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Peter Culpepper, MBA, CPAChief Operating Officer, and Interim Chief Executive Officer
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Laura Panjwani @OncEditorLauraPublished Online: Monday, Oct 31, 2016
James Allison, PhD
Web Exclusives >
James Allison Says Rational Combinations Key toImmunotherapy Success in "Cold" Tumors
Immunotherapy has been a game changer in oncology,improving survival and providing long, durable responses inmelanoma, lung, head and neck cancer, and others.
The success of immunotherapies in those cancers—whichare likely seeing a better rate of response due to their highmutational burden—is now paving the way for what areknown as “cold” tumors, those that don’t have a heavymutational burden or significant T-cell infiltration, said JamesAllison, PhD.
“There is enough progress being made across the board thatI think we can start thinking about some of the colder tumorsresponding if we just keep studying and making rationalcombination decisions,” said Allison, professor and chair of
Immunology at MD Anderson Cancer Center. “As we understand this better, we canrationally put two things together that won’t just duplicate or cancel each other out, butwill do different things that can at least be additive, if not synergistic.”
In an interview with OncLive, Allison discussed exciting advancements in immunotherapycombinations, the potential synergistic effects of radiation with immunotherapies, andconsiderations that must be made when combining other agents with immunotherapy.
OncLive: What are you most excited about right now in the immunotherapy space?
Allison: Some people are afraid to use the word “cure,” preferring to say that we’veturned it into a chronic disease instead, but I know patients who are 10 or 15 years outafter a single round of treatment with immunotherapy that considered themselves cured.So I don’t think it is so much of a stretch. The realization that we can cure even a fractionof people is exciting. It shows that it can at least be done. The most exciting single thingright now is the fact that the 2-year survival of patients with metastatic melanoma whohave received both ipilimumab (Yervoy) and nivolumab (Opdivo) is 60%. If theipilimumab response is any indication, it’s going to be much longer than that. Thatprovides the excitement that we can get it to work in other cancer types.
We are also gaining insight on how to rationally combine agents. Mechanist studies arereally showing us a lot with these different checkpoint blockers and different mechanisms
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Figure 5. Composite MPE image showing a cross-section of murine tissue spanning the transition from normal skin (leftmost edge ofimage) to a subcutaneously implanted hepatoma tumor (area at right hand third of image). The sample was prepared by fresh frozensectioning following systemic administration of RB.
Figure 6. Detailed view ofmurine hepatoma tumorillustrated in Figure 5. RBlocalization within tumor cells,especially within sub-cellularorganelles, is evident.
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/"-#)3+%%#256 72)'(%%8,59 :).21$!%$5; 2$!#$4 <9
6/=>?=@%-ABCDE>BFG%HI%/D@BJ=K%#JBDAJD5/=>?=@5%+E=AL5%9-ABCDE>BFG%HI%'DMJ=>FKD5'#:5)N>FE=KB=5%;<EHCDJFN>%<?=EO=JDNFBJ>5PAHQCBKKD51DAAD>DD5-#)5%
!H>D%&DAR=K%?=>%SDDA%N>D@%FH%=>>D>>%?DT=FBJ%INAJFBHA5HTF?=KOBJ @B>HE@DE>%=A@%=>%=%T?HFH>DA>BFB>DE%BA%FED=FODAF%HI%>UBA%KD>BHA>L%+A%F?B>%>FN@G%!&%M=>%FD>FD@%BAVCBFEH%BA%F?D%=S>DAJD%HI%KBR?F%IHE%BF>%DIIDJF>%HA%ODK=AHO=%JDKK>%=A@%ODJ?=AB>O%HI%BF>%DIIDJF>L%+A%=@@BFBHA%=%T=FBDAF%M=>%FED=FD@%SG%BAFE=KD>BHA=K%BAWDJFBHA%HI =%>J%ODF=>F=>B>%MBF?%<3V6XY!&ZL
/DF?H@>)%T=ADK%HI%ODK=AHO=%JDKK>%MDED%DQTH>D@%FH%!&%=F%@BIIDEDAF%JHAJDAFE=FBHA>%=A@%IHE%C=EGBAR%TDEBH@>%=A@%DIIDJF>%>FN@BD@%SG%OHET?HKHRG%=THTFH>B>%=>>=G>%=A@%/11%=>>=G>
!D>NKF>L%!&%BA@NJD@%JDKK%@D=F?%BA%ODK=AHO=%JDKK>%SNF%AHF%IBSEHSK=>F>L%,D=F?%M=>%TED@HOBA=AFKG%@ND%FH%ADJEH>B>%SNF%9%KBAD>%=K>H NA@DEMDAF%=THTFH>B>%F?=F%M=>%@DTDA@DAF%HA%=JFBC=FBHA%HI%J=>T=>D>L%.DKK%@D=F?%M=>%AHF%@ND%FH%EDKD=>D%HI%!D=JFBCD%HQGRDA%>TDJBD>L!& B>%F=UDA%NT%BAFH%KG>H>HOD>%=A@%BF%B>%TEHS=SKD%YSNF%AHF%TEHCDAZ%F?=F%JDKK%@D=F?%ED>NKF>%IEHO%EDKD=>D%HI%.=F?DT>BA>L1?D%T=FBDAF%M?H%?=@%J?DOHF?DE=TG%E=@BHED>B>F=AF%EDJNEEDAF%@B>D=>D%BA%F?D%?D=@%=A@%ADJU%=ED=%?=@%E=TB@%ADJEH>B>%HI%F?D%BAWDJFD@%ODF=>F=>B>%=A@%F?B>%=>%MDKK%=>%;%NABAWDJFD@ >J%ODF=>F=>D>%NA@DE%MDAF%JHOTKDFD%EDOB>>BHA%HCDE%=%TDEBH@%HI%>DCDE=K%OHAF?>
.HAJKN>BHA0NEF?DE%>FN@BD>%FH%NA@DE>F=A@%F?D%ODJ?=AB>O%HI%=JFBHA%%=A@%BF>%EHKD BA%JKBABJ=K%%O=A=RDODAF%HI%ODK=AHO=%%=ED%M=EE=AFD@
)S>FE=JF
Me 4405
SK 28
Fibroblast
Control
6h
24h
24h
A B
C
!
"!
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A
B
Me 4405 SK 28
Control
RB
Florescence Intensity
Rel
ativ
e C
ell N
umbe
r
239:":,,CR,>?,B"!!,S*D,M@EK=+(,468),Q6@0CA6A8683=,7@E,CA6A8683=,F+11,T+78),3@,*+17@6I7,F+11(,7<8+5,"#)
?R,216U,FH86I+85H,O3(86957I(,6<,CA6A86(3(,C((7H(,4H,8)+,VM,*+8)6E,3@:,.P,"%,7@E,,*+,##!-,F+11(,J5+78+E,U38),>?,B"!!,S*D,<65,"#,)
!-&!&-"!"-'!'-#!#--!
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239:':,>?,B"!!,S*D,M@EK=+(,CA6A86(3(,6<,*+17@6I7,8)56K9)F7(A7(+0T+A+@E+@8,7@E,0M@E+A+@E+@8,V78)U7H(
Control
30 min
6 hours
Florescence Intensity
Rel
ativ
e C
ell N
umbe
r
Me 4405 SK 28 B
C
)
!
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CR,216U,FH86I+85H,O3(86957I(,6<,>[.,,V56EK=836@?R,F+11,T+78),3@,8)+,V5+(+@=+,6<,Z.O
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.KBABJ=K%$IIDJF>%HI%+AFE=KD>BHA=K%%%<3V6X%Y%!&Z
J)+,<6116U3@9,A)686(,311K(8578+,=6IA1+8+,5+I3((36@,6<,(=,I+87(87(+(,8)78,5+=K55+E,<6116U3@9,(K59+5H,7@E,3557E37836@:,
)IFDE%+AWDJFBHA%HI%<3V6X
1MH%OHAF?>%L'DJEH>B>%HI%BAWDJFD@%KD>BHA
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0.0
0.5
1.0
1.5
2.0
2.5
0 4 8 12 16 20 24
Weeks
Les
ion
Vol
ume
(cc)
Lesion 1
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
0 4 8 12 16 20 24
Weeks
Les
ion
Vol
ume
(cc)
Lesion ALesion BLesion C
VG0&!0**0!&.K4\+=8,!&!]J5+78+E,1+(36@
?H(87@E+5,1+(36@(
/DJ?=AB>O%HI%)JFBHA^
^ >?,3(,87/+@,KA,4H,8KI65 =+11(,7@E,@68,@65I71,=+11(:
^ >?,16=713_+(,3@,V17(I7,I+I457@+(`,@K=1+75,I+I457@+(`1H(6(6I+( 7@E,a,Z6193,
• Internalization in membranes of tumor cells
• Accumulation in perinuclear organelles -lysosomes
• Exclusion from nucleus
/DJ?=AB>O%HI%)JFBHARed Channel : PV-10 + ED-1
Blue Channel : LSG
• PV-10 activation precipitates lysosomal release• Loss of cell morphology occurs within 5-10 min
Pre-Rx 0 min Post 2 min Post 5 min Post 10 min Post
bOc,C>;,?c.JCQT;>,*;JC.JC.;.,>;.V[QTMQZa
V+5)7A(,EK+,86,MIIK@+,5+(A6@(+(a
<HFDAFB=K%#G>FDOBJ%&DADIBF
<3V6X%)NFHKG>B>%J=A%>FBONK=FD%)AFBV1NOHE%+OONABFG
^ !"#$%&'$()*+'#,&-',+)./*+.0#,)*%.,1
2%*3*+)&'%*0$/+.).$+'$(',4*%%,+)'0*11&
511$6&')"#$%&')$'/%$6'"+07*0-*8
5")$19&.&'$(')"#$%'0*11&'1*,8&')$'.#:"1&*'*;:$&"%*'$('.##"+*'&9&)*#')$',+)./*+.0')"#$%'#,)*%.,1
<##"+*'&9&)*#'+$)'0$#:%$#.&*8'49'1$0,1.=*8'>;!"#$%?&:*0.(.0'.##"+*'%*&:$+&*'0,+'%*&"1)
2@?AB'8$*&+C)'8*+,)"%*')"#$%',+)./*+&
!D>HKNFBHA%HI%.HAFE=K=FDE=K1NOHE%
Y)IFDE%1ED=FODAF%HI%<EBO=EG%1NOHEZ
0%
20%
40%
60%
80%
100%
Immune-Deficient Immune-Competent
Primary HCC
Secondary HCC
Primary TumorPrimary Tumor(Treated)(Treated)
Secondary TumorSecondary Tumor(Untreated)(Untreated)
Secondary HCCSecondary HCCResolvedResolved
(N=5) (N=6)
Res
pons
e R
ate
Res
pons
e R
ate
<H>>BSKD%/DJ?=AB>O
��Untreated tumors exhibit high levels Untreated tumors exhibit high levels of granulocytes (of granulocytes (basophilsbasophils, , eosinophilseosinophils and mast cells) in tissue and mast cells) in tissue surrounding tumorssurrounding tumors
��PVPV--10 treatment results in increased 10 treatment results in increased levels of mononuclear tumorlevels of mononuclear tumor--infiltrating lymphocytesinfiltrating lymphocytes
��Release of tumor antigens to local Release of tumor antigens to local antigenantigen--presenting cells may facilitate presenting cells may facilitate presentation of appropriate antigenic presentation of appropriate antigenic targets to T and Btargets to T and B--cells cells
��Collateral destruction of granulocytes Collateral destruction of granulocytes surrounding the tumor may surrounding the tumor may precipitate precipitate chemokinechemokine release and release and local inflammation, and could serve local inflammation, and could serve an adjuvant role in promoting an adjuvant role in promoting specific antispecific anti--tumor responsetumor response
#-//)!4
!H>D%&DAR=K%Y<3V6XZB>%F=UDA%NT%SG%KG>H>HOD>%BA%/DK=AHO=%JDKK>%SNF%AHF%AHEO=K%IBSEHSK=>F>L
+F%FEBRRDE>%TED@HOBA=AFKG%ADJEHFBJ%JDKK%@D=F?%TDE?=T>%SG%EDKD=>D%HI%J=F?DT>BA>L%!"#%=TTD=E%AHF%FH%SD%BACHKCD@L
+AFE=KD>BHA=K%BAWDJFBHA%HI%=%ODK=AHO=%ODF=>F=>B>%ED>NKFD@%BA%ADJEHFBJ%@D=F?%HI%F?D%BAWDJFD@%KD>BHA%%=A@%;%SG>F=A@DE%KD>BHA>L%1?D%&G>F=A@DE%ED>THA>D%O=G%SD%%FH%BA@NJFBHA%HI%%BOONAD%ED>THA>D^
0NEF?DE%BACD>FBR=FBHA%HI%F?D%EHKD%HI%!&%BA%FED=FODAF%HI%ODK=AHO=%B>%M=EE=AFD@L
0">?2#8+(\<237+(]811$?;8$+(V26<@$'(23=(^$'?$B+(0$1W(R$?W((,--.Y().(4?>;1W()9F(IZ(
58>($@(21W+(:36"@2'7$@((,-).
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58>($@(21W+(:36"@2'7$@((,-).
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/"-#)3+%%#256 72)'(%%8,59 :).21$!%$5; 2$!#$4 <9
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!H>D%&DAR=K%?=>%SDDA%N>D@%FH%=>>D>>%?DT=FBJ%INAJFBHA5HTF?=KOBJ @B>HE@DE>%=A@%=>%=%T?HFH>DA>BFB>DE%BA%FED=FODAF%HI%>UBA%KD>BHA>L%+A%F?B>%>FN@G%!&%M=>%FD>FD@%BAVCBFEH%BA%F?D%=S>DAJD%HI%KBR?F%IHE%BF>%DIIDJF>%HA%ODK=AHO=%JDKK>%=A@%ODJ?=AB>O%HI%BF>%DIIDJF>L%+A%=@@BFBHA%=%T=FBDAF%M=>%FED=FD@%SG%BAFE=KD>BHA=K%BAWDJFBHA%HI =%>J%ODF=>F=>B>%MBF?%<3V6XY!&ZL
/DF?H@>)%T=ADK%HI%ODK=AHO=%JDKK>%MDED%DQTH>D@%FH%!&%=F%@BIIDEDAF%JHAJDAFE=FBHA>%=A@%IHE%C=EGBAR%TDEBH@>%=A@%DIIDJF>%>FN@BD@%SG%OHET?HKHRG%=THTFH>B>%=>>=G>%=A@%/11%=>>=G>
!D>NKF>L%!&%BA@NJD@%JDKK%@D=F?%BA%ODK=AHO=%JDKK>%SNF%AHF%IBSEHSK=>F>L%,D=F?%M=>%TED@HOBA=AFKG%@ND%FH%ADJEH>B>%SNF%9%KBAD>%=K>H NA@DEMDAF%=THTFH>B>%F?=F%M=>%@DTDA@DAF%HA%=JFBC=FBHA%HI%J=>T=>D>L%.DKK%@D=F?%M=>%AHF%@ND%FH%EDKD=>D%HI%!D=JFBCD%HQGRDA%>TDJBD>L!& B>%F=UDA%NT%BAFH%KG>H>HOD>%=A@%BF%B>%TEHS=SKD%YSNF%AHF%TEHCDAZ%F?=F%JDKK%@D=F?%ED>NKF>%IEHO%EDKD=>D%HI%.=F?DT>BA>L1?D%T=FBDAF%M?H%?=@%J?DOHF?DE=TG%E=@BHED>B>F=AF%EDJNEEDAF%@B>D=>D%BA%F?D%?D=@%=A@%ADJU%=ED=%?=@%E=TB@%ADJEH>B>%HI%F?D%BAWDJFD@%ODF=>F=>B>%=A@%F?B>%=>%MDKK%=>%;%NABAWDJFD@ >J%ODF=>F=>D>%NA@DE%MDAF%JHOTKDFD%EDOB>>BHA%HCDE%=%TDEBH@%HI%>DCDE=K%OHAF?>
.HAJKN>BHA0NEF?DE%>FN@BD>%FH%NA@DE>F=A@%F?D%ODJ?=AB>O%HI%=JFBHA%%=A@%BF>%EHKD BA%JKBABJ=K%%O=A=RDODAF%HI%ODK=AHO=%%=ED%M=EE=AFD@
)S>FE=JF
Me 4405
SK 28
Fibroblast
Control
6h
24h
24h
A B
C
!
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3B=SBKBFG%Y.HAFEHK[
23456417(8*+,##!-./,"%.+53+(#
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A
B
Me 4405 SK 28
Control
RB
Florescence Intensity
Rel
ativ
e C
ell N
umbe
r
239:":,,CR,>?,B"!!,S*D,M@EK=+(,468),Q6@0CA6A8683=,7@E,CA6A8683=,F+11,T+78),3@,*+17@6I7,F+11(,7<8+5,"#)
?R,216U,FH86I+85H,O3(86957I(,6<,CA6A86(3(,C((7H(,4H,8)+,VM,*+8)6E,3@:,.P,"%,7@E,,*+,##!-,F+11(,J5+78+E,U38),>?,B"!!,S*D,<65,"#,)
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239:':,>?,B"!!,S*D,M@EK=+(,CA6A86(3(,6<,*+17@6I7,8)56K9)F7(A7(+0T+A+@E+@8,7@E,0M@E+A+@E+@8,V78)U7H(
Control
30 min
6 hours
Florescence Intensity
Rel
ativ
e C
ell N
umbe
r
Me 4405 SK 28 B
C
)
!
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CR,216U,FH86I+85H,O3(86957I(,6<,>[.,,V56EK=836@?R,F+11,T+78),3@,8)+,V5+(+@=+,6<,Z.O
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J)+,<6116U3@9,A)686(,311K(8578+,=6IA1+8+,5+I3((36@,6<,(=,I+87(87(+(,8)78,5+=K55+E,<6116U3@9,(K59+5H,7@E,3557E37836@:,
)IFDE%+AWDJFBHA%HI%<3V6X
1MH%OHAF?>%L'DJEH>B>%HI%BAWDJFD@%KD>BHA
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0.0
0.5
1.0
1.5
2.0
2.5
0 4 8 12 16 20 24
Weeks
Les
ion
Vol
ume
(cc)
Lesion 1
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
0 4 8 12 16 20 24
Weeks
Les
ion
Vol
ume
(cc)
Lesion ALesion BLesion C
VG0&!0**0!&.K4\+=8,!&!]J5+78+E,1+(36@
?H(87@E+5,1+(36@(
/DJ?=AB>O%HI%)JFBHA^
^ >?,3(,87/+@,KA,4H,8KI65 =+11(,7@E,@68,@65I71,=+11(:
^ >?,16=713_+(,3@,V17(I7,I+I457@+(`,@K=1+75,I+I457@+(`1H(6(6I+( 7@E,a,Z6193,
• Internalization in membranes of tumor cells
• Accumulation in perinuclear organelles -lysosomes
• Exclusion from nucleus
/DJ?=AB>O%HI%)JFBHARed Channel : PV-10 + ED-1
Blue Channel : LSG
• PV-10 activation precipitates lysosomal release• Loss of cell morphology occurs within 5-10 min
Pre-Rx 0 min Post 2 min Post 5 min Post 10 min Post
bOc,C>;,?c.JCQT;>,*;JC.JC.;.,>;.V[QTMQZa
V+5)7A(,EK+,86,MIIK@+,5+(A6@(+(a
<HFDAFB=K%#G>FDOBJ%&DADIBF
<3V6X%)NFHKG>B>%J=A%>FBONK=FD%)AFBV1NOHE%+OONABFG
^ !"#$%&'$()*+'#,&-',+)./*+.0#,)*%.,1
2%*3*+)&'%*0$/+.).$+'$(',4*%%,+)'0*11&
511$6&')"#$%&')$'/%$6'"+07*0-*8
5")$19&.&'$(')"#$%'0*11&'1*,8&')$'.#:"1&*'*;:$&"%*'$('.##"+*'&9&)*#')$',+)./*+.0')"#$%'#,)*%.,1
<##"+*'&9&)*#'+$)'0$#:%$#.&*8'49'1$0,1.=*8'>;!"#$%?&:*0.(.0'.##"+*'%*&:$+&*'0,+'%*&"1)
2@?AB'8$*&+C)'8*+,)"%*')"#$%',+)./*+&
!D>HKNFBHA%HI%.HAFE=K=FDE=K1NOHE%
Y)IFDE%1ED=FODAF%HI%<EBO=EG%1NOHEZ
0%
20%
40%
60%
80%
100%
Immune-Deficient Immune-Competent
Primary HCC
Secondary HCC
Primary TumorPrimary Tumor(Treated)(Treated)
Secondary TumorSecondary Tumor(Untreated)(Untreated)
Secondary HCCSecondary HCCResolvedResolved
(N=5) (N=6)
Res
pons
e R
ate
Res
pons
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��PVPV--10 treatment results in increased 10 treatment results in increased levels of mononuclear tumorlevels of mononuclear tumor--infiltrating lymphocytesinfiltrating lymphocytes
��Release of tumor antigens to local Release of tumor antigens to local antigenantigen--presenting cells may facilitate presenting cells may facilitate presentation of appropriate antigenic presentation of appropriate antigenic targets to T and Btargets to T and B--cells cells
��Collateral destruction of granulocytes Collateral destruction of granulocytes surrounding the tumor may surrounding the tumor may precipitate precipitate chemokinechemokine release and release and local inflammation, and could serve local inflammation, and could serve an adjuvant role in promoting an adjuvant role in promoting specific antispecific anti--tumor responsetumor response
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Results
Efficacy of PV-10 in the MT-901 Breast Cancer ModelBALB/c mice were injected on each flank with MT-901 cells.
Seven days later, mice received IL injection of the right flanklesion with 50 ml PV-10. As shown in Figure 1A, significantlysmaller tumors were observed in mice treated with PV-10(p,0.001 compared to PBS-treated mice). In addition, smallertumor sizes were measured in the bilateral (untreated) tumors ofmice treated with PV-10 (Figure 1B, p,0.05 compared to PBStreated mice).
We next examined induction of tumor-specific immunity inMT901-bearing mice. Mice were injected s.c. with MT901 cellson the flank. On day 7, mice received an intralesional injection ofPBS or PV-10. Spleens were collected on day 21 after treatment.Splenocytes were re-stimulated with MT-901 cells for 48 hours,and supernatants were collected. As shown in Figure 2, mice thatreceived PV-10 produced increased IFN-c in response to MT-901cells compared to PBS-treated mice (p,0.05). No IFN-cproduction was measured in response to irrelevant control CT-26 cells.
Efficacy of PV-10 in the B16 Melanoma ModelIn a more aggressive model, mice received 16105 B16 cells s.c.
on day 0 to establish a solitary tumor on the flank and 56105 B16cells intravenously (i.v.) to establish multiple lung lesions. On day
7, mice were treated with 50 ml PBS or PV-10 IL to the flanktumor. Growth of s.c. tumors was measured until day 21 whenmice were sacrificed to enumerate lung lesions. All mice thatreceived PBS treatments displayed growth of tumor in the flankand had more than 250 lung lesions. In contrast, mice thatreceived IL PV-10 demonstrated fewer lung lesions (Figure 3A,p,0.01 compared to PBS-treated mice) and had significantlysmaller subcutaneous tumors (Figure 3B, p,0.05 compared tomice treated with IL PBS). Representative lungs are shown inFigure 3C. To determine whether injection of the s.c. B16 tumorwas required for the observed regression of tumor in the lungs,mice bearing B16 lung lesions were injected s.c. with 50 ml of PV-10 into a non-tumor bearing flank. No difference was measured inthe number of B16 lung lesions in mice treated s.c. with PBS orPV-10 (not shown). This indicates that direct injection of PV-10into a tumor lesion is required for the observed systemic effect ofPV-10.
To determine whether PV-10 has a direct effect on thefrequency of immune cell subsets, mice bearing B16 tumor inthe flank were treated on day 7 with a single IL injection of 50 mLPBS or PV-10. Spleens were collected 7 days after injection. Asshown in Figure 4, no differences were measured in the percentageof T cells (CD8+ or CD4+), regulatory T cells (CD4+ CD25+
Foxp3+), NK cells, B cells (CD19+), myeloid derived suppressorcells (CD11b+Gr1+) or macrophages (F4/80+) in splenocytes ofPV-10 treated mice. No differences were observed in cell subsetson day 21 (not shown).
To determine whether B16-specific T cells were induced afterinjection with PV-10, mice bearing B16 tumor were treated on day7 with a single IL injection of 50 mL PBS or PV-10. One weeklater, spleens were collected and splenocytes were restimulatedwith B16 or MC-38 cells. IFN-c was measured in the supernatantsafter 48 hours. Splenocytes isolated from B16-bearing mice treatedwith PV-10 demonstrated a significant increase in production ofIFN-c compared to PBS-treated mice (Figure 5A). No IFN-cproduction was demonstrated in response to irrelevant controlMC-38 cells in either treatment group. Flow cytometric analysisdemonstrated that CD8+ T cells were producing IFN-c (notshown).
Next, we measured the ability of T cells in splenocytes to lyseB16 cells. As shown in Figure 5B, T cells from PV-10 treated mice
Figure 1. Treatment with PV-10 leads to tumor regression inMT-901 breast cancer model. BALB/c mice (n = 8 mice per group)were injected on each flank with MT-901 cells. Seven days later, micereceived intratumoral injection of the right flank lesion with 50 ml PV-10.Tumor growth was measured in (A) treated and (B) untreated tumors.Data shows the mean 6 SEM for each time point. Experiment wasrepeated two times with similar results. *indicates p,0.001, **indicatesp,0.05.doi:10.1371/journal.pone.0068561.g001
Figure 2. PV-10 treatment leads to tumor-specific IFN-gammaresponses in mice bearing MT-901 breast cancer. Splenocyteswere plated at 26106, co-cultured with 26105 irradiated MT-901 tumorcells, and incubated for 48 hours. Culture supernatants were analyzedfor IFN-c production using commercially available ELISA kit. Data showsthe mean 6 SD of triplicates. *indicates p,0.05.doi:10.1371/journal.pone.0068561.g002
Intralesional PV-10 Therapy
PLOS ONE | www.plosone.org 3 July 2013 | Volume 8 | Issue 7 | e68561
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Summary
Purpose
Results Introduction
Intralesional Injection of Rose Bengal Induces an Anti-tumor Immune Response and Potent Tumor Regressions in a Murine
Model of Colon Cancer
• Rose Bengal (RB) is a synthetic garment dye thathas been used in humans safely for nearly 100 years
• Treatment of patients with advanced melanoma withintratumoral injection of PV-10 (10% formulation ofRB) has shown regression of in-transit melanomadeposits and non-treated tumors
• The effect of RB on colorectal cancer (CRC) isunknown
To study effects of RB on the growth and viability of colorectal cancer cells
Krunal H. Pardiwala1, Guilin Qiao1, Jayraman Sundararajan3, Bellur Prabhakar3, and Ajay V. Maker1,2
Dept. of Surgery1, Division of Surgical Oncology2; Dept. of Microbiology and Immunology3; University of Illinois at Chicago, Chicago, IL
Acknowledgements The authors wish to thank Provectus providing PV-10 for the experiments
References 1.! Siegel R et al. CA 64(1):20. 2014.2.! Edwards, BK et al. Cancer 116(3)544-73. 2010.3.! Alexander W. ASCO meeting 2010. 35(8):469-78. 2010.4.! Ito A et al. J Natl Cancer Inst 77(1):277-81. 1986.5.! Thompson JF et al. Melanoma Res 18(6)405-11. 2008.6.! Thompson JF et al. Annals of Surg Oncol. E-pub. 2014.
"Methods
• Murine (CT26) and human (HT29) colorectal cell lineswere treated in vitro with various concentrations ofRB. 50µM 5-FU was used as a positive control
• Cell viability was assessed by the MTS assay andTrypan Blue staining
• SNARF-1 and DAF-FM staining were performed toassess cellular stability
• CT26 cells were inoculated in syngeneic mice toestablish two subcutaneous tumors. Onceestablished, one of the tumors was injected withPV-10 at a dose equal to half the calculated tumorvolume
• Tumor dimensions were measured daily• Splenocytes from treated and control animals were
collected and co-cultured with irradiated CT26 cells.Supernatants were collected and INF! levels assessedby ELISA
!"#$%&'()*+,(-.!"#$!%&'()*+&,-.!/0*'!'1+-!,0-2,-3!41,)!56789!.-+0-,-!:;<7/*=3!'*0-! >?@A! 1B!,)-!(0-.-B+-!*/! ,C'*0!+*'(20-3!,*!.)2'7,0-2,-3!2B1'2=.!"DE!F.;!GE!(HI'%J!(!K!9;9L$!!%
/-0'1201)%3*4('%5'(607.!MN-0!1B,02,C'*02=!1BO-+P*B!41,)!56789J!CB,0-2,-3!+*B,02=2,-02=! #Q#! ,C'*0.! 3-'*B.,02,-3! 2! B*B7.1HB1R+2B,! ,0-B3! ,*4203.!.=*4-0! ,C'*0!H0*4,);!SB-!*/! .1T! 2B1'2=.!-T(-01-B+-3!2! +*'(=-,-!U&.,2B3-0!0-.(*B.-!1B!,)-!CB,0-2,-3!,C'*0;!!%
• Both murine and human colorectal cancer cellsshow near complete cell death within hours ofexposure to Rose Bengal
• Decrease in intracellular pH and increase inintracellular nitric oxide , as seen with the SNARF-1and DAF-FM staining reflect PV-10 induced celldeath
• Five of 6 CRC tumors treated intralesionally withPV-10 experienced tumor ablation. 40% of tumorsthat experienced a partial or complete responselater demonstrated secondary tumor remodelingand growth, while 60% maintained a durable CR upto 9 days
• Untreated contralateral tumors in animals treatedwith PV-10 may trend towards a slower growthrate. One in six of the animals demonstrated acomplete bystander response in an untreatedtumor
• Splenocytes from treated mice produce greateramount of INF! when re-exposed to irradiatedCT26 cells
3'1201)% 3*4('% 5'(607.! "#)20,$! MN-0! 1B,02,C'*02=! 1BO-+P*B! 41,)! 56789J! RF-!,0-2,-3! #Q#! ,C'*0.! -T(-01-B+-3! 2B! 2BP7,C'*0! 0-.(*B.-! 1B! ,)-! R0.,! :L7<:!)*C0.;!V4*!*/! ,)-.-! ,C'*0.! 0-'*3-=-3!2B3!+*BPBC-3!,*!H0*4;!V)0--! ,C'*0.!)23! 2! 3C02U=-! +*'(=-,-! 0-.(*B.-;! Q-(0-.-B,2PF-! 1'2H-! */! (0-7,0-2,'-B,!">'2H-!MJ!<!32&.!(*.,!1B*+C=2P*B$J!56789!,0-2,-3!"W2&!:!(*.,!,0-2,'-B,$!"1'2H-!X$ 2B3!2!+*'(=-,-!0-.(*B3-0!"1'2H-!#J!W2&!D$!20-!.)*4B!2U*F-;
A B C
n = 6 p = 0.42
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0
20
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120
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8199% ><2?<9<0@% A==2@=! "M$! ?-20! +*'(=-,-! +-==! 3-2,)! 1.! .--B! 2,! :L! )*C0.! 2N-0!2331P*B!*/!QX! ,*!'C01B-!#V:\!#Q#! +-==.! 2,! +*B+-B,02P*B.! H0-2,-0! ,)2B!:E9!][;! ! "X$! ZV:D! )C'2B! #Q#! +-==.! 20-! 2=.*! .-B.1PF-! ,*! QX! 2,! +*B+-B,02P*B.!2U*F-! 899C[;! ! #-==! 3-2,)! 2,! )1H)-0! +*B+-B,02P*B.! 42.! .1'1=20! ,*! E7@^!,0-2,'-B,;!Y1'1=20!0-.C=,.!4-0-!.--B!41,)!V0&(2B!U=C-!.,21B1BH!2B3!U-H1BB1BH!2.!-20=&!2,!8!)*C0!2N-0!2331P*B!*/!QX!,*!+-==.!"B*,!.)*4B$%
A B
** ** **
*
** ** **
*
A B C
• Rose Bengal induced potent cell death in humanand murine colon cancer cells in vitro
• Intralesional injection in established tumorsinduced an anti-tumor immune response andsignificant tumor regressions in vivo
• These studies establish that intralesional PV-10therapy warrants further study as a potentialimmunotherapeutic agent in colorectal cancerand metastases
Conclusions
Summary
Purpose
Results Introduction
Intralesional Injection of Rose Bengal Induces an Anti-tumor Immune Response and Potent Tumor Regressions in a Murine
Model of Colon Cancer
• Rose Bengal (RB) is a synthetic garment dye thathas been used in humans safely for nearly 100 years
• Treatment of patients with advanced melanoma withintratumoral injection of PV-10 (10% formulation ofRB) has shown regression of in-transit melanomadeposits and non-treated tumors
• The effect of RB on colorectal cancer (CRC) isunknown
To study effects of RB on the growth and viability of colorectal cancer cells
Krunal H. Pardiwala1, Guilin Qiao1, Jayraman Sundararajan3, Bellur Prabhakar3, and Ajay V. Maker1,2
Dept. of Surgery1, Division of Surgical Oncology2; Dept. of Microbiology and Immunology3; University of Illinois at Chicago, Chicago, IL
Acknowledgements The authors wish to thank Provectus providing PV-10 for the experiments
References 1.! Siegel R et al. CA 64(1):20. 2014.2.! Edwards, BK et al. Cancer 116(3)544-73. 2010.3.! Alexander W. ASCO meeting 2010. 35(8):469-78. 2010.4.! Ito A et al. J Natl Cancer Inst 77(1):277-81. 1986.5.! Thompson JF et al. Melanoma Res 18(6)405-11. 2008.6.! Thompson JF et al. Annals of Surg Oncol. E-pub. 2014.
"Methods
• Murine (CT26) and human (HT29) colorectal cell lineswere treated in vitro with various concentrations ofRB. 50µM 5-FU was used as a positive control
• Cell viability was assessed by the MTS assay andTrypan Blue staining
• SNARF-1 and DAF-FM staining were performed toassess cellular stability
• CT26 cells were inoculated in syngeneic mice toestablish two subcutaneous tumors. Onceestablished, one of the tumors was injected withPV-10 at a dose equal to half the calculated tumorvolume
• Tumor dimensions were measured daily• Splenocytes from treated and control animals were
collected and co-cultured with irradiated CT26 cells.Supernatants were collected and INF! levels assessedby ELISA
!"#$%&'()*+,(-.!"#$!%&'()*+&,-.!/0*'!'1+-!,0-2,-3!41,)!56789!.-+0-,-!:;<7/*=3!'*0-! >?@A! 1B!,)-!(0-.-B+-!*/! ,C'*0!+*'(20-3!,*!.)2'7,0-2,-3!2B1'2=.!"DE!F.;!GE!(HI'%J!(!K!9;9L$!!%
/-0'1201)%3*4('%5'(607.!MN-0!1B,02,C'*02=!1BO-+P*B!41,)!56789J!CB,0-2,-3!+*B,02=2,-02=! #Q#! ,C'*0.! 3-'*B.,02,-3! 2! B*B7.1HB1R+2B,! ,0-B3! ,*4203.!.=*4-0! ,C'*0!H0*4,);!SB-!*/! .1T! 2B1'2=.!-T(-01-B+-3!2! +*'(=-,-!U&.,2B3-0!0-.(*B.-!1B!,)-!CB,0-2,-3!,C'*0;!!%
• Both murine and human colorectal cancer cellsshow near complete cell death within hours ofexposure to Rose Bengal
• Decrease in intracellular pH and increase inintracellular nitric oxide , as seen with the SNARF-1and DAF-FM staining reflect PV-10 induced celldeath
• Five of 6 CRC tumors treated intralesionally withPV-10 experienced tumor ablation. 40% of tumorsthat experienced a partial or complete responselater demonstrated secondary tumor remodelingand growth, while 60% maintained a durable CR upto 9 days
• Untreated contralateral tumors in animals treatedwith PV-10 may trend towards a slower growthrate. One in six of the animals demonstrated acomplete bystander response in an untreatedtumor
• Splenocytes from treated mice produce greateramount of INF! when re-exposed to irradiatedCT26 cells
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A B C
n = 6 p = 0.42
n = 6 p = 0.027
n= 3
C
A
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A B
** ** **
*
** ** **
*
A B C
• Rose Bengal induced potent cell death in humanand murine colon cancer cells in vitro
• Intralesional injection in established tumorsinduced an anti-tumor immune response andsignificant tumor regressions in vivo
• These studies establish that intralesional PV-10therapy warrants further study as a potentialimmunotherapeutic agent in colorectal cancerand metastases
Conclusions
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Assessment of Immune and Clinical Efficacy after Intralesional PV-10 in Injected and Uninjected Metastatic Melanoma Lesions
Amod A Sarnaik, Georgina Crago, Hao Liu, Krithika Kodumudi, Amy Weber, Timothy McCardle, Jeffrey S Weber, Shari Pilon-Thomas H. Lee Moffitt Cancer Center, Tampa, FL
Prior Treatment Abstract (updated)
Summary •! IL PV-10 can induce regression of injected and
uninjected metastatic melanoma •! IL PV-10 can enhance tumor-specific reactivity in
circulating T cells •! IL PV-10 leads to responses in treatment-refractory
tumors •! IL PV-10 may be rationally combined with systemic
immunotherapy for the treatment of metastatic melanoma
Intralesional (IL) therapy is under investigation to treat dermal and subcutaneous metastatic cancer. In our murine model, IL injection of PV-10 (10% Rose Bengal) induced regression of injected and uninjected “bystander” melanomas. We observed a consistent increase in anti-tumor T cell responses following IL PV-10 . We translated these findings into a pilot clinical trial that enrolled 13 patients with dermal and/or subcutaneous metastatic melanoma. Two study lesions in each patient were sampled by biopsy pre-treatment; one of the two lesions was injected with IL PV-10, then both residual sites were completely excised. We compared tumors before and after treatment with H&E staining to determine pathologic complete response (pCR), and we confirmed results with MelanA immunohistochemistry. Peripheral blood mononuclear cells (PBMC) before and after IL PV-10 were phenotyped for activation markers by flow cytometry. Of the evaluable patients to date, treatment with IL PV-10 led to pCR in the post-treatment biopsies of both PV10-injected and uninjected study lesions in 4 of the 8 patients, and all 8 exhibited at least partial regression of the injected lesion. IL PV-10 was associated with an increase in circulating cytotoxic CD3+/CD8+ T cells (n=10, paired t test, p=0.03). Pre and post PV-10 treated CD8+ PBMC from one patient were re-stimulated with autologous tumor in vitro. Compared to pre-treatment, PV-10 treatment produced an increase in tumor-specific interferon-gamma release by ELISA. Six of 8 patients had metastatic disease refractory to previous ipilimumab, anti-PD-1 and/or vemurafenib therapy. Four of these 6 patients exhibited pCR to PV10 in both the injected and uninjected lesions. IL PV-10 treatment can lead to systemic anti-melanoma immunity and pCR in injected and uninjected lesions including treatment-refractory tumors. Further studies are ongoing to determine the mechanism by which PV-10 increases tumor-specific T cell responses as well as to establish the interaction of intralesional PV-10 with combination checkpoint protein inhibition.
Change in Melanin A IHC (cont)
Clinical Trial Design
Partial biopsy of two lesions and PBMC collection pretreatment
Excision of 2 lesions and PBMC collection post treatment
IL PV-10 of one of the two lesions
PBMC collection post treatment
patients
Day 7 Day 14 Day 28
PV-10 alters T cell immunity
Of the 13 consented patients, 5 had no previous treatment, 6 received ILI or ipilimumab, and 2 received PD-1 blocking antibody; 6 received two or more prior systemic therapy.
PV001
Change in Melanin A IHC
PV10-Injected Lesion Uninjected Lesion
PV004
PV002
PV003
PV10-Injected Lesion Uninjected Lesion
Pre-Rx Post-RX Pre-Rx Post-Rx
PV006
PV005
PV007
PV008
Pre-Rx Post-RX Pre-Rx Post-Rx
0
20
40
60
80
100
post pre
p=0.004 p=0.004
Injected Lesion
Uninjected Lesion
Quantitation of Melanin A Change
Day 0
CD8+ T cells CD4+ T cells
CD3+ T cells NKT cells
PBMC were purified before and after IL PV-10. CD3, CD8 and NKT cells significantly increased after IL PV-10. For one patient, CD8 cells were purified from PBMC and restimulated with autologous tumor. There was a significant increase in tumor-specific IFN-! release measured by ELISA."
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H. LEE MOFFITT CANCER CENTER & RESEARCH INSTITUTE,
AN NCI COMPREHENSIVE CANCER CENTER – Tampa, FL 1-888-MOFFITT (1-888-663-3488) www.MOFFITT.org
© 2010 H. Lee Moffitt Cancer Center and Research Institute,
Inc.
Introduction Rose Bengal is a water-soluble xanthene dye that has been previously used in liver function studies and is still in use by ophthalmologists. PV-10 is a 10% solution of Rose Bengal formulated for intralesional (IL) injection. In clinical trials, IL PV-10 therapy induced regression of both injected lesions and uninjected bystander lesions in patients with melanoma. We have previously shown that IL injection of PV-10 into a single subcutaneous B16 melanoma tumor led to regression of both the injected tumor and uninjected B16 lung lesions. Tumor regression correlated with the induction of systemic anti-tumor T cell immunity. In this study, we have measured whether IL PV-10 and co-inhibitory blockade improves anti-tumor immunity and regression of melanoma.
Efficacy of Intralesional Injection with PV-10 in Combination with Co-Inhibitory Blockade in a Murine Model of Melanoma
Shari Pilon-Thomas, Hao Liu, Krithika Kodumudi, Ellen Moore, Amy Weber, and Amod A. Sarnaik
H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
Methods Single flank model: C57BL/6 mice were injected subcutaneously (s.c.) on with B16 or B16-OVA cells. Tumor was injected IL with 50 uL of PV-10 or PBS on day 7-14 after tumor induction. Three days after IL injection, mice received intraperitoneal (i.p.) injections of 20 mg/kg anti-CTLA4, anti-PD1 or anti-PDL1 antibodies. NrIgG antibodies were used as a control. Antibody injections were continued every 3-4 days.
Bilateral model: C57BL/6 mice were injected s.c. on both flanks with B16 cells. The tumor on the right flanks was injected IL with 50 uL of PV-10 or PBS on day 7 after tumor induction. Three days after IL injection, mice received i.p. injections of 20 mg/kg NrIgG or anti-PDL1 antibodies. Antibody injections were continued every 3-4 days. Tumor sizes were measured.
For analysis of T cell activation, splenocytes were collected on day 7-14 after PV-10 injection after at least 2 i.p. antibody treatments. Splenocytes were co-cultured with irradiated B16 or B16-OVA cells and MC-38 colorectal cells. Supernatants were collected after 48 hours IFN-gamma was measured by ELISA.
!! These murine studies support combination therapy with IL PV-10 and co-inhibitory blockade.
!! Combination therapy with IL PV-10 and anti-PDL1 antibodies led to reduced tumor growth in both injected and uninjected bystander lesions. Increased anti-tumor immunity was measured in mice treated with anti-PDL1 antibodies.
!! Combination therapy with IL PV-10 and anti-PD1 antibodies led to a reduction in tumor growth and increased tumor-specific T cell activity.
!! Combination therapy with IL PV-10 and anti-CTLA4 antibodies led to a trend in tumor reduction and increased tumor-specific T cell activity.
Acknowledgement: Provectus Pharmaceuticals, Inc. provided PV-10 for these experiments.
Combination Therapy with IL PV-10 and anti-CTLA-4 antibodies in B16-OVA bearing mice
Combination Therapy with IL PV-10 and anti-PD-1 antibodies in B16-OVA bearing mice
Results
Conclusions
Combination Therapy with IL PV-10 and anti-PD-L1 antibodies in B16 bearing mice
0
10
20
30
40
50
60
0 5 10 15 20 25
Days
Mea
n Tu
mor
siz
e (m
m2 )
PBS + NrIgG PBS + anti-PDL1 PV10 + NrIgG PV10 + anti-PDL1
p<0.05
Mea
n Tu
mor
Siz
e (m
m2 )
0
10 20 30
40
50 60
70 80
0 5 10 15 20 25
PBS PV10 anti-PD1 PV10+anti-PD1
Days
p<0.05
0
500
1000
1500
2000
2500
3000
3500
PBS PV10 anti-PD1 PV10+anti-PD1
IFN
-gam
ma
(pg/
ml)
0
10
20
30
40
50
60
70
PBS PV10 anti-CTLA4 PV10+anti-CTLA4
Mea
n Tu
mor
Wei
ght (
mg)
0
200
400
600
800
1000
1200
1400
1600
PBS
PV10
anti-CTLA4 PV10+anti-CTLA4
IFN
-gam
ma
(pg/
ml)
B16 MC38
p=0.05
0
500
1000
1500
2000
2500
PBS PV10 anti-PDL1 PV10+anti-PDL1
IFN
-gam
ma
(pg/
ml)
* *
*p<0.05 compared to PV-10 treated mice
Treatment with anti-PD-L1 antibodies alone or in combination with PV-10 leads to the induction of B16-specific T cells
Treatment with IL PV-10 and anti-PD-L1 antibodies slows growth of injected and uninjected tumors in a bilateral B16 tumor model
Treatment with anti-PD-L1 antibodies in combination with IL PV-10 results in reduced growth of B16 tumor
Treatment with anti-PD-1 antibodies in combination with IL PV-10 results in reduced growth of B16-OVA tumor
Treatment with anti-CTLA-4 antibodies in combination with IL PV-10 leads to the induction of B16-specific T cells
Treatment with anti-CTLA-4 antibodies in combination with IL PV-10 results in smaller B16-OVA tumors
Treatment with anti-PD-1 antibodies in combination with IL PV-10 leads to the induction of B16-OVA-specific T cells
PBS + NrIgG PV-10 + NrIgG PBS + anti-PDL1 PV-10 + anti-PDL1
0 50 100 150 200 250 300
0 5 10 15 20 25 30 35
Tum
or S
ize
(mm
2 )
PV10 Treated Tumor
*p<0.01
*p<0.01
Untreated Tumor
0 50 100 150 200 250 300
0 5 10 15 20 25 30 35 Days
Tum
or S
ize
(mm
2 )
p<0.05
D81"3OU<"%2?($@(21W+(IJUP(,-)*
anti-CTLA4 PV10+anti-CTLA4 anti-CTLA4 PV10+anti-CTLA4
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H. LEE MOFFITT CANCER CENTER & RESEARCH INSTITUTE,
AN NCI COMPREHENSIVE CANCER CENTER – Tampa, FL 1-888-MOFFITT (1-888-663-3488) www.MOFFITT.org
© 2010 H. Lee Moffitt Cancer Center and Research Institute,
Inc.
Introduction Rose Bengal is a water-soluble xanthene dye that has been previously used in liver function studies and is still in use by ophthalmologists. PV-10 is a 10% solution of Rose Bengal formulated for intralesional (IL) injection. In clinical trials, IL PV-10 therapy induced regression of both injected lesions and uninjected bystander lesions in patients with melanoma. We have previously shown that IL injection of PV-10 into a single subcutaneous B16 melanoma tumor led to regression of both the injected tumor and uninjected B16 lung lesions. Tumor regression correlated with the induction of systemic anti-tumor T cell immunity. In this study, we have measured whether IL PV-10 and co-inhibitory blockade improves anti-tumor immunity and regression of melanoma.
Efficacy of Intralesional Injection with PV-10 in Combination with Co-Inhibitory Blockade in a Murine Model of Melanoma
Shari Pilon-Thomas, Hao Liu, Krithika Kodumudi, Ellen Moore, Amy Weber, and Amod A. Sarnaik
H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
Methods Single flank model: C57BL/6 mice were injected subcutaneously (s.c.) on with B16 or B16-OVA cells. Tumor was injected IL with 50 uL of PV-10 or PBS on day 7-14 after tumor induction. Three days after IL injection, mice received intraperitoneal (i.p.) injections of 20 mg/kg anti-CTLA4, anti-PD1 or anti-PDL1 antibodies. NrIgG antibodies were used as a control. Antibody injections were continued every 3-4 days.
Bilateral model: C57BL/6 mice were injected s.c. on both flanks with B16 cells. The tumor on the right flanks was injected IL with 50 uL of PV-10 or PBS on day 7 after tumor induction. Three days after IL injection, mice received i.p. injections of 20 mg/kg NrIgG or anti-PDL1 antibodies. Antibody injections were continued every 3-4 days. Tumor sizes were measured.
For analysis of T cell activation, splenocytes were collected on day 7-14 after PV-10 injection after at least 2 i.p. antibody treatments. Splenocytes were co-cultured with irradiated B16 or B16-OVA cells and MC-38 colorectal cells. Supernatants were collected after 48 hours IFN-gamma was measured by ELISA.
Results
!! These murine studies support combination therapy with IL PV-10 and co-inhibitory blockade.
!! Combination therapy with IL PV-10 and anti-PDL1 antibodies led to reduced tumor growth in both injected and uninjected bystander lesions. Increased anti-tumor immunity was measured in mice treated with anti-PDL1 antibodies.
!! Combination therapy with IL PV-10 and anti-PD1 antibodies led to a reduction in tumor growth and increased tumor-specific T cell activity.
!! Combination therapy with IL PV-10 and anti-CTLA4 antibodies led to a trend in tumor reduction and increased tumor-specific T cell activity.
Acknowledgement: Provectus Pharmaceuticals, Inc. provided PV-10 for these experiments.
Combination Therapy with IL PV-10 and anti-CTLA-4 antibodies in B16-OVA bearing mice
Combination Therapy with IL PV-10 and anti-PD-1 antibodies in B16-OVA bearing mice
Results
Conclusions
Combination Therapy with IL PV-10 and anti-PD-L1 antibodies in B16 bearing mice
0
10
20
30
40
50
60
0 5 10 15 20 25
Days
Mea
n Tu
mor
siz
e (m
m2 )
PBS + NrIgG PBS + anti-PDL1 PV10 + NrIgG PV10 + anti-PDL1
p<0.05
Mea
n Tu
mor
Siz
e (m
m2 )
0
10 20 30
40
50 60
70 80
0 5 10 15 20 25
PBS PV10 anti-PD1 PV10+anti-PD1
Days
p<0.05
0
500
1000
1500
2000
2500
3000
3500
PBS PV10 anti-PD1 PV10+anti-PD1
IFN
-gam
ma
(pg/
ml)
0
10
20
30
40
50
60
70
PBS PV10 anti-CTLA4 PV10+anti-CTLA4
Mea
n Tu
mor
Wei
ght (
mg)
0
200
400
600
800
1000
1200
1400
1600
PBS
PV10
anti-CTLA4 PV10+anti-CTLA4
IFN
-gam
ma
(pg/
ml)
B16 MC38
p=0.05
0
500
1000
1500
2000
2500
PBS PV10 anti-PDL1 PV10+anti-PDL1
IFN
-gam
ma
(pg/
ml)
* *
*p<0.05 compared to PV-10 treated mice
Treatment with anti-PD-L1 antibodies alone or in combination with PV-10 leads to the induction of B16-specific T cells
Treatment with IL PV-10 and anti-PD-L1 antibodies slows growth of injected and uninjected tumors in a bilateral B16 tumor model
Treatment with anti-PD-L1 antibodies in combination with IL PV-10 results in reduced growth of B16 tumor
Treatment with anti-PD-1 antibodies in combination with IL PV-10 results in reduced growth of B16-OVA tumor
Treatment with anti-CTLA-4 antibodies alone in combination with IL PV-10 leads to the induction of B16-specific T cells
Treatment with anti-CTLA-4 antibodies in combination with IL PV-10 results in smaller B16-OVA tumors
Treatment with anti-PD-1 antibodies in combination with IL PV-10 leads to the induction of B16-OVA-specific T cells
PBS + NrIgG PV-10 + NrIgG PBS + anti-PDL1 PV-10 + anti-PDL1
0 50 100 150 200 250 300
0 5 10 15 20 25 30 35
Tum
or S
ize
(mm
2 )
PV10 Treated Tumor
*p<0.01
*p<0.01
Untreated Tumor
0 50 100 150 200 250 300
0 5 10 15 20 25 30 35 Days
Tum
or S
ize
(mm
2 )
p<0.05
25
p<0.05
anti-PD1 PV10+anti-PD1
\_
V#3V#3;V#3;E0+3+&.04E#/P+3.'+#34F.'.H. LEE MOFFITT CANCER CENTER & RESEARCH
INSTITUTE, AN NCI COMPREHENSIVE CANCER CENTER – Tampa, FL
1-888-MOFFITT (1-888-663-3488) www.MOFFITT.org
© 2010 H. Lee Moffitt Cancer Center and Research Institute, Inc.
Introduction Rose Bengal is a water-soluble xanthene dye that has been previously used in liver function studies and is still in use by ophthalmologists. PV-10 is a 10% solution of Rose Bengal formulated for intralesional (IL) injection. In clinical trials, IL PV-10 therapy induced regression of both injected lesions and uninjected bystander lesions in patients with melanoma. We have previously shown that IL injection of PV-10 into a single subcutaneous B16 melanoma tumor led to regression of both the injected tumor and uninjected B16 lung lesions. Tumor regression correlated with the induction of systemic anti-tumor T cell immunity. In this study, we have measured whether IL PV-10 and co-inhibitory blockade improves anti-tumor immunity and regression of melanoma.
Efficacy of Intralesional Injection with PV-10 in Combination with Co-Inhibitory Blockade in a Murine Model of Melanoma
Shari Pilon-Thomas, Hao Liu, Krithika Kodumudi, Ellen Moore, Amy Weber, and Amod A. Sarnaik
H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
Methods Single flank model: C57BL/6 mice were injected subcutaneously (s.c.) on with B16 or B16-OVA cells. Tumor was injected IL with 50 uL of PV-10 or PBS on day 7-14 after tumor induction. Three days after IL injection, mice received intraperitoneal (i.p.) injections of 20 mg/kg anti-CTLA4, anti-PD1 or anti-PDL1 antibodies. NrIgG antibodies were used as a control. Antibody injections were continued every 3-4 days.
Bilateral model: C57BL/6 mice were injected s.c. on both flanks with B16 cells. The tumor on the right flanks was injected IL with 50 uL of PV-10 or PBS on day 7 after tumor induction. Three days after IL injection, mice received i.p. injections of 20 mg/kg NrIgG or anti-PDL1 antibodies. Antibody injections were continued every 3-4 days. Tumor sizes were measured.
For analysis of T cell activation, splenocytes were collected on day 7-14 after PV-10 injection after at least 2 i.p. antibody treatments. Splenocytes were co-cultured with irradiated B16 or B16-OVA cells and MC-38 colorectal cells. Supernatants were collected after 48 hours IFN-gamma was measured by ELISA.
!! These murine studies support combination therapy with IL PV-10 and co-inhibitory blockade.
!! Combination therapy with IL PV-10 and anti-PDL1 antibodies led to reduced tumor growth in both injected and uninjected bystander lesions. Increased anti-tumor immunity was measured in mice treated with anti-PDL1 antibodies.
!! Combination therapy with IL PV-10 and anti-PD1 antibodies led to a reduction in tumor growth and increased tumor-specific T cell activity.
!! Combination therapy with IL PV-10 and anti-CTLA4 antibodies led to a trend in tumor reduction and increased tumor-specific T cell activity.
Acknowledgement: Provectus Pharmaceuticals, Inc. provided PV-10 for these experiments.
Combination Therapy with IL PV-10 and anti-CTLA-4 antibodies in B16-OVA bearing mice
Combination Therapy with IL PV-10 and anti-PD-1 antibodies in B16-OVA bearing mice
Results
Conclusions
Combination Therapy with IL PV-10 and anti-PD-L1 antibodies in B16 bearing mice
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Days
Mea
n Tu
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m2 )
PBS + NrIgG PBS + anti-PDL1 PV10 + NrIgG PV10 + anti-PDL1
p<0.05
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m2 )
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PBS PV10 anti-PD1 PV10+anti-PD1
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1000
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3500
PBS PV10 anti-PD1 PV10+anti-PD1
IFN
-gam
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ml)
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PBS PV10 anti-CTLA4 PV10+anti-CTLA4
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ght (
mg)
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200
400
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PBS
PV10
anti-CTLA4 PV10+anti-CTLA4
IFN
-gam
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ml)
B16 MC38
p=0.05
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500
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PBS PV10 anti-PDL1 PV10+anti-PDL1 IF
N-g
amm
a (p
g/m
l)
* *
*p<0.05 compared to PV-10 treated mice
Treatment with anti-PD-L1 antibodies alone or in combination with PV-10 leads to the induction of B16-specific T cells
Treatment with IL PV-10 and anti-PD-L1 antibodies slows growth of injected and uninjected tumors in a bilateral B16 tumor model
Treatment with anti-PD-L1 antibodies in combination with IL PV-10 results in reduced growth of B16 tumor
Treatment with anti-PD-1 antibodies in combination with IL PV-10 results in reduced growth of B16-OVA tumor
Treatment with anti-CTLA-4 antibodies alone in combination with IL PV-10 leads to the induction of B16-specific T cells
Treatment with anti-CTLA-4 antibodies in combination with IL PV-10 results in smaller B16-OVA tumors
Treatment with anti-PD-1 antibodies in combination with IL PV-10 leads to the induction of B16-OVA-specific T cells
PBS + NrIgG PV-10 + NrIgG PBS + anti-PDL1 PV-10 + anti-PDL1
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0 5 10 15 20 25 30 35
Tum
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ize
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