Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.

CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

%URDGHQLQJVSHFLILFLW\DQGHQKDQFLQJF\WRWR[LFLW\RIDGRSWLYH7FHOOVIRUQDVRSKDU\QJHDO
FDUFLQRPDLPPXQRWKHUDS\

'DPLDQD$QWRQLD)Dq'HERUD0DUWRUHOOL.DW\0DVWRUFL(OHQD0XUDUR-HVVLFD'DO&RO
*LRYDQQL)UDQFKLQ/XLJL%DU]DQ(OLVD&RPDUR(PDQXHOD9DFFKHU$QWRQLR5RVDWR5LFFDUGR
'ROFHWWL

&DQFHU%LR,PPXQRWKHUDS\8QLW'LYLVLRQRI5DGLRWKHUDS\0HGLFDO2QFRORJ\'LYLVLRQ$
&52,5&&61DWLRQDO&DQFHU,QVWLWXWH$YLDQR,WDO\2WRODU\QJRORJ\$]LHQGD2VSHGDOLHUD6
0DULDGHJOL$QJHOL3RUGHQRQH,WDO\,VWLWXWR2QFRORJLFR9HQHWR,29,5&&63DGXD,WDO\

5XQQLQJWLWOH,PSURYHGDGRSWLYHFHOOWKHUDS\IRUQDVRSKDU\QJHDOFDUFLQRPD

.H\ZRUGV$GRSWLYHFHOOWKHUDS\(SVWHLQ%DUUYLUXVGR[RUXELFLQLPPXQRJHQLFFHOOGHDWK
QDVRSKDU\QJHDOFDUFLQRPD

)LQDQFLDOVXSSRUW7KLVVWXG\ZDVVXSSRUWHGE\DJUDQWIURPWKH$VVRFLD]LRQH,WDOLDQDSHUOD
5LFHUFDVXO&DQFURFRQWUDFWWR5'ROFHWWL(OHQD0XUDURZDVVXSSRUWHGE\D
)RQGD]LRQH8PEHUWR9HURQHVL)HOORZVKLS


3RWHQWLDOFRQIOLFWVRILQWHUHVW7KH$XWKRUVGHFODUHQRSRWHQWLDOFRQIOLFWRILQWHUHVW


:RUGFRXQWH[FOXGLQJUHIHUHQFHV

7RWDOQXPEHURIILJXUHVDQGWDEOHV

&RUUHVSRQGLQJ$XWKRU5LFFDUGR'ROFHWWL0'&DQFHU%LR,PPXQRWKHUDS\8QLW&HQWURGL5LIHULPHQWR
2QFRORJLFR,5&&61DWLRQDO&DQFHU,QVWLWXWH9LD)*DOOLQL$YLDQR31,WDO\3K
ID[HPDLOUGROFHWWL#FURLW


1

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

$EVWUDFW

$OWKRXJKSURPLVLQJFOLQLFDOUHVSRQVHVWRDGRSWLYHLPPXQRWKHUDS\IRUQDVRSKDU\QJHDOFDUFLQRPD

13&DUHVWLOOOLPLWHGE\WKHUHVWULFWHGQXPEHURI(SVWHLQ%DUUYLUXV(%9DQWLJHQVWKDWFDQEH

WDUJHWHGDQGWKHLUSRRULPPXQRJHQLFLW\2XUSUHYLRXVZRUNLQGLFDWHGWKDWWKHLPPXQRJHQLF

IHDWXUHVRIWKH13&DVVRFLDWHGYLUDODQWLJHQ%$5)PD\EHH[SORLWHGIRULPPXQRWKHUDSHXWLF

SXUSRVHV1HYHUWKHOHVV7FHOOOLQHVREWDLQHGZLWKFXUUHQWSURWRFROVLQFOXGHRQO\QHJOLJLEOH

QXPEHUVRI%$5)VSHFLILFF\WRWR[LF7O\PSKRF\WHVSRLQWLQJWRWKHQHHGWRHQULFKWKHVHHIIHFWRUV

LQ%$5)VSHFLILFLWLHV&RQVLGHULQJWKDWLQ%O\PSKRF\WHV%$5)LVPDLQO\DO\WLF(%9DQWLJHQ

ZHWHVWHGGLIIHUHQW(%9O\WLFF\FOHLQGXFHUV73$EXW\ULFDFLGGR[RUXELFLQDQGFLVSODWLQXVHGDW

VXERSWLPDOFRQFHQWUDWLRQVIRUWKHLUDELOLW\WRXSUHJXODWH%$5)H[SUHVVLRQLQO\PSKREODVWRLG%

FHOOOLQHV/&/VWKHFRPPRQO\XVHGDQWLJHQSUHVHQWLQJFHOOVZLWKRXWFRPSURPLVLQJWKHLU

VXUYLYDO7KH/&/VWUHDWHGZLWKGR[RUXELFLQ';/&/VFDQUHSURGXFLEO\DQGHIILFLHQWO\JHQHUDWH

(%9VSHFLILFHIIHFWRUVHQULFKHGLQ%$5)VSHFLILFLWLHVIURPERWKKHDOWK\GRQRUVDQG13&

SDWLHQWV7KHVH';/&/VDOVRKDGPRUHSURQRXQFHGLPPXQRJHQLFSURSHUWLHVLQFOXGLQJ+/$FODVV

,XSUHJXODWLRQDQGH[SUHVVLRQRILPPXQRJHQLFFHOOGHDWKPDUNHUVVXFKDVHQKDQFHGFDOUHWLFXOLQ

H[SRVXUHDQG+0*%UHOHDVH,QSDUWLFXODUGR[RUXELFLQWULJJHUVDQ+0*%DXWRFULQHSDUDFULQH

ORRSZLWKLWVUHFHSWRU7/5ZKLFKLVDOVRXSUHJXODWHGLQ';/&/VDQGLVUHVSRQVLEOHIRU1)κ%

DFWLYDWLRQDQGDGHOD\HGDSRSWRVLVWKDWDOORZVDSURORQJHGVWLPXODWLRQRI(%9VSHFLILF7FHOO

SUHFXUVRUV7KLVSURWRFROPD\WKXVFRQVWLWXWHDYDOLGDOWHUQDWLYHWRWKHXVHRIHQJLQHHUHG/&/VWR

JHQHUDWH(%9VSHFLILF7FHOOOLQHVIRUDGRSWLYHLPPXQRWKHUDS\EHLQJUHODWLYHO\VLPSOHHDVLO\

XSJUDGDEOHWR*03VWDQGDUGVDQGWKHUHIRUHPRUHEURDGO\DSSOLFDEOH

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

,QWURGXFWLRQ

1DVRSKDU\QJHDOFDUFLQRPD13&LVDPDOLJQDQWWXPRUDULVLQJIURPWKHPXFRVDOHSLWKHOLXPRIWKH

QDVRSKDU\Q[ZKLFKLVFKDUDFWHUL]HGE\DPXOWLIDFWRULDOHWLRORJ\DQGDSHFXOLDUJHRJUDSKLFDO

GLVWULEXWLRQ,QIDFW13&LVUHODWLYHO\UDUHLQPRVWSDUWVRIWKHZRUOGSDUWLFXODUO\LQ(XURSHDQG

1RUWK$PHULFDZKHUHDVLWKDVDKLJKLQFLGHQFHLQ6RXWKHUQ&KLQDDQGVRXWKHDVW$VLDZKHUHLW

FRQVWLWXWHVDVLJQLILFDQWKHDOWKSUREOHP(1, 2). 13&LVFKDUDFWHUL]HGE\VHYHUDOKLVWRSDWKRORJLFDO

HQWLWLHVWKHXQGLIIHUHQWLDWHGIRUPEHLQJWKHPRVWIUHTXHQW(3, 4)7KHVWDQGDUGWUHDWPHQWIRU13&LV

UDGLRWKHUDS\ZLWKRUZLWKRXWFKHPRWKHUDS\GHSHQGLQJRQGLVHDVHVWDJH$OWKRXJKH[FHOOHQWFRQWURO

FDQEHDFKLHYHGZLWKDFRPELQDWLRQRIUDGLRDQGFKHPRWKHUDS\ORFDOUHFXUUHQFHVWLOORFFXUVLQ

RIFDVHV(3, 4)DQGSURJQRVLVRISDWLHQWVZLWKPHWDVWDWLFGLVHDVHUHPDLQVSRRU(3, 4)+HQFH

QHZORZWR[LFLW\WKHUDSHXWLFVWUDWHJLHVDUHVWURQJO\QHHGHGWRLPSURYHWKHFOLQLFDOFRQWURORI13&

,QDGGLWLRQWRGLHWDU\HQYLURQPHQWDODQGJHQHWLFULVNIDFWRUVWKLVPDOLJQDQF\VKRZVD

VWURQJDVVRFLDWLRQZLWK(SVWHLQ%DUUYLUXV(%9DVVXSSRUWHGE\WKHREVHUYDWLRQWKDWWKH(%9

JHQRPHLVPRQRFORQDODQGGHWHFWHGLQWXPRUFHOOVRIYLUWXDOO\DOOFDVHVRI13&(5, 6)(%9

,QIHFWLRQVRI(%9LQ13&FHOOVDUHFKDUDFWHUL]HGE\WKHVRFDOOHGW\SH,,ODWHQF\ZLWKWKH

H[SUHVVLRQRIDOLPLWHGVHWRIODWHQWSURWHLQVLQFOXGLQJ(%1$/03/03DQG%$5)

ZKLFKPD\FRRSHUDWLYHO\FRQWULEXWHWR(%9GULYHQHSLWKHOLDOFHOOWUDQVIRUPDWLRQ7KHVHYLUDO

DQWLJHQVDUHDOPRVWFRQVWDQWO\DOEHLWKHWHURJHQHRXVO\H[SUHVVHGE\13&FHOOVDQGKDYHEHFRPH

DWWUDFWLYHWDUJHWVIRULPPXQRWKHUDS\$GRSWLYHLQIXVLRQRIDXWRORJRXV(%9VSHFLILF7FHOOVKDV

UHFHQWO\HPHUJHGDVDQHIIHFWLYHWRROIRUWKHWUHDWPHQWRI(%9GULYHQPDOLJQDQFLHVDQGLWVHIILFDF\

LQO\PSKRLGWXPRUVVXFKDVSRVWWUDQVSODQWDWLRQO\PSKRSUROLIHUDWLYHGLVHDVHKDVEHHQFRQYLQFLQJO\

GHPRQVWUDWHG(7)3KDVH,,,FOLQLFDOVWXGLHVKDYHDOVRSURYLGHGHYLGHQFHVXSSRUWLQJWKHSRWHQWLDO

DSSOLFDWLRQRIF\WRWR[LF7O\PSKRF\WH&7/±EDVHGWKHUDS\WR13&SDWLHQWVHLWKHUDVVLQJOH

WUHDWPHQWIRUUHFXUUHQWRUPHWDVWDWLFGLVHDVHRULQFRPELQDWLRQZLWKFKHPRWKHUDS\(7-10)

1HYHUWKHOHVVWKHUDWHRIFOLQLFDOUHVSRQVHVREWDLQDEOHLQ13&SDWLHQWVE\DGRSWLYHLPPXQRWKHUDS\
3

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

LVXQVDWLVIDFWRU\7KLVPD\EHGXHWRWKHOLPLWHGVHWRIYLUDODQWLJHQVH[SUHVVHGE\13&FHOOVDV

ZHOODVWRWKHLUSRRULPPXQRJHQLFLW\DVVKRZQE\WKHVXEGRPLQDQFHRIVSHFLILF7FHOOUHVSRQVHV

XVXDOO\HOLFLWHGE\/03DQG/03(11)7KHVHOLPLWDWLRQVDUHVWLPXODWLQJWKHH[SORUDWLRQRI

QRYHOVWUDWHJLHVWRLPSURYHWKHHIILFDF\RIDGRSWLYHLPPXQRWKHUDS\IRU13&*HQHWLFPDQLSXODWLRQ

RIGHQGULWLFFHOOVDQGO\PSKREODVWRLGFHOOOLQHV/&/VWKHFRQYHQWLRQDODQWLJHQSUHVHQWLQJFHOOV

$3&VXVHGLQFXUUHQWSURWRFROVZDVUHFHQWO\VKRZQWRLQGXFH(%9VSHFLILF&7/FXOWXUHV

HQULFKHGLQVSHFLILFLWLHVIRU/03DQWLJHQVDEOHWRPHGLDWHHQKDQFHGFOLQLFDOUHVSRQVHVLQO\PSKRPD

SDWLHQWV(12)KRZHYHUWKHDSSOLFDELOLW\RIVXFKSURFHGXUHLVOLPLWHGWRYHU\IHZVSHFLDOL]HGFHQWHUV

2QHRIWKHSRVVLEOHVWUDWHJLHVWRRYHUFRPHWKHVHOLPLWDWLRQVLVWKHH[SORLWDWLRQRIDGGLWLRQDO

YLUDOSURWHLQVH[SUHVVHGE\13&FHOOVZKLFKPD\VHUYHDVWXPRUDVVRFLDWHGDQWLJHQVWREHWDUJHWHG

IRULPSURYHG&7/LQGXFWLRQDQGH[SDQVLRQ7KHRQFRJHQLF(%9SURWHLQ%$5)LVH[SUHVVHGLQWKH

PDMRULW\RI13&FDVHV(13-16)DQGPD\FRQVWLWXWHDQDWWUDFWLYHWKHUDSHXWLFWDUJHW,QSDUWLFXODUZH

KDYHSUHYLRXVO\GHPRQVWUDWHGWKDW13&SDWLHQWVVKRZVWURQJVSRQWDQHRXV&'DQG&'7FHOO

UHVSRQVHVDJDLQVW%$5)SURWHLQDQGGHULYHGHSLWRSHVLQGLFDWLQJWKDW%$5)LVDQDWXUDOO\

LPPXQRJHQLFSURWHLQWKDWPD\EHWDUJHWHGE\VSHFLILF&7/V(17),QGHHGZHKDYHDOVRVKRZQWKDW

%$5)VSHFLILF&7/VFDQEHHDVLO\JHQHUDWHGIURP(%9GRQRUVDQLPSRUWDQWSUHUHTXLVLWHWR

H[SORLW%$5)LPPXQRJHQLFLW\IRULPPXQRWKHUDSHXWLFSXUSRVHV1HYHUWKHOHVV(%9VSHFLILF7

FHOOOLQHVJHQHUDWHGE\FXUUHQWSURWRFROVEDVHGRQWKHXVHRIDXWRORJRXV(%9/&/VDV$3&VVKRZ

RQO\YHU\OLPLWHGVSHFLILFLWLHVIRU%$5)LIDQ\(17)

2QWKHVHJURXQGVZHSHUIRUPHGDVWXG\DLPLQJDWGHYHORSLQJDQHZDQGEURDGO\DSSOLFDEOH

SURWRFROIRUDGRSWLYHLPPXQRWKHUDS\RI13&EDVHGRQWKHJHQHUDWLRQRI7FHOOOLQHVHQULFKHGLQ

%$5)VSHFLILFHIIHFWRUV&RQVLGHULQJWKDWLQ%O\PSKRF\WHV%$5)LVPDLQO\DO\WLF(%9

DQWLJHQ(18)ZHGHYLVHGDVWUDWHJ\EDVHGRQWKHXVHRIORZGRVHVRI(%9O\WLFF\FOHLQGXFHUVWR

XSUHJXODWH%$5)LQ/&/VZLWKRXWLQGXFLQJPDVVLYHFHOODSRSWRVLVRUDFRPSOHWH(%9O\WLF

UHSOLFDWLRQWRDOORZWKHVHFHOOVWRHIIHFWLYHO\SUHVHQW%$5)SHSWLGHVWRJHWKHUZLWKRWKHUWDUJHW

HSLWRSHV7RWKHVHHQGVZHLQYHVWLJDWHGGLIIHUHQW(%9O\WLFF\FOHLQGXFHUVVXFKDV73$ZLWK
4

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

EXW\ULFDFLGGR[RUXELFLQDQGFLVSODWLQ(19, 20)ZKLFKZHUHXVHGDWVXERSWLPDOFRQFHQWUDWLRQVWR

XSUHJXODWH%$5)H[SUHVVLRQLQ/&/VZLWKRXWFRPSURPLVLQJFHOOVXUYLYDO2XUUHVXOWV

GHPRQVWUDWHWKDW/&/VWUHDWHGZLWKGR[RUXELFLQFDQUHSURGXFLEO\DQGHIILFLHQWO\JHQHUDWH(%9

VSHFLILFHIIHFWRUVHQULFKHGLQ%$5)VSHFLILFLWLHVIURPERWKKHDOWK\GRQRUVDQG13&SDWLHQWV

0RUHRYHUGR[RUXELFLQWUHDWHG/&/VDUHDOVRHQGRZHGZLWKPRUHSURQRXQFHGLPPXQRJHQLF

SURSHUWLHVDOORZLQJWKHJHQHUDWLRQRI(%9VSHFLILF&7/VZLWKHQKDQFHGHIIHFWRUIXQFWLRQV2XU

SURWRFROPD\WKHUHIRUHFRQVWLWXWHDYDOLGDOWHUQDWLYHWRWKHXVHRIHQJLQHHUHG/&/VWRJHQHUDWH

(%9VSHFLILF7FHOOOLQHVIRUDGRSWLYHLPPXQRWKHUDS\EHLQJUHODWLYHO\VLPSOHDQGGHVLJQHGWR

IXOO\H[SORLWWKHLPPXQRJHQLFSURSHUWLHVRIGR[RUXELFLQDGUXJODUJHO\XVHGLQWKHFOLQLF7KHVH

FKDUDFWHULVWLFVPDNHRXUSURWRFROHDVLO\XSJUDGDEOHWR*03VWDQGDUGVDQGWKHUHIRUHPRUHEURDGO\

DSSOLFDEOH

0DWHULDODQGPHWKRGV

&HOOOLQHVDQGFXOWXUHFRQGLWLRQV

7KHIROORZLQJFHOOOLQHVDOOREWDLQHGLQZHUHXVHGF13&REWDLQHGIURP7

2RND/\RQ*UDQWDPDQWOHFHOOO\PSKRPD$&&'06=7$NLQGJLIWIURP0*

0DVXFFL6WRFNKROP.$&&'06=GRQRUGHULYHG(%9LQIHFWHG/&/VJHQHUDWHGE\in

vitroWUDQVIRUPDWLRQRI%VZLWKWKH%(%9VWUDLQ6KRUWWDQGHPUHSHDW675ORFLIRUFHOOOLQHV

DXWKHQWLFDWLRQZHUHHYDOXDWHGIRUDOOFHOOOLQHVLQRXUODELQDQGDWWKHHQGRIWKHVWXG\LQ

E\XVLQJWKH*HQH3ULQW6\VWHP3URPHJD$OOFHOOOLQHVZHUHFXOWXUHGLQ530,*LEFR

FRQWDLQLQJIHWDOERYLQHVHUXP*LEFRP0/JOXWDPLQHμJPOVWUHSWRP\FLQDQG

,8POSHQLFLOOLQ6LJPDZLWKWKHH[FHSWLRQRI*UDQWDFXOWXUHGLQFRPSOHWH'XOEHFFR¶V

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

0RGLILHG(DJOH¶V0HGLXP&DPEUH['RQRUDQGSDWLHQWGHULYHG%$5)VSHFLILF&7/OLQHVZHUH

FXOWXUHGLQ&HOO*URŠ*03'&&HOO*HQL[VXSSOHPHQWHGZLWKμJPOVWUHSWRP\FLQDQG

,8POSHQLFLOOLQ6LJPD/&/VZHUHVHHGHGLQFRPSOHWH530,PHGLXP[FHOOVPO

DQGWUHDWHGZLWKHLWKHUQJPO2WHWUDGHFDQR\OSKRUERODFHWDWH73$DQG—0VRGLXP

EXW\UDWH1D%6LJPDIRUKRXUV'R[RUXELFLQ';Q0IRUKRXUVFLVSODWLQ&63—0

IRUKRXUV$IWHU';DQG&63WUHDWPHQWFHOOVZHUHZDVKHGRQFHDQGFXOWXUHGLQIUHVKFRPSOHWH

PHGLXPIRUIXUWKHUKRXUV';DQG&63ZHUHSURYLGHGE\WKHDQWLFDQFHUGUXJXQLWRIRXU

LQVWLWXWLRQ

*HQHUDWLRQRI(%9VSHFLILF&7/OLQHV

'RQRUDQGSDWLHQWGHULYHG%$5)VSHFLILF&7/VZHUHJHQHUDWHGDQGZHHNO\UHVWLPXODWHGXVLQJ

DV$3&VDXWRORJRXV/&/VWUHDWHGRUQRWZLWKVXERSWLPDOFRQFHQWUDWLRQVRI73$1D%';RU&63

$IWHUWUHDWPHQW/&/VZHUHȖLUUDGLDWHG*\EHIRUHWKHILUVWVWLPXODWLRQDQG*\EHIRUHHDFK

RWKHUUHVWLPXODWLRQ,/QJPOZDVDGGHGWRWKHFXOWXUHVWDUWLQJIURPGD\WKDQGIUHVKPHGLXP

ZDVUHSODFHGHYHU\GD\V(IIHFWRUVZHUHFRFXOWXUHGZLWK$3&VDWD7FHOO/&/UDWLR

0XOWLVSHFWUDOLPDJLQJIORZF\WRPHWU\

7KHIROORZLQJIOXRUHVFHQWFRQMXJDWHGPRQRFORQDODQWLERGLHVZHUHXVHGDQWL±&'3&PRXVH

,J*FORQH6)&,7K\'%HFNPDQ&RXOWHUDQWL±&'3(PRXVH,J*FORQH+,%

H%LRVFLHQFHDQWL±JUDQ]\PHȕ±),7&PRXVH,J*FORQH*%%'3KDUPLQJHQ7RGHWHUPLQH

WKH7$3&FRQMXJDWHIRUPDWLRQ&'7FHOOVDQGDXWRORJRXV%/&/DQGWRHQXPHUDWHJUDQ]\PH

ȕJUDQXOHVDF\WRWR[LFLW\DVVD\ZDVSHUIRUPHG%ULHIO\DIWHUKRXUVFRFXOWXUHDXWRORJRXV/&/7

FHOOV[FHOOVFRQGLWLRQZHUHVWDLQHGZLWKDQWL±&'3&DQGDQWL±&'3(DQWLERGLHVLQDQ

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

DSSURSULDWHYROXPHRIUDEELWVHUXPDQG3%67KHQFHOOVZHUHLQFXEDWHGZLWK

IL[DWLRQSHUPHDELOL]DWLRQEXIIHUIRUPLQXWHVDWƒ&ZDVKHGWZLFHDQGODEHOHGZLWKanti–

JUDQ]\PHȕDQWLERG\LQWKHSUHVHQFHRIUDEELWVHUXPLQ3%6DWƒ&IRUPLQXWHVDQGDIWHU

WZRZDVKHVFHOOVZHUHUHVXVSHQGHGLQ3%6ZLWKSDUDIRUPDOGHK\GH&HOOVZHUHUXQRQ

,PDJH6WUHDP;F\WRPHWHUXVLQJWKH,163,5(VRIWZDUHDQGLPDJHVZHUHDQDO\]HGXVLQJWKH,'($6

VRIWZDUH$PQLV&HOOVZHUHH[FLWHGZLWKDQPODVHUP:LQWHQVLW\%ULJKWILHOGVLGH

VFDWWHUDQGIOXRUHVFHQWFHOOLPDJHVZHUHDFTXLUHGDWîPDJQLILFDWLRQ2QO\HYHQWVZLWKEULJKWILHOG

DUHDV!ȝPH[FOXGLQJGHEULVDQGQRQVDWXUDWLQJSL[HOVZHUHFROOHFWHG,Q7$3&ELQGLQJ

H[SHULPHQWV[HYHQWVZHUHFROOHFWHGIRUHDFKVDPSOH&HOOVZHUHJDWHGIRUIRFXVHGSRSXODWLRQV

DQGGRXEOHWVFRQWDLQLQJDWOHDVWRQH7FHOOZHUHJDWHGIURPDPRQJDOOFHOOV,QWUDFHOOXODUJUDQ]\PH

ȕJUDQXOHVIRUPDWLRQZDVGHWHUPLQHGE\VX%XODUORFDOL]DWLRQDQGVSRWFRXQWH[SHULPHQWV

HYHQWVZHUHFROOHFWHGIRUHDFKVDPSOH&\WRSODVPLFORFDOL]DWLRQRIWKHJUDQXOHVZDVPHDVXUHGXVLQJ

WKH,'($6VRIWZDUH³LQWHUQDOL]DWLRQDOJRULWKP´GHILQHGDVWKHUDWLREHWZHHQLQWHQVLW\LQVLGHWKH

FHOODQGWKHLQWHQVLW\RIWKHHQWLUHFHOO7KHLQVLGHRIWKHFHOOLVGHILQHGE\WKH³HURVLRQPDVN´WKDW

ILWVWKHFHOOPHPEUDQH&HOOVFRQWDLQLQJVPDOOFRQFHQWUDWHGIOXRUHVFHQWVSRWVKDYHSRVLWLYHVFRUHV

ZKHUHDVFHOOVVKRZLQJOLWWOHGLIIXVHIOXRUHVFHQFHKDYHQHJDWLYHVFRUHV2QO\YLDEOHFHOOVZHUH

VHOHFWHGRQWKHEDVLVRIPRUSKRORJ\6LQJOHVWDLQHGFRQWUROVZHUHXVHGWRFRPSHQVDWHIOXRUHVFHQFH

EHWZHHQFKDQQHOLPDJHVRQDSL[HOE\SL[HOEDVLV

)RUSDWLHQWVDQGGRQRUVJHQHUDWLRQRI+/$$ H[SUHVVLQJF13&FHOOV

TXDQWLWDWLYH5HDOWLPH3&5LPPXQREORWWLQJF\WRWR[LFLW\DVVD\V+0*%UHOHDVHTXDQWLILFDWLRQ

DQG7/5QHXWUDOL]DWLRQH[SHULPHQWVUHIHUWR6XSSOHPHQWDU\PDWHULDO

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

5HVXOWV

'R[RUXELFLQXSUHJXODWHV%$5)P51$H[SUHVVLRQ

'LIIHUHQW(%9O\WLFF\FOHLQGXFHUVZHUHXVHGDWVXERSWLPDOFRQFHQWUDWLRQVWRLQGXFHDPDQO\

DERUWLYHO\WLFF\FOHDQGXSUHJXODWH%$5)H[SUHVVLRQLQ/&/VZLWKRXWLQGXFLQJPDMRUDSRSWRWLF

HIIHFWV$VDILUVWVWHSZHXVHGWKH73$1D%FRPELQDWLRQDVWKHPRVWFRPPRQO\XVHGWUHDWPHQWWR

WULJJHU(%9O\WLFUHDFWLYDWLRQ(2, 21)+HDOWK\GRQRUGHULYHG/&/VWUHDWHGIRUKRXUVZLWK

73$1D%VKRZHGDVLJQLILFDQWO\LQFUHDVHG%$5)P51$H[SUHVVLRQPHDQIROGLQFUHDVH

S”)LJ$1HYHUWKHOHVVWKHWUHDWPHQWDOVRLQGXFHGDEURDGXSUHJXODWLRQRIERWKODWHQW

/03(%1$(%1$DQGO\WLF=(%5$($(%9JHQHVWRJHWKHUZLWKDKLJKO\SURGXFWLYH

O\WLFF\FOH)LJ$6XSSOHPHQWDU\7DEOHDQG)LJ6$ZKLFKZDVLQYDULDEO\DVVRFLDWHGZLWK

PDUNHGOHYHOVRI/&/DSRSWRVLV)LJ%$VWURQJ(%9UHDFWLYDWLRQZDVLQGXFHGDOVRE\&63

ZKHUHDV';WUHDWHG/&/VVKRZHGDPDUNHGLQFUHDVHLQ%$5)P51$H[SUHVVLRQOHYHOVDQGD

ORZHUEXWVWLOOVLJQLILFDQWS”XSUHJXODWLRQRIRWKHU(%9JHQHVZLWKRXWLQGXFLQJDPDVVLYH

(%9O\WLFUHSOLFDWLRQ)LJ$6XSSOHPHQWDU\)LJ$1RWDEO\';KDGOHVVSURQRXQFHGSUR

DSRSWRWLFHIIHFWVDVFRPSDUHGWRWKHRWKHUGUXJVLQYHVWLJDWHGDVVKRZQE\3$53FOHDYDJHDQDO\VLV

)LJ%

(%9VSHFLILF&7/GLIIHUHQWLDWLRQXQDOWHUHGE\';WUHDWHG/&/V

(%9VSHFLILF&7/OLQHVZHUHJHQHUDWHGE\SULPLQJKHDOWK\GRQRUGHULYHGSHULSKHUDOEORRG

PRQRQXFOHDUFHOOV3%0&VZLWKDXWRORJRXV/&/VHLWKHUXQWUHDWHGRUWUHDWHGZLWK73$1D%';

RU&63&'&'UDWLRDQG7FHOOGLIIHUHQWLDWLRQSURILOHZHUHPRQLWRUHGE\PXOWLSDUDPHWULFIORZ

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

F\WRPHWU\DQDO\VLVDWGD\DQG)LJ&7KHSHUFHQWDJHRI&'7FHOOVUDQJHGIURPWR

EHWZHHQWKHILUVWDQGWKHODVWVWLPXODWLRQ&RPSDUDEOHLQFUHDVHVLQ&'7FHOOVZHUHREVHUYHG

DWGD\“ZKHUHDV&'FHOOVUDQJHGEHWZHHQ“)LJ'&RPELQHG

DQDO\VLVRI&&5DQG&'5$GHPRQVWUDWHGWKDWRI&'DQGRI&'7FHOOV

GLVSOD\HGDQ(IIHFWRU0HPRU\SKHQRW\SH(0&&5&'5$)LJ'$QDO\VLVRIDGGLWLRQDO

(0PDUNHUV&'DQG&'/GHPRQVWUDWHGDKLJKSHUFHQWDJHRI&'&'7FHOOVLQDOO

FXOWXUHVLQGLFDWLQJWKDW&'7FHOOVDUHQRWH[KDXVWHG$ERXWRI&'7FHOOVH[SUHVVDOVR

WKH&'//VHOHFWLQ$QDO\VLVRI&7/FXOWXUHVREWDLQHGIURPGLIIHUHQWGRQRUVGLGQRWGLVFORVH

DQ\VLJQLILFDQWGLIIHUHQFHLQWKHGLVWULEXWLRQRI&'DQG&'/LQWKH&&5&'5$&'(0

SRSXODWLRQ6XSSOHPHQWDU\)LJ6%6LPLODUILQGLQJVZHUHREVHUYHGIRU&'7FHOOVQRW

VKRZQ7KHUHIRUHQRVLJQLILFDQWGLIIHUHQFHVZHUHREVHUYHGZLWKUHJDUGWRWKHGLIIHUHQWLDWLRQ

SURILOHRIWKHIRXU&7/FXOWXUHV

';&7/VNLOO7$FHOOVZLWKH[RJHQRXVRUHQGRJHQRXV%$5)SHSWLGHV

(%9VSHFLILF&7/FXOWXUHVZHUHWHVWHGIRUWKHLUDELOLW\WRUHFRJQL]HDQGNLOO7$WDUJHWFHOOV

ORDGHGZLWKNQRZQ(%9GHULYHGSHSWLGHVLQD+/$$ UHVWULFWHGIDVKLRQ)LJ$%,Q

SDUWLFXODU7$FHOOVZHUHORDGHGZLWKWZR%$5)GHULYHGSHSWLGHVSDQGS(17)RUZLWK

WKH/03GHULYHG</4SHSWLGH(22)';&7/VZHUHDEOHWRVSHFLILFDOO\NLOOERWK%$5)DQG

/03ORDGHG7$LQGXFLQJDKLJKHUSHUFHQWDJHRIVSHFLILFO\VLVDVFRPSDUHGWR73$1D%DQG

&63&7/V)LJ%&7/UHVSRQVHVDJDLQVW(%1$)09DQG/03&/*(%9SHSWLGH

HSLWRSHVZHUHDOVRLQYHVWLJDWHG:KLOH/03WDUJHWVZHUHHIILFLHQWO\UHFRJQL]HGE\ERWK';DQG

&63&7/VSEXWQRWE\&75/RU73$1D%&7/V)09ORDGHGFHOOVZHUHO\VHGDW

FRPSDUDEOHOHYHOVE\DOOGLIIHUHQW&7/FXOWXUHVLQYHVWLJDWHG6XSSOHPHQWDU\)LJ6&7RDVVHVV

WKHDELOLW\RI';&7/VWRVSHFLILFDOO\UHFRJQL]HDQGNLOO13&FHOOVVSRQWDQHRXVO\H[SUHVVLQJ(%9

DQWLJHQVWKH+/$$QHJDWLYH13&FFHOOOLQHZDVHQJLQHHUHGWRVWDEO\H[SUHVV+/$$ 
9

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

F$6XSSOHPHQWDU\)LJ6$$VVKRZQLQ)LJXUH&'';&7/VZHUHWKHPRUH

HIILFLHQWDQGVSHFLILFFXOWXUHVLQNLOOLQJ13&FHOOVH[SUHVVLQJ%$5)HQGRJHQRXVO\ZKHUHDVQR

NLOOLQJZDVREVHUYHGDJDLQVWWKHSDUHQWDO+/$$QHJDWLYHF:WFHOOV&RQYHUVHO\FRQWURO

73$1D%DQG&63&7/VLQGXFHGRQO\ORZOHYHOVRIVSHFLILFO\VLVDJDLQVWF$)LJ'

7KHOLPLWHGH[WHQWRI.FHOONLOOLQJIXUWKHUDVVXUHGDERXWWKHVSHFLILFLW\RIO\VLV)LJ(

(%9VSHFLILFF\WRWR[LFDFWLYLW\ZDVDOVRDVVHVVHGE\HQXPHUDWLQJWKHIRUPDWLRQRI/&/7

FHOOGRXEOHWVE\PXOWLVSHFWUDOLPDJLQJIORZF\WRPHWU\$IWHULQFXEDWLRQRIHIIHFWRU7FHOOVZLWK

SHSWLGHSXOVHGDXWRORJRXV/&/VZHREVHUYHGWKDW';&7/VDQGSDUWLFXODUO\%$5)SHSWLGH

ORDGHG/&/VJHQHUDWHGWKHKLJKHVWIUHTXHQF\RIHIIHFWRUWDUJHWGRXEOHWV)LJ$ S”';

&7/VvsDOORWKHU&7/FXOWXUHV6WLPXODWLRQZLWKWKH/03</4SHSWLGHHOLFLWHGFRPSDUDEOH

QXPEHUVRIGRXEOHWVLQDOOFXOWXUHVH[FHSWIRU&7/VJHQHUDWHGZLWKXQWUHDWHG/&/VZKLFKVKRZHG

DORZHUQXPEHURIGRXEOHWV)LJ$

,QWKHVDPHH[SHULPHQWDOFRQGLWLRQVWKHLQWUDFHOOXODUFRQWHQWRIJUDQ]\PHȕJUDQXOHVZDV

HYDOXDWHGLQDOO&7/FXOWXUHVE\VSRWFRXQWLQJDQDO\VLV$VVKRZQLQ)LJXUH%LIFRPSDUHGZLWK

&75/73$1D%DQG&63&7/VDVLJQLILFDQWO\KLJKHUDEVROXWHQXPEHURIJUDQ]\PHȕJUDQXOHV

ZDVGHWHFWHGLQ';&7/VS”VWLPXODWHGZLWKHPSW\/&/VRUZLWK/&/VSXOVHGZLWKYLUDO

SHSWLGHVLQGLFDWLQJWKDW';/&/VDUHJHQHUDOO\PRUHHIILFLHQWO\DUPHGVHHWKHVFDOHRIGLIIHUHQW

JUDSKVLQ)LJ%1RWDEO\';&7/VVWLPXODWHGZLWK%$5)SHSWLGHSXOVHG/&/VVKRZHGDQ

HYHQKLJKHUQXPEHURIJUDQ]\PHȕJUDQXOHVDVFRPSDUHGWRWKRVHVWLPXODWHGZLWK/&/VORDGHG

ZLWK/03SHSWLGHVS”)LJXUH%

';HQKDQFHV/&/LPPXQRJHQLFLW\

7RFKDUDFWHUL]HWKHPHFKDQLVPVXQGHUO\LQJWKHHQKDQFHGIXQFWLRQDOSURSHUWLHVRI';WUHDWHG/&/V

DV$3&VZHLQYHVWLJDWHGWKHSRVVLEOHDELOLW\RI';WRXSUHJXODWH+/$FODVV,H[SUHVVLRQ

&RQVLGHULQJWKDWȖLUUDGLDWLRQLVFXUUHQWO\XVHGWRLQDFWLYDWH/&/VEHIRUH&7/VWLPXODWLRQDQGWKDW
10

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

WKLVSURFHGXUHZDVVKRZQWRLQFUHDVH+/$H[SUHVVLRQ(23, 24)ZHLQYHVWLJDWHGWKHSRVVLEOH

V\QHUJLVPEHWZHHQ';DQGȖLUUDGLDWLRQ+/$$ P51$H[SUHVVLRQZDVWKHUHIRUHPRQLWRUHG

E\T573&5LQ/&/VEHIRUHDQGKRXUVDIWHUȖLUUDGLDWLRQ)LJ$';WUHDWHG/&/VUHYHDOHGD

PDUNHGLQFUHDVHLQ+/$$ H[SUHVVLRQPHDQ IROGDQHIIHFWWKDWSHUVLVWHGDOVRDIWHU

KRXUV)LJ$1RVLJQLILFDQWFKDQJHVZHUHGHWHFWHGLQ73$1D%DQG&63/&/VFRPSDUHGWR

FRQWURO/&/VPHDQ DQGIROGH[SUHVVLRQUHVSHFWLYHO\DWERWKWLPHSRLQWV+/$$ 

XSUHJXODWLRQLQ';/&/VZDVDOVRFRQILUPHGE\IORZF\WRPHWU\DQDO\VLVDWKRXUVDIWHUȖ

LUUDGLDWLRQ)LJ%

5HFHQWHYLGHQFHLQGLFDWHVWKDW';LVDEOHWRLQGXFHLPPXQRJHQLFFHOOGHDWK,&'ERWKin

vitro (25)DQGin vivo (26, 27)DQHIIHFWPDLQO\PHGLDWHGE\GDPDJHDVVRFLDWHGPROHFXODUSDWWHUQV

'$03VLQFOXGLQJFHOOVXUIDFHH[SRVHGFDOUHWLFXOLQ&57VHFUHWHG$73DQGUHOHDVHGKLJK

PRELOLW\JURXSER[SURWHLQ+0*%(28):HWKHUHIRUHH[SORUHGWKHSRVVLELOLW\WKDW';

WUHDWPHQWPD\LQGXFHWKHH[SUHVVLRQRIPROHFXOHVSRWHQWLDOO\DEOHWRHQKDQFH/&/

LPPXQRJHQLFLW\HYHQDWGRVHVLQGXFLQJRQO\DOLPLWHGFHOOGHDWK$QDO\VLVRIFULWLFDO,&'PDUNHUV

GHPRQVWUDWHGWKDW';LQGXFHVDQHDUO\(29)&57H[SRVXUHKRXUVZLWKDERXWRIFHOOV

GLVSOD\LQJD&57PHPEUDQHORFDOL]DWLRQ&RQYHUVHO\RQO\VOLJKWLQFUHDVHVLQ&57H[SRVXUHZHUH

REVHUYHGLQ73$1D%DQG&63/&/VDVFRPSDUHGWRFRQWURO/&/V)LJ%5HOHDVHRI+0*%

LQ/&/FXOWXUHPHGLXPZDVPHDVXUHGE\(/,6$DWWKHHQGRIWUHDWPHQWDVDODWH,&'PDUNHU(30)

)LJ&1RWDEO\WKHH[WHQWRI+0*%UHOHDVHE\';/&/VZDVDERXWIROGKLJKHUWKDQWKDWRI

FRQWURO/&/VZKHUHDVDPRGHUDWHGHFUHDVHZDVREVHUYHGLQERWK73$1D%DQG&63WUHDWHG

/&/VDERXWIROGFKDQJH

7RYHULI\ZKHWKHUWKHREVHUYHG,&'HIIHFWVSHUVLVWHGRUZHUHHQKDQFHGXSRQȖLUUDGLDWLRQ

H[SUHVVLRQRIWKH+63DQG+63SURWHLQVZDVDVVHVVHGE\LPPXQREORWWLQJLPPHGLDWHO\DIWHUȖ

LUUDGLDWLRQEXWEHIRUHFRFXOWXUHZLWK&7/VWDQGDIWHUKRXUVW(31))LJ'1RWDEO\LQ

';/&/V+63SURWHLQOHYHOVDUHXQFKDQJHGDIWHUȖLUUDGLDWLRQDQGVKRZDPDUNHGLQFUHDVHDWW

11

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

2QWKHFRQWUDU\H[SUHVVLRQOHYHOVRIERWK+63DQG+63SURWHLQVGHFUHDVHLQ73$1D%DQG

&63/&/VDWWDQGWUHVSHFWLYHO\)LJ'7RYHULI\ZKHWKHUWKHHQKDQFHG+0*%UHOHDVH

VKRZHGE\';/&/VFRXOGWULJJHUUHFHSWRUGHSHQGHQWUHVSRQVHVLQDQDXWRFULQHSDUDFULQHIDVKLRQ

ZHHYDOXDWHGWKHH[SUHVVLRQRILWVIXQFWLRQDOUHFHSWRU7/5DQGRILWVGRZQVWUHDPDGDSWRU

PROHFXOH0\HORLG'LIIHUHQWLDWLRQSULPDU\UHVSRQVH0\'(27))ORZF\WRPHWU\DQDO\VLV

VKRZHGWKDW';DQG&63PDUNHGO\LQFUHDVHGWKHQXPEHURI7/5H[SUHVVLQJFHOOVDQG

UHVSHFWLYHO\FRPSDUHGWRFRQWURO/&/VRISRVLWLYHFHOOVZKHUHDV73$1D%RQO\

VOLJKWO\DIIHFWHG7/5H[SUHVVLRQ)LJ%,QDGGLWLRQLPPXQREORWWLQJDQDO\VLVUHYHDOHGWKDW';

WUHDWPHQWXSUHJXODWHV0\'H[SUHVVLRQLQ/&/VDWWDQHIIHFWPXFKPRUHHYLGHQWDQGW)LJ

'ZKHUHDVDVLJQLILFDQWGHFUHDVHLQ0\'SURWHLQOHYHOVZDVREVHUYHGLQ73$1D%DQG&63

/&/VDWWZKLFKEHFDPHDOPRVWXQGHWHFWDEOHDWW)LJXUH'0RUHRYHU3$53FOHDYDJH

DQDO\VLVGHPRQVWUDWHGWKDWWKHODWHSKDVHVRIDSRSWRWLFFDVFDGHLQ';/&/VRFFXUUHGRQO\K

DIWHUȖLUUDGLDWLRQZKHUHDVDFOHDYHG3$53LVDOUHDG\GHWHFWDEOHDWWLQ73$1D%DQG&63

/&/VZKLFKLVDOPRVWFRPSOHWHO\IUDJPHQWHGDWW7RDVVHVVWKHSRVVLEOHUROHRIWKH

7/50\'SDWKZD\LQWKHGHOD\HGRQVHWRIDSRSWRVLVREVHUYHGIRU';/&/VZHLQYHVWLJDWHG

FDVSDVHDQG3$53FOHDYDJH0\'H[SUHVVLRQDQG1)ț%DFWLYDWLRQLQ';WUHDWHG/&/VLQWKH

SUHVHQFHRUDEVHQFHRID7/5QHXWUDOL]LQJDQWLERG\,QWHUHVWLQJO\WKHSUHVHQFHRIWKHDQWL7/5

DQWLERG\ZDVDEOHWRFRXQWHUDFWQRWRQO\WKH';GHSHQGHQW0\'XSUHJXODWLRQEXWDOVRWKH

DFWLYDWLRQRIVRPHGRZQVWUHDPWUDQVFULSWLRQIDFWRUVVXFKDV1)κ%DVVKRZQE\DQDO\VLVRIWKH

H[SUHVVLRQRIWKH1)κ%SDQGSKRVSKRSVXEXQLWV)LJXUH()XUWKHUPRUHDKLJKHU

H[SUHVVLRQRIFOHDYHGFDVSDVHWRJHWKHUZLWKVOLJKWO\GHFUHDVHGOHYHOVRIWKHIXOOOHQJWK3$53

IRUPZDVREVHUYHGLQFHOOVH[SRVHGWR';IRUKRXUVDVFRPSDUHGZLWK';RQO\/&/V

FRQVLVWHQWO\ZLWKDGHOD\HGRQVHWRIWKHDSRSWRWLFFDVFDGHLQWKHSUHVHQFHRIDIXQFWLRQDO7/5

GHSHQGHQWVLJQDOLQJ)LJ(1RWHZRUWK\DVLJQLILFDQWLQFUHDVHRI3$53FOHDYDJHZDVREVHUYHG

RQO\KRXUVDIWHU';H[SRVXUHDQHIIHFWWKDWZDVPDUNHGO\PRUHHYLGHQWLQWKHSUHVHQFHRIDQ

12

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

DQWL7/5DQWLERG\VXSSRUWLQJDUROHIRU7/5GHSHQGHQWVLJQDOLQJLQVOLJKWO\VXVWDLQLQJ/&/

VXUYLYDODIWHU';WUHDWPHQW)LJ()

/\WLFDFWLYLW\RI';&7/VJHQHUDWHGIURP13&SDWLHQWV

7RYDOLGDWHRXUUHVXOWVLQWKH13&VHWWLQJZHYHULILHGZKHWKHU';ZDVDOVRDEOHWRHQKDQFHWKH

LPPXQRJHQLFLW\RI/&/VJHQHUDWHGIURP13&SDWLHQWV$VIRUKHDOWK\GRQRUGHULYHG/&/V';

WUHDWPHQWVLJQLILFDQWO\XSUHJXODWHG%$5)DQG/03P51$OHYHOV6XSSOHPHQWDU\)LJ6%

DQGHQKDQFHG+/$$ 7/5H[SUHVVLRQDQG&57H[SRVXUHDOVRLQ/&/VGHULYHGIURP

GLIIHUHQW13&SDWLHQWV6XSSOHPHQWDU\)LJ6&)XUWKHUPRUHDVWURQJXSUHJXODWLRQRIWKH,&'

PDUNHUV+63+63DQG0\'ZDVREVHUYHGLQ';WUHDWHG/&/VIURP13&SDWLHQWVDWW

6XSSOHPHQWDU\)LJ6',QDGGLWLRQ+0*%UHOHDVHZDVDOVRVLJQLILFDQWO\LQFUHDVHGE\';LQ

SDWLHQWGHULYHG/&/VDVFRPSDUHGWRXQWUHDWHG/&/V6XSSOHPHQWDU\)LJ6(7RDVVHVVWKH

DELOLW\RI';/&/VWRHIILFLHQWO\SULPHDQGLQGXFH(%9VSHFLILFHIIHFWRUFHOOVZHJHQHUDWHG&7/V

IURPGLIIHUHQW13&SDWLHQWVXVLQJDXWRORJRXVXQWUHDWHGDQG';WUHDWHG/&/VDVVWLPXODWRUV

&\WRWR[LFLW\DVVD\VZHUHSHUIRUPHGXVLQJ7$FHOOVORDGHGZLWK%$5)RU/03GHULYHG

SHSWLGHV$VVKRZQLQ)LJXUH';&7/VGLVSOD\HGKLJKHUVSHFLILFLW\DQGF\WRWR[LFDFWLYLW\WR

%$5)SHSWLGHVFRPSDUHGWR&7/VLQGXFHGE\XQWUHDWHG/&/VDQGVSHFLILF

O\VLVUHVSHFWLYHO\1RWDEO\';&7/VIURP13&SDWLHQWVDOVRVKRZHGDQHQKDQFHGNLOOLQJDELOLW\

DJDLQVW/03ORDGHGWDUJHWV)LJ$IXUWKHUVXSSRUWLQJWKHHQKDQFHGLPPXQRJHQLFIHDWXUHVRI

';WUHDWHG/&/V0RUHLPSRUWDQWO\';&7/VZHUHDEOHWRUHFRJQL]HDQGVSHFLILFDOO\NLOOWXPRU

FHOOVH[SUHVVLQJ%$5)HQGRJHQRXVO\LQSDUWLFXODUWKHF$13&FHOOVDQG*UDQWD

O\PSKRPDFHOOVDQGVSHFLILFO\VLVUHVSHFWLYHO\)LJ%

13

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

'LVFXVVLRQ

,QWKHSUHVHQWVWXG\ZHUHSRUWWKHGHYHORSPHQWRIDQHZDQGEURDGO\DSSOLFDEOHSURWRFRO

DOORZLQJDQHIILFLHQWJHQHUDWLRQDQGH[SDQVLRQRI(%9VSHFLILF&7/VHQULFKHGLQVSHFLILFLWLHVIRU

%$5)WREHXVHGIRUDGRSWLYHLPPXQRWKHUDS\RI13&([SORLWDWLRQRIWKHLPPXQRJHQLFLW\RI

WKLVYLUDOSURWHLQFRQVWLWXWHVDUHOHYDQWLPSURYHPHQWRIFXUUHQWSURWRFROVZKLFKZHUHVRIDU

GHVLJQHGWRWDUJHWPDLQO\/03DQG/03SURWHLQVEXWQRW%$5),QGHHG7FHOOFXOWXUHV

JHQHUDWHGZLWKFRQYHQWLRQDOSURWRFROVDUHYLUWXDOO\GHYRLGRI%$5)VSHFLILFHIIHFWRUV

&RQVLGHULQJWKDW%$5)LVH[SUHVVHGE\WKHPDMRULW\RI13&VIURPERWKHQGHPLFDQGQRQ

HQGHPLFDUHDV(13-16)DYDLODELOLW\RIDSURWRFRODEOHWRJHQHUDWH(%9VSHFLILF&7/VHIILFLHQWO\

WDUJHWLQJDOVR%$5)PD\HQKDQFHWKHVWLOOXQVDWLVIDFWRU\UDWHVRIFOLQLFDOUHVSRQVHVREWDLQDEOHE\

DGRSWLYHLPPXQRWKHUDS\LQWKLVVHWWLQJ(9, 10)

2XUSURWRFROH[SORLWVWKHSURSHUWLHVRI';DFRPPRQO\XVHGDQWLQHRSODVWLFGUXJWRSURPRWH

DQDERUWLYH(%9UHDFWLYDWLRQDQGLQGXFH,&'LQWUHDWHG/&/V(32, 33),QIDFWZHGHPRQVWUDWHWKDW

/&/WUHDWPHQWZLWKVXEF\WRWR[LFFRQFHQWUDWLRQVRI';LVDEOHWRXSUHJXODWH%$5)H[SUHVVLRQ

ZLWKRXWLQGXFLQJPDVVLYH(%9O\WLFUHSOLFDWLRQZKLOHSUHVHUYLQJWKHH[SUHVVLRQRIRWKHUUHOHYDQW

(%9ODWHQF\WDUJHWVSDUWLFXODUO\/03&RQVLVWHQWO\';/&/VHIILFLHQWO\JHQHUDWHDQGH[SDQG

7FHOOHIIHFWRUVDEOHWRVSHFLILFDOO\UHFRJQL]HDQGNLOOERWKDXWRORJRXVWDUJHWVSUHVHQWLQJ%$5)

GHULYHGHSLWRSHVDQG13&FHOOVHQGRJHQRXVO\H[SUHVVLQJ%$5)LQDQ+/$FODVV,UHVWULFWHG

IDVKLRQ1RWDEO\';&7/VQRWRQO\UHWDLQWKHDELOLW\WRUHFRJQL]HVXEGRPLQDQW/03HSLWRSHV

EXWDOVRVKRZDQLQFUHDVHGO\WLFHIILFLHQF\DJDLQVW/03H[SUHVVLQJWDUJHWVDVFRPSDUHGWR

HIIHFWRUVJHQHUDWHGE\FRQYHQWLRQDOSURWRFROV

14

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

,QWHUHVWLQJLQVLJKWVZHUHREWDLQHGE\JUDQ]\PHȕJUDQXOHVFRXQWLQJLQ(%9VSHFLILF7FHOOV

ZKLFKGHPRQVWUDWHGWKDW';&7/VLQFOXGHDVXEVWDQWLDOIUDFWLRQRIHIIHFWRUVUHDGLO\DFWLYDWHGDQG

³DUPHG´XSRQVWLPXODWLRQZLWK%$5)HSLWRSHSHSWLGHV,QWULJXLQJO\DOVR73$1D%RU&63

&7/VGLVSOD\HGDUHODWLYHO\KLJKHUQXPEHURIJUDQ]\PHȕJUDQXOHSURGXFLQJ7FHOOVZKHQWKHVH

HIIHFWRUVZHUHFXOWXUHGLQWKHSUHVHQFHRI%$5)ORDGHG/&/VFRQVLVWHQWZLWKWKHUHODWLYH

LPPXQRGRPLQDQFHRI%$5)ZLWKUHVSHFWWR/030RUHRYHULQWKHSUHVHQFHRI%$5)

SUHVHQWLQJWDUJHWV';&7/VIRUPHGWKHKLJKHVWQXPEHURIGRXEOHWVZLWKWDUJHW/&/V7KHVH

ILQGLQJVWRJHWKHUVXSSRUWWKHFRQFOXVLRQWKDW';&7/VDUHKLJKO\DFWLYDWHGHIIHFWRUVZLWKDEURDGHU

DQGPRUHHIILFLHQWNLOOLQJSRWHQWLDODVFRPSDUHGWR7FHOOFXOWXUHVJHQHUDWHGZLWKXQWUHDWHG

73$1D%RU&63WUHDWHG/&/V

7KHLQFUHDVHGDELOLW\RI';/&/VWRJHQHUDWH(%9VSHFLILF&7/VZLWKKLJKHUHIILFLHQF\

FRXOGEHDWOHDVWLQSDUWUHODWHGWRWKHSURORQJHGVXUYLYDORI/&/VDVFRPSDUHGWRWKRVHWUHDWHGZLWK

RWKHUO\WLFF\FOHLQGXFHUV1RWDEO\SHUVLVWHQFHRIOLYH';/&/VLQFRFXOWXUHVZLWKDXWRORJRXV7

O\PSKRF\WHVLVFRPSDUDEOHWRWKDWRIXQWUHDWHG/&/VVXJJHVWLQJWKDWWUHDWPHQWZLWKVXEF\WRWR[LF

GRVHVRI';SUHVHUYHVWKH/&/LQWHJULW\QHFHVVDU\IRUDQRSWLPDOLQWHUDFWLRQZLWK(%9VSHFLILF7

FHOOSUHFXUVRUV1HYHUWKHOHVVRXUUHVXOWVDOVRLQGLFDWHWKDWRWKHUPHFKDQLVPVDUHSUREDEO\LQYROYHG

LQWKHLQFUHDVHGLPPXQRJHQLFLW\RI';/&/V,QWKLVUHVSHFWRXUILQGLQJWKDWRQO\';/&/VVKRZ

LQFUHDVHG+/$$ P51$DQGSURWHLQOHYHOVLVRISDUWLFXODUUHOHYDQFHFRQVLGHULQJWKHFHQWUDO

UROHRIWKHVHPROHFXOHVLQDQWLJHQSUHVHQWDWLRQ,QWULJXLQJO\QDQRPRODUFRQFHQWUDWLRQVRI';ZHUH

VKRZQWRLQGXFHDFRPSOH[SURWHLQXELTXLWLQDWLRQUHVSRQVHZLWKRXWLQKLELWLQJSURWHDVRPHDFWLYLWLHV

(34)VXJJHVWLQJWKDWWKHUHVXOWLQJLQWUDFHOOXODUDFFXPXODWLRQRIGDPDJHGSURWHLQVPD\LQGXFH

TXDQWLWDWLYHDQGRUTXDOLWDWLYHFKDQJHVLQWKHLPPXQRJHQLFHSLWRSHUHSHUWRLUH2XUUHVXOWVDUH

WKHUHIRUHFRQVLVWHQWZLWKWKHSRVVLELOLW\WKDW';/&/VKDYHHQKDQFHGLPPXQRJHQLFSURSHUWLHVDOVR

EHFDXVHRIWKHLUDELOLW\WRLQGXFHDPRUHHIILFLHQWDQGSURORQJHGSUHVHQWDWLRQRIWXPRUDVVRFLDWHG

DQWLJHQHSLWRSHV&RQVLGHULQJWKDW';LVDEOHWRLQGXFH,&'(35-37)ZHH[SORUHGWKHK\SRWKHVLV

WKDW';FRXOGLQGXFHIXQFWLRQDOFKDQJHVSUHVXPDEO\UHVSRQVLEOHIRUWKHHQKDQFHGLPPXQRJHQLF
15

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

IHDWXUHVRI/&/VHYHQDWGRVHVXQDEOHWRLQGXFHHDUO\DSRSWRWLFHIIHFWV&RQVLVWHQWZLWKWKHDELOLW\

RI';WRPRGXODWHWKHH[SUHVVLRQUHOHDVHRI'$03VERWKin vitroDQGin vivo (27, 38)ZH

GHPRQVWUDWHWKDWRQO\';ZDVDEOHWRHOLFLWDQHDUO\&57H[SRVXUHDW/&/VXUIDFHRQHRIWKH

IXQFWLRQDOKDOOPDUNRI,&'(36, 39),QWKHVHWWLQJRI,&'&57VHUYHVDVDQ³HDWPHVLJQDO´DQG

SURPRWHVWKHLPPXQRJHQLFLW\RIG\LQJWXPRUFHOOV,QOLQHZLWKWKHQRWLRQWKDW&57H[SRVXUHLVDQ

HDUO\HYHQWLQ,&'';HQKDQFHG&57H[SRVXUHZDVREVHUYHGDWDWLPHSRLQWZKHQQRHYLGHQFHRI

RYHUWDSRSWRVLVZDVSUHVHQW([WUDFHOOXODU+0*%LVDQRWKHUFULWLFDOPHGLDWRURI,&'EHLQJ

UHOHDVHGE\FHOOVLQUHVSRQVHWRYDULRXVVWLPXOLLQFOXGLQJSURDSRSWRWLFGUXJV(25, 36, 40)DQG

H[HUWLQJGLUHFWHIIHFWVRQ&'7FHOOVSURPRWLQJ7KSRODUL]DWLRQ7FHOOH[SDQVLRQDQGVXUYLYDO

1RQHWKHOHVV+0*%PD\DOVRDFWLQGLUHFWO\E\LQGXFLQJIXQFWLRQDOFKDQJHVRQ$3&V,Q

SDUWLFXODULWKDVEHHQVKRZQWKDW+0*%UHOHDVHGE\GDPDJHGRUDSRSWRWLFFHOOVPD\ELQGWR

7/5RQGHQGULWLFFHOOVWKHUHE\PHGLDWLQJGRZQVWUHDPVLJQDOLQJWKURXJK0\'WKDWUHVXOWVLQ

LPSURYHGWXPRUDQWLJHQSUHVHQWDWLRQDQGHQKDQFHG&7/DFWLYLW\(40)2XUUHVXOWVLQGLFDWHWKDW

EHVLGHVUHOHDVLQJKLJKHUDPRXQWVRI+0*%';WUHDWHG/&/VDOVRVKRZLQFUHDVHGVXUIDFH

H[SUHVVLRQRIWKHFRJQDWH7/5UHFHSWRUDQGXSUHJXODWLRQRIWKHGRZQVWUHDPVLJQDOLQJHIIHFWRU

0\'VXJJHVWLQJWKHRFFXUUHQFHRIDQDXWRFULQHSDUDFULQHORRSLQWKHin vitro/&/FXOWXUH7KLV

ORFDOVWLPXODWLRQOHDGVWRHQKDQFHG1)ț%DFWLYDWLRQDVVKRZQE\XSUHJXODWLRQRIWKHSVXEXQLW

DQGHQKDQFHGOHYHOVRIWKHSSKRVSKRU\ODWHGIRUP1RWDEO\H[SHULPHQWVFDUULHGRXWZLWKD7/5

QHXWUDOL]LQJDQWLERG\GHPRQVWUDWHGWKDWERWKWKH1)ț%DFWLYDWLRQDQGWKHGHOD\HGRQVHWRI

DSRSWRVLVREVHUYHGLQ';WUHDWHG/&/VEXWQRWLQ73$1D%RU&63WUHDWHG/&/VDUH

GHSHQGHQWRQ7/5WULJJHULQJ2QWKHVHJURXQGVRXUUHVXOWVVXSSRUWWKHSRVVLELOLW\WKDWWKH

HQKDQFHG+0*%UHOHDVHLQGXFHGE\';PD\UHVXOWLQWKHDFWLYDWLRQRIDQDXWRFULQHSDUDFULQH

ORRSLQ/&/VDEOHWRSURPRWHDSURORQJHGVXUYLYDODQGDQHQKDQFHGLPPXQRJHQLFLW\RIWKHVH$3&V

)LJ

:HKDYHDOVRLQYHVWLJDWHGWKHHIIHFWVRI';RQWKHH[SUHVVLRQRI+63DQG+63NH\

FKDSHURQHPROHFXOHVSOD\LQJLPSRUWDQWUROHVLQVLJQDOLQJSURWHLQIXQFWLRQWUDIILFNLQJDQGWXUQRYHU
16

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

(41)DQGEHLQJDOVRIXQFWLRQDOO\LQYROYHGLQSURWHLQDQGHSLWRSHIROGLQJ2XUILQGLQJVLQGLFDWHWKDW

RQO\';EXWQRW73$1D%RU&63LQGXFHVDPDUNHGXSUHJXODWLRQRIWKHVHSURWHLQVHYHQLQWKH

DEVHQFHRIRYHUWDSRSWRVLV7KLVFRXOGEHDQDGGLWLRQDOPHFKDQLVPSUREDEO\XQGHUO\LQJWKH

HQKDQFHGLPPXQRJHQLFLW\RI';/&/VDQGWKHLUDQWLJHQV

2QHRIWKHPDMRUDGYDQWDJHVRIRXULQQRYDWLYHSURWRFROUHVLGHVLQLWVHDV\XSJUDGDELOLW\WR

*03VWDQGDUGVFRQVLGHULQJWKDWQRPROHFXODUHQJLQHHULQJRIFHOOVLVUHTXLUHGDQGWKDW';LVD

GUXJDOUHDG\DQGEURDGO\XVHGLQWKHFOLQLFV,WLVQRWHZRUWK\WKDWRXUSURWRFROGRHVQRWDOWHUWKH

GLIIHUHQWLDWLRQSKHQRW\SHRI(%9VSHFLILF&7/VWKDWDUHJHQHUDWHGDQGVKRZHGDKLJKHIILFLHQF\

DQGUHSURGXFLELOLW\DOVRZKHQ3%0&VIURP13&SDWLHQWVZHUHXVHG7KHVHIHDWXUHVDUHRI

UHOHYDQFHWRDVVXUHWKHIHDVLELOLW\DQGWKHEURDGDSSOLFDELOLW\RIRXUSURWRFRO:HDUHFXUUHQWO\

LQYHVWLJDWLQJZKHWKHUWKHEURDGHUDQWLJHQVSHFLILFLW\RI';&7/VLVQRWUHVWULFWHGWRYLUDOSURWHLQV

EXWDOVRLQYROYHVFHOOXODUDQWLJHQVZKRVHWDUJHWLQJPD\IXUWKHUHQKDQFHWKHWKHUDSHXWLFSRWHQWLDORI

WKHVHHIIHFWRUV

,QFRQFOXVLRQRXUUHVXOWVSURYLGHWKHUDWLRQDOHWRLQYHVWLJDWHWKHVDIHW\DQGHIILFDF\RI';

/&/LQGXFHG(%9VSHFLILF&7/VLQDSKDVH,,,FOLQLFDOVWXG\RIDGRSWLYHLPPXQRWKHUDS\IRU13&

SDWLHQWV7KLVZLWKWKHILQDOJRDOWRDVVHVVZKHWKHURXUSURWRFROLVDEOHWRHQKDQFHWKHUDWHRI

FOLQLFDOUHVSRQVHVWRDGRSWLYHLPPXQRWKHUDS\LQ13&SDWLHQWVSDUWLFXODUO\IRUWKRVHZLWKUHODSVHG

RUUHIUDFWRU\GLVHDVH




5HIHUHQFHV

<X0&<XDQ-0(SLGHPLRORJ\RIQDVRSKDU\QJHDOFDUFLQRPD6HPLQ&DQFHU%LRO


17

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

<X;:DQJ=0HUW]-(=(%UHJXODWHVWKHODWHQWO\WLFVZLWFKLQLQIHFWLRQE\(SVWHLQ%DUU
YLUXV3/R63DWKRJH

&KDQ-.&%UD\)0F&DUURQ3)RR:/HH$:0<LS7HWDO:+2FODVVLILFDWLRQRIWXPRUV
SDWKRORJ\ JHQHWLFVRIKHDGDQGQHFNWXPRXUV1DVRSKDU\QJHDO&DUFLQRPD,QWHUQDWLRQDO
$JHQF\IRU5HVHDUFKRQ&DQFHU,$5&3

6SDQR-3%XVVRQ3$WODQ'%RXUKLV-3LJQRQ-3(VWHEDQ&HWDO1DVRSKDU\QJHDO
FDUFLQRPDVDQXSGDWH(XU-&DQFHU

/R.:7R.)+XDQJ'3)RFXVRQQDVRSKDU\QJHDOFDUFLQRPD&DQFHU&HOO

+XDQJ'3/.$HWLRORJLFDOIDFWRUVDQGSDWKRJHQHVLV1DVRSKDU\QJHDOFDUFLQRPDQGHGLWLRQ
&KLQHVHXQLYHUVLW\3UHVV3S

0HUOR$7XUULQL5'ROFHWWL5=DQRYHOOR3$PDGRUL$5RVDWR$$GRSWLYHFHOOWKHUDS\
DJDLQVW(%9UHODWHGPDOLJQDQFLHVDVXUYH\RIFOLQLFDOUHVXOWV([SHUW2SLQ%LRO7KHU


&KLD:.7HR0:DQJ::/HH%$QJ6)7DL:0HWDO$GRSWLYH7FHOOWUDQVIHUDQG
FKHPRWKHUDS\LQWKHILUVWOLQHWUHDWPHQWRIPHWDVWDWLFDQGRUORFDOO\UHFXUUHQWQDVRSKDU\QJHDO
FDUFLQRPD0RO7KHU

&RPROL33HGUD]]ROL30DFFDULR5%DVVR6&DUPLQDWL2/DELULR0HWDO&HOOWKHUDS\RI
VWDJH,9QDVRSKDU\QJHDOFDUFLQRPDZLWKDXWRORJRXV(SVWHLQ%DUUYLUXVWDUJHWHGF\WRWR[LF7
O\PSKRF\WHV-&OLQ2QFRO

6WUDDWKRI.&%ROODUG&03RSDW8+XOV0+/RSH]70RUULVV0&HWDO7UHDWPHQWRI
QDVRSKDU\QJHDOFDUFLQRPDZLWK(SVWHLQ%DUUYLUXVVSHFLILF7O\PSKRF\WHV%ORRG


.KDQQD5%XUURZV651LFKROOV-3RXOVHQ/0,GHQWLILFDWLRQRIF\WRWR[LF7FHOOHSLWRSHV
ZLWKLQ(SVWHLQ%DUUYLUXV(%9RQFRJHQHODWHQWPHPEUDQHSURWHLQ/03HYLGHQFHIRU+/$
$VXSHUW\SHUHVWULFWHGLPPXQHUHFRJQLWLRQRI(%9LQIHFWHGFHOOVE\/03VSHFLILFF\WRWR[LF7
O\PSKRF\WHV(XU-,PPXQRO

%ROODUG&0*RWWVFKDON67RUUDQR9'LRXI2.X6+D]UDW<HWDO6XVWDLQHGFRPSOHWH
UHVSRQVHVLQSDWLHQWVZLWKO\PSKRPDUHFHLYLQJDXWRORJRXVF\WRWR[LF7O\PSKRF\WHVWDUJHWLQJ
(SVWHLQ%DUUYLUXVODWHQWPHPEUDQHSURWHLQV-&OLQ2QFRO

'HFDXVVLQ*6ELK/DPPDOL)GH7XUHQQH7HVVLHU0%RXJXHUPRXK$2RND7([SUHVVLRQRI
%$5)JHQHHQFRGHGE\(SVWHLQ%DUUYLUXVLQQDVRSKDU\QJHDOFDUFLQRPDELRSVLHV&DQFHU5HV


+D\HV'3%ULQN$$9HUYRRUW0%0LGGHOGRUS-00HLMHU&-YDQGHQ%UXOH$-([SUHVVLRQ
RI(SVWHLQ%DUUYLUXV(%9WUDQVFULSWVHQFRGLQJKRPRORJXHVWRLPSRUWDQWKXPDQSURWHLQVLQ
GLYHUVH(%9DVVRFLDWHGGLVHDVHV0RO3DWKRO

%ULQN$$9HUYRRUW0%0LGGHOGRUS-00HLMHU&-YDQGHQ%UXOH$-1XFOHLFDFLGVHTXHQFH
EDVHGDPSOLILFDWLRQDQHZPHWKRGIRUDQDO\VLVRIVSOLFHGDQGXQVSOLFHG(SVWHLQ%DUUYLUXVODWHQW
WUDQVFULSWVDQGLWVFRPSDULVRQZLWKUHYHUVHWUDQVFULSWDVH3&5-&OLQ0LFURELRO
18

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

6WHYHQV6-9HUNXLMOHQ6$+DULZL\DQWR%+DULMDGL3DUDPLWD'.)DFKLURK-HWDO
1RQLQYDVLYHGLDJQRVLVRIQDVRSKDU\QJHDOFDUFLQRPDQDVRSKDU\QJHDOEUXVKLQJVUHYHDOKLJK
(SVWHLQ%DUUYLUXV'1$ORDGDQGFDUFLQRPDVSHFLILFYLUDO%$5)P51$,QW-&DQFHU


0DUWRUHOOL'+RXDOL.&DJJLDUL/9DFFKHU(%DU]DQ/)UDQFKLQ*HWDO6SRQWDQHRXV7FHOO
UHVSRQVHVWR(SVWHLQ%DUUYLUXVHQFRGHG%$5)SURWHLQDQGGHULYHGSHSWLGHVLQSDWLHQWVZLWK
QDVRSKDU\QJHDOFDUFLQRPDEDVHVIRULPSURYHGLPPXQRWKHUDS\,QW-&DQFHU

=KDQJ&;'HFDXVVLQ*'DLOOLH-2RND7$OWHUHGH[SUHVVLRQRIWZR(SVWHLQ%DUUYLUXVHDUO\
JHQHVORFDOL]HGLQ%DP+,$LQQRQSURGXFHU5DMLFHOOV-9LURO

/LPD576HFD+%UDV61DVFLPHQWR069DVFRQFHORV0+7UHDWPHQWRI$NDWD(%9SRVLWLYH
FHOOVZLWKGR[RUXELFLQFDXVHVPRUH(%9UHDFWLYDWLRQWKDQWUHDWPHQWZLWKHWRSRVLGH&KHPRWKHUDS\


+VX&++HUJHQKDKQ0&KXDQJ6(<HK3<:X7&*DR0HWDO,QGXFWLRQRI(SVWHLQ%DUU
YLUXV(%9UHDFWLYDWLRQLQ5DMLFHOOVE\GR[RUXELFLQDQGFLVSODWLQ$QWLFDQFHU5HV


&RXQWU\PDQ-.*UDGRYLOOH/0LOOHU*+LVWRQHK\SHUDFHW\ODWLRQRFFXUVRQSURPRWHUVRIO\WLF
F\FOHUHJXODWRU\JHQHVLQ(SVWHLQ%DUUYLUXVLQIHFWHGFHOOOLQHVZKLFKDUHUHIUDFWRU\WRGLVUXSWLRQRI
ODWHQF\E\KLVWRQHGHDFHW\ODVHLQKLELWRUV-9LURO

.KDQQD5%XUURZV651LFKROOV-3RXOVHQ/0,GHQWLILFDWLRQRIF\WRWR[LF7FHOOHSLWRSHV
ZLWKLQ(SVWHLQ%DUUYLUXV(%9RQFRJHQHODWHQWPHPEUDQHSURWHLQ/03HYLGHQFHIRU+/$
$VXSHUW\SHUHVWULFWHGLPPXQHUHFRJQLWLRQRI(%9LQIHFWHGFHOOVE\/03VSHFLILFF\WRWR[LF7
O\PSKRF\WHV(XU-,PPXQRO

&KLULYD,QWHUQDWL0*UL]]L)3LQNVWRQ-0RUURZ.-'
&XQKD1)UH]]D((HWDO*DPPD
UDGLDWLRQXSUHJXODWHV0+&FODVV,,,DQG,&$0,PROHFXOHVLQPXOWLSOHP\HORPDFHOOOLQHVDQG
SULPDU\WXPRUV,Q9LWUR&HOO'HY%LRO$QLP

5HLWV($+RGJH-:+HUEHUWV&$*URRWKXLV7$&KDNUDERUW\0:DQVOH\(.HWDO
5DGLDWLRQPRGXODWHVWKHSHSWLGHUHSHUWRLUHHQKDQFHV0+&FODVV,H[SUHVVLRQDQGLQGXFHV
VXFFHVVIXODQWLWXPRULPPXQRWKHUDS\-([S0HG

$SHWRK/0LJQRW*3DQDUHWDNLV7.URHPHU*=LWYRJHO/,PPXQRJHQLFLW\RIDQWKUDF\FOLQHV
PRYLQJWRZDUGVPRUHSHUVRQDOL]HGPHGLFLQH7UHQGV0RO0HG

&DVDUHV13HTXLJQRW027HVQLHUH$*KLULQJKHOOL)5RX[6&KDSXW1HWDO&DVSDVH
GHSHQGHQWLPPXQRJHQLFLW\RIGR[RUXELFLQLQGXFHGWXPRUFHOOGHDWK-([S0HG


.U\VNR'9.DF]PDUHN$.U\VNR2+H\QGULFN[/:R]QLFNL-%RJDHUW3HWDO7/5DQG
7/5DUHVHQVRUVRIDSRSWRVLVLQDPRXVHPRGHORIGR[RUXELFLQLQGXFHGDFXWHLQIODPPDWLRQ&HOO
'HDWK'LIIHU

0DFFXEELQ'/&RKHQ6$(KUNH0-,QGRPHWKDFLQPRGXODWLRQRIDGULDP\FLQLQGXFHGHIIHFWV
RQPXOWLSOHF\WRO\WLFHIIHFWRUIXQFWLRQV&DQFHU,PPXQRO,PPXQRWKHU

19

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

2EHLG07HVQLHUH$*KLULQJKHOOL))LPLD*0$SHWRK/3HUIHWWLQL-/HWDO&DOUHWLFXOLQ
H[SRVXUHGLFWDWHVWKHLPPXQRJHQLFLW\RIFDQFHUFHOOGHDWK1DW0HG

$SHWRK/*KLULQJKHOOL)7HVQLHUH$2EHLG02UWL]&&ULROOR$HWDO7ROOOLNHUHFHSWRU
GHSHQGHQWFRQWULEXWLRQRIWKHLPPXQHV\VWHPWRDQWLFDQFHUFKHPRWKHUDS\DQGUDGLRWKHUDS\1DW
0HG

*DUJ$'0DUWLQ6*RODE-$JRVWLQLV3'DQJHUVLJQDOOLQJGXULQJFDQFHUFHOOGHDWKRULJLQV
SODVWLFLW\DQGUHJXODWLRQ&HOO'HDWK'LIIHU

9DFFKHOOL($UDQGD)(JJHUPRQW$*DORQ-6DXWHV)ULGPDQ&&UHPHU,HWDO7ULDO:DWFK
&KHPRWKHUDS\ZLWKLPPXQRJHQLFFHOOGHDWKLQGXFHUV2QFRLPPXQRORJ\H

0DWWDUROOR65/RL6'XUHW+0D<=LWYRJHO/6P\WK0-3LYRWDOUROHRILQQDWHDQGDGDSWLYH
LPPXQLW\LQDQWKUDF\FOLQHFKHPRWKHUDS\RIHVWDEOLVKHGWXPRUV&DQFHU5HV

0DQGLOL*.KDGMDYL$*DOOR90LQHUR9*%HVVRQH/&DUWD)HWDO&KDUDFWHUL]DWLRQRIWKH
SURWHLQXELTXLWLQDWLRQUHVSRQVHLQGXFHGE\'R[RUXELFLQ)(%6-

8MKD]\3=DOHVNLV*0LKLFK((KUNH0-%HUOHWK(6'R[RUXELFLQLQGXFHVVSHFLILFLPPXQH
IXQFWLRQVDQGF\WRNLQHH[SUHVVLRQLQSHULWRQHDOFHOOV&DQFHU,PPXQRO,PPXQRWKHU


=LWYRJHO/.HSS26HQRYLOOD/0HQJHU/&KDSXW1.URHPHU*,PPXQRJHQLFWXPRUFHOO
GHDWKIRURSWLPDODQWLFDQFHUWKHUDS\WKHFDOUHWLFXOLQH[SRVXUHSDWKZD\&OLQ&DQFHU5HV


9DFFKHOOL(6HQRYLOOD/(JJHUPRQW$)ULGPDQ:+*DORQ-=LWYRJHO/HWDO7ULDOZDWFK
&KHPRWKHUDS\ZLWKLPPXQRJHQLFFHOOGHDWKLQGXFHUV2QFRLPPXQRORJ\H

&DVDUHV13HTXLJQRW027HVQLHUH$*KLULQJKHOOL)5RX[6&KDSXW1HWDO&DVSDVH
GHSHQGHQWLPPXQRJHQLFLW\RIGR[RUXELFLQLQGXFHGWXPRUFHOOGHDWK-([S0HG


*DUJ$''XGHN$0$JRVWLQLV3&DQFHULPPXQRJHQLFLW\GDQJHUVLJQDOVDQG'$03VZKDW
ZKHQDQGKRZ"%LRIDFWRUV

/L*/LDQJ;/RW]H07+0*%7KH&HQWUDO&\WRNLQHIRU$OO/\PSKRLG&HOOV)URQW
,PPXQRO

3UDWW:%0RULVKLPD<3HQJ+02VDZD<3URSRVDOIRUDUROHRIWKH+VS+VSEDVHG
FKDSHURQHPDFKLQHU\LQPDNLQJWULDJHGHFLVLRQVZKHQSURWHLQVXQGHUJRR[LGDWLYHDQGWR[LF
GDPDJH([S%LRO0HG0D\ZRRG

 

20

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

)LJXUHOHJHQGV

)LJXUH$';HQKDQFHV%$5)H[SUHVVLRQT573&5IRU(%9O\WLFDQGODWHQWJHQH

H[SUHVVLRQSHUIRUPHGRQ/&/VWUHDWHGZLWK73$1D%';RU&63P51$IROGH[SUHVVLRQZDV

QRUPDOL]HGUHODWLYHWRFRQWURO/&/V&WUO/&/V0HDQRILQGHSHQGHQWH[SHULPHQWV P ”

VLJQLILFDQFHIRUIROGLQFUHDVHRI%$5)P51$H[SUHVVLRQvs.RWKHU(%9JHQHV P ”

VLJQLILFDQFHRIIROGLQFUHDVHvs.&WUO/&/V6WXGHQWtWHVW%';/&/VVKRZDOLPLWHGODWH

DSRSWRVLV3$53FOHDYDJHZDVDVVHVVHGDWWKHHQGRIWUHDWPHQW:KROHFHOOO\VDWHVȝJZHUH

DQDO\]HGE\LPPXQREORWWLQJIRUWKHLQGLFDWHGSURWHLQV*$3'+LVDORDGLQJFRQWURO';/&/VGR

QRWDIIHFW7FHOOGLIIHUHQWLDWLRQ&'LIIHUHQWLDWLRQVWDWXVRI&'&'DQG&'&'ZDV

DVVHVVHGE\DQDO\VLVRI&&5DQG&'5$H[SUHVVLRQLQHDUO\VWDJHFXOWXUHVGD\DQGDIWHU

WKHODVWVWLPXODWLRQGD\'3HUFHQWDJHVRI7HUPLQDOO\GLIIHUHQWLDWHG7HPUD&&5±

&'5$(IIHFWRU0HPRU\(0&&5±&'5$±&HQWUDO0HPRU\&0&&5&'5$±

DQGQDwYH&&5&'5$DUHVKRZQ/HIWKLVWRJUDP&'&'UDWLRZLWKLQ&'

O\PSKRF\WHVDWGD\&HQWUDODQGULJKWKLVWRJUDPVUHSRUW&'DQG&'7FHOOSKHQRW\SHV

UHVSHFWLYHO\0HDQRILQGHSHQGHQWH[SHULPHQWV

)LJXUH$';&7/VVSHFLILFDOO\NLOO7$FHOOVORDGHGZLWK%$5)GHULYHGSHSWLGHV

,QGXFWLRQRI%$5)SDQGSDQG/03</4SHSWLGHVSHFLILF7FHOOUHVSRQVHVIURPWKUHH

UHSUHVHQWDWLYHRXWRIILYH+/$$ GRQRUV(7UDWLR6SHFLILFO\VLVZDVFDOFXODWHGE\

VXEWUDFWLQJWKHO\VLVRIHPSW\7$FHOOV%%R[SORWUHSUHVHQWLQJF\WRWR[LFLW\DVVD\SHUIRUPHG

RQ7$FHOOVORDGHGZLWK%$5)RU/03SHSWLGHV P”vs.WKHRWKHU&7/OLQHVLQ

H[SHULPHQWVFDUULHGRXWRQLQGHSHQGHQW+/$$ GRQRUV&';&7/VVKRZKLJKVSHFLILF

NLOOLQJDJDLQVW%$5)±HQGRJHQRXVO\H[SUHVVLQJWXPRUFHOOV&\WRWR[LFDFWLYLW\RI&7/VGHULYHG

IURPWZRUHSUHVHQWDWLYHRXWRIILYH+/$$ GRQRUVDJDLQVWF:WQHJDWLYHFRQWURODQG

F$*UDQWDDQG.VSHFLILFLW\FRQWUROFHOOV(7UDWLR+/$$ 

21

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

UHVWULFWLRQZDVFRQILUPHGZLWKWKHDQWLERG\WR+/$$ FU'%R[SORWRIF\WRWR[LFLW\

DVVD\UHVXOWVIURP&7/FXOWXUHVRIILYHLQGHSHQGHQW+/$$ GRQRUVWHVWHGDJDLQVW%$5)±

HQGRJHQRXVO\H[SUHVVLQJWXPRUFHOOOLQHV P”vs.RWKHUWDUJHWV P”vs.O\VLVRIRWKHU

&7/VDJDLQVWF$((IILFLHQF\RI&7/VDWGLIIHUHQW(7UDWLR

)LJXUH(QXPHUDWLRQRI/&/7FHOOGRXEOHWVDQGJUDQ]\PHȕJUDQXOHV3HSWLGHORDGHG

DXWRORJRXV/&/VZHUHFRFXOWXUHGZLWK';&7/VDQGODEHOHGZLWKDQWLERGLHVWR&'&'DQG

JUDQ]\PHȕWRHQXPHUDWH/&/&7/GRXEOHWVDQGJUDQ]\PHȕ±SRVLWLYHVSRWV$4XDQWLWDWLRQRI

/&/7FHOOGRXEOHWV7KHy-D[LVGLVSOD\VWKHQRUPDOL]HGIUHTXHQF\RI/&/&7/GRXEOHWV

FRQWDLQLQJDWOHDVWRQH7FHOO0HDQRILQGHSHQGHQWH[SHULPHQWVSHUIRUPHGZLWKGLIIHUHQW

GRQRUGHULYHG&7/FXOWXUHV P ”';&7/VvsDOORWKHU&7/FXOWXUHV5LJKWSDQHO

UHSUHVHQWDWLYHLPDJHV&'3(DQG&'3&îPDJQLILFDWLRQ%(QXPHUDWLRQRI

JUDQ]\PHȕVSRWVZLWKLQ&'7FHOOV/HIWSDQHOH[SHULPHQWVZHUHSHUIRUPHGXVLQJDVHIIHFWRU

FHOOV';&7/VDQGHQXPHUDWLQJERWKWKHFHOOVDQGJUDQ]\PHȕVSRWIRUPDWLRQZLWKLQWKHVHFHOOV

5LJKWSDQHOUHSUHVHQWDWLYHLPDJHVRIJUDQ]\PHȕ±),7&&'3(DQG&'3&FHOOV/RZHU

KLVWRJUDPVJUDQ]\PHȕJUDQXOHFRQWHQWLQ&WUO73$1D%±DQG&63&7/V0HDQRI

LQGHSHQGHQWH[SHULPHQWV


)LJXUH$';XSUHJXODWHV+/$$ H[SUHVVLRQLQ/&/V5HODWLYHTXDQWLWDWLRQE\T57

3&5RI+/$$ P51$H[SUHVVLRQLQ/&/VEHIRUH1,1RW,UUDGLDWHGt DQGKDIWHU

ȖLUUDGLDWLRQW'DWDDUHQRUPDOL]HGUHODWLYHWR&WUO/&/V%+/$$ &57DQG7/5FHOO

VXUIDFHH[SUHVVLRQLVLQFUHDVHGLQ';/&/V([SUHVVLRQRI+/$$ XSSHUSDQHOVDQG7/5

ORZHUSDQHOVZDVDQDO\]HGE\IORZF\WRPHWU\DWWZKHUHDV&57H[SUHVVLRQFHQWUDOSDQHOV

ZDVLQYHVWLJDWHGDIWHUKUV,VRW\SHFRQWUROGDVKHGOLQH&WUO/&/VJUD\DUHDDQGWUHDWHG/&/V

EODFNOLQHDUHVKRZQ&';LQFUHDVHV+0*%UHOHDVH+LVWRJUDPVUHSUHVHQWWKHQRUPDOL]HG

IROGLQFUHDVHRI+0*%UHOHDVHGE\WUHDWHGvs. XQWUHDWHG/&/V([SHULPHQWVZHUHSHUIRUPHGLQ
22

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

WULSOLFDWHDQGSHUIRUPHGDWOHDVWWKUHHWLPHVRQGLIIHUHQWGRQRUGHULYHG/&/V'';XSUHJXODWHV

+63+63DQG0\'$QDO\VLVZDVSHUIRUPHGZLWK3$53FOHDYDJHLPPHGLDWHO\DIWHUȖ

LUUDGLDWLRQWDQGKUVDIWHUȖLUUDGLDWLRQW*$3'+VKRZVHTXDOSURWHLQORDGLQJ()';

DFWLYDWHV1)κ%DQGGHOD\VDSRSWRVLVRQVHW([SUHVVLRQRISSSKRVSKRS0\'

FOHDYHGFDVSDVHZDVHYDOXDWHGDWWKHHQGRI';WUHDWPHQWFOHDYHG3$53ZDVDVVHVVHGDWWKHHQG

RI';WUHDWPHQWDQGDWKUV

)LJXUH';&7/VHOLFLWVWURQJ(%9VSHFLILFUHVSRQVHVWRERWK%$5)DQG/03GHULYHG

SHSWLGHVDQGWR%$5)HQGRJHQRXVO\H[SUHVVLQJ13&FHOOV$3HSWLGHVSHFLILFUHVSRQVHVRI

&7/VJHQHUDWHGE\';/&/VIURP+/$$ 13&SDWLHQWV&\WRWR[LFLW\DVVD\VZHUH

SHUIRUPHGDJDLQVW7$FHOOVORDGHGZLWKWKHLQGLFDWHGSHSWLGHV(7UDWLRVSHFLILFO\VLV

ZDVREWDLQHGE\VXEWUDFWLQJHPSW\7$O\VLV%&\WRWR[LFDFWLYLW\RISDWLHQWGHULYHG&7/V

DJDLQVWWKHF:WF$*UDQWDDQG.FHOOV+/$$ UHVWULFWLRQZDV

FRQILUPHGZLWKWKHFUDQWLERG\QRWVKRZQ*UDQWDFHOOVPD\EHPRUHYDULDEO\O\VHG

DQGJHQHUDOO\WRDOHVVHUH[WHQWWKDQF$FRQVLVWHQWO\ZLWKWKHQRQFRQVWLWXWLYHDQGPDUNHGO\

UHGXFHGDERXWIROGQRWVKRZQH[SUHVVLRQRI%$5)LQWKHVHFHOOV

)LJXUH3URSRVHGPHFKDQLVPVXQGHUO\LQJWKHHQKDQFHGLPPXQRJHQLFLW\RI';LQGXFHG

/&/V';DFWVDWGLIIHUHQWOHYHOVWRLPSURYHWKHIXQFWLRQRI/&/VDV$3&VIRUWKHJHQHUDWLRQRI

(%9VSHFLILF&7/V';XSUHJXODWHV+/$$ VXUIDFHH[SUHVVLRQDQGLQGXFHV,&'DVVKRZQE\

HQKDQFHG&57VXUIDFHH[SRVXUHDQG+0*%UHOHDVH';DOVRXSUHJXODWHVWKHH[SUHVVLRQRI7/5

WKXVDFWLYDWLQJDQDXWRFULQHSDUDFULQH+0*%7/5ORRSWKDWHQKDQFHV/&/VXUYLYDOWKURXJK1)

κ%DFWLYDWLRQ7KHVHVXUYLYDOVLJQDOVFRXQWHUDFWIRUDFHUWDLQSHULRGRIWLPHWKHSURDSRSWRWLF

VLJQDOVLQGXFHGE\WKHGUXJUHVXOWLQJLQDGHOD\HGDSRSWRVLVRI';/&/VZKLFKPD\WKXVKDYHD

SURORQJHGLQWHUDFWLRQVZLWK(%9VSHFLILF7FHOOSUHFXUVRUV*OREDOO\WKHVHHIIHFWVUHVXOWLQ
23

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

HQKDQFHGVWLPXODWLRQDQGDFWLYDWLRQRI7FHOOVZKLFKDOVRVKRZWKHDELOLW\WRVSHFLILFDOO\UHFRJQL]H

DQGNLOO%$5)H[SUHVVLQJWXPRUFHOOV

24

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.
 Author Manuscript Published OnlineFirst on March 23, 2016; DOI: 10.1158/2326-6066.CIR-15-0108
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Broadening specificity and enhancing cytotoxicity of adoptive T

cells for nasopharyngeal carcinoma immunotherapy
Damiana Antonia Faè, Debora Martorelli, Katy Mastorci, et al.

Cancer Immunol Res Published OnlineFirst March 23, 2016.

Updated version Access the most recent version of this article at:
doi:10.1158/2326-6066.CIR-15-0108

Supplementary Access the most recent supplemental material at:

Material http://cancerimmunolres.aacrjournals.org/content/suppl/2016/03/23/2326-6066.CIR-15-0108.DC1.
html

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been
Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.

Permissions To request permission to re-use all or part of this article, contact the AACR Publications
Department at permissions@aacr.org.

Downloaded from cancerimmunolres.aacrjournals.org on March 26, 2016. © 2016 American Association for Cancer Research.