLVRISHSWLGHVWLPXODWHG3%0&IRU71)ĮDQG

,)1ȖVHFUHWLRQ,QHDFKDQLPDO7FHOOUHVSRQVHVDJDLQVWHDFKRIIUDFWLRQVRIWKH

FRPSOHWHSHSWLGHSRROZHUHWHVWHGWRJHWKHUFRYHULQJWKHZKROHYDFFLQH)RUHDFK

DQLPDOWKHSORWLQWKHULJKWFROXPQGLVSOD\VF\WRNLQHVHFUHWLRQXSRQVWLPXODWLRQ

ZLWKWKHPRVWVWLPXODQWSHSWLGHIUDFWLRQRIWKHSRRO$OOSORWVDUHJDWHGRQ&'

7FHOOV7KHQDPHRIWKLVIUDFWLRQLVSULQWHGLQVLGHHDFKSORWLQGLFDWLQJWKH+,9

DQWLJHQWKDWLWSDUWO\UHSUHVHQWV7KHSORWVLQWKHOHIWFROXPQGLVSOD\VF\WRNLQH

VHFUHWLRQZLWKRXWSHSWLGHVWLPXODWLRQ6HHVXSSOHPHQWDU\GDWDIRUWHFKQLFDOGHWDLOV

RIWKHDVVD\

,)1Ȗ

71)Į

 



















 



$QLPDO

$QLPDO

$QLPDO

$QLPDO

$QLPDO

8QVWLPXODWHG

&RQWURO

3HSWLGHSRRO 6WLPXODWHG

3RO

1HI

3RO

1HI

3RO

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^

^

^

^

^

^ ^ ^ ^



,175$'(50$/'1$7$7722,1*35,0(652%8677&(//5(63216(6,10$&&$&$08/$77$

,)1Ȗ

*DJ

3RO

1HI

(QY

3HSWLGH3RROV

,QWUDPXVFXODU '1$LQMHFWLRQ

,QWUDPXVFXODU 1<9$&LQMHFWLRQ ,QWUDGHUPDO

'1$7DWWRR

,/

:HHNVSRVWYDFFLQDWLRQ 6)&3%0&











  











  











  











  











  











  











  











  











  











  











  











  

,)1DŽ











  











  











  











  











  











  











  











  











  











  











  











  

*DJ

3RO

1HI

(QY

6)&3%0&

:HHNVSRVWYDFFLQDWLRQ



6XSSO )LJ  8SSHU SDQHO ,)1Ȗ SURGXFWLRQ E\ SHULSKHUDO EORRG PRQRQXFOHDU

FHOOV3%0&WR(QY*DJ3RODQG1HISHSWLGHVDVPHDVXUHGE\(/,VSRWDVVD\

%DFNJURXQGUHVSRQVHVPHDQQXPEHURI6)&SOXV[6'RIWULSOLFDWHVDPSOHVZLWK

PHGLXPDORQHZHUHVXEWUDFWHG7KHVROLGOLQHUHSUHVHQWVWKHPHDQUHVSRQVHRIDOO

DQLPDOVZLWKLQRQHJURXS/RZHUSDQHOWKHVDPHGLVSOD\EXWIRU,/SURGXFWLRQ

5HIHUHQFHV

 *XUXQDWKDQ 6 ' 0 .OLQPDQ DQG 5$ 6HGHU  '1$ YDFFLQHV

LPPXQRORJ\DSSOLFDWLRQDQGRSWLPL]DWLRQ $QQX5HY,PPXQRO



 5RWKHQIXVVHU6(7XPD0:DJQHU6(QGUHVDQG*+DUWPDQQ

5HFHQW DGYDQFHV LQ LPPXQRVWLPXODWRU\ &S* ROLJRQXFOHRWLGHV &XUU 2SLQ 0RO

7KHU

 -RKQVWRQ 6 $ $ 0 7DODDW DQG 0 - 0F*XLUH  *HQHWLF

LPPXQL]DWLRQZKDW¶VLQDQDPH"$UFK0HG5HV

 %LQV $ ' $ -RUULWVPD 0 & :RONHUV & ) +XQJ 7 & :X 7 1

6FKXPDFKHUDQG-%+DDQHQ$UDSLGDQGSRWHQW'1$YDFFLQDWLRQVWUDWHJ\

GHILQHGE\LQYLYRPRQLWRULQJRIDQWLJHQH[SUHVVLRQ1DW0HG

 6X/0*UDI<=KDQJ+YRQ%ULHVHQ+;LQJ-.RVWOHU+0HO]O

+:ROI<6KDRDQG5:DJQHU&KDUDFWHUL]DWLRQRIDYLUWXDOO\IXOOOHQJWK

KXPDQLPPXQRGHILFLHQF\YLUXVW\SHJHQRPHRIDSUHYDOHQWLQWHUVXEW\SH&%¶ UHFRPELQDQWVWUDLQLQ&KLQD-9LURO

 %DOOD-KDJMKRRUVLQJK 6 6 * .RRSPDQ 3 0RRLM : .RRUQVWUD 6

0F&RUPDFN-:HEHU*3DQWDOHRDQG-/+HHQH\/RQJWHUPSHUVLVWHQFH

RI+,9YDFFLQHLQGXFHG&'&'5$&'/&&5PHPRU\7KHOSHUFHOOV

$,'6