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`httpsdoiorg101172JC115223
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`Improving chemotherapeutic drug
`penetration in tumors by vascular
`targeting and barrier alteration
`
`Flavio Curnis Angelina Sacchi and Angelo Corti
`
`See the related Commentary beginning on page 433
`
`Department of Biological and Technological Research San Raffaele Hospital Scientific Institute Milan Italy
`
`Drug delivery and penetration into neoplastic cells distant from tumor vessels are critical
`for the
`effectiveness of solid tumor chemotherapy We have found that targeted delivery to tumor vessels of
`picogram doses of TNFoc TNF a cytokine able to alter endothelial barrier function and tumor inter
`stitial pressure enhances the penetration of doxorubicin in tumors in murine models Vascular tar
`geting was achieved by coupling TNF with CNGRC a peptide that targets the tumor neovasculature
`This treatment enhanced eight to tenfold the therapeutic efficacy of doxorubicin with no evidence
`of increased toxicity Similarly vascular targeting enhanced the efficacy of melphalan a different
`chemotherapeutic drug Synergy with chemotherapy was observed with 35 ngkg of targeted TNF
`intraperitoneally about 106fold lower than the LD50 and 105 fold lower than the dose required for
`nontargeted TNF In addition we have also found that targeted delivery of low doses of TNF to tumor
`vessels does not induce the release of soluble TNF receptors into the circulation The delivery of
`minute amounts ofTNF to tumor vessels represents a new approach for avoiding negative feedback
`mechanisms and preserving its ability to alter drug penetration barriers Vascular targeting could be
`a novel strategy for increasing the therapeutic index of chemotherapeutic drugs
`J Clin Invest 110475482 2002 doi101172JCI200215223
`
`Introduction
`To reach cancer cells in solid tumors chemotherapeu
`tic drugs must enter the tumor blood vessels cross the
`vessel wall and finally migrate through the intersti
`tium Heterogeneous
`tumor perfusion vascular per
`meability and cell density and increased interstitial
`pressure could represent critical barriers that may limit
`the penetration of drugs into neoplastic cells distant
`from tumor vessels and consequently
`the effectiveness
`
`of chemotherapy 1 Strategies aimed at improving
`drug penetration in tumors are therefore of great
`experimental and clinical
`interest
`A growing body of evidence suggests that TNFoc
`TNF an inflammatory cytokine endowed with potent
`antitumor activity could be exploited for this purpose
`For example the addition of TNF to regional isolated
`limb perfusion with melphalan or doxorubicin has pro
`duced higher response rates in patients with extremity
`softtissue sarcomas or melanomas than those obtained
`drugs alone 26 TNFinduced
`with chemotherapeutic
`
`Received for publication February 7 2002 and accepted
`June 18 2002
`
`in revised form
`
`Address correspondence to Angelo Corti
`Immunobiotechnology Unit Department of Biological and
`Technological Research San Raffaele Hospital Scientific
`Institute via Olgettina 58 20132 Milan Italy Phone 3902
`26434802 Fax 390226434786 Email cortiangelohsrit
`Conflict of interest No conflict of interest has been declared
`Nonstandard abbreviations used TNFa TNF Thy 11
`RMA cells RMAT murine TNF mTNF
`cDNAtransfected
`human TNF hTNF soluble TNF receptors sTNFRs soluble
`p55TNF receptor 5TNFR1 soluble p75TNF receptor
`sTNFR2
`
`alteration of endothelial barrier function reduction of
`tumor interstitial pressure increased chemotherapeu
`tic drug penetration and tumor vessel damage are
`believed to be important mechanisms of the synergy
`between TNF and chemotherapy 3 4 710 Unfortu
`nately systemic administration of TNF is accompanied
`by prohibitive toxicity the maximum tolerated dose
`810 µgkg being 1050 times lower than the estimat
`ed effective dose 11 12 For this reason systemic
`administration of TNF has been abandoned
`and the
`clinical use of this cytokine is limited to locoregional
`treatments Nevertheless some features of the TNF
`activity in particular the selectivity for tumorassociat
`ed vessels and the synergy with chemotherapeutic
`drugs have continued to nourish hopes regarding the
`possibility of wider therapeutic applications 13
`The vascular effects ofTNF provide the rationale for
`a vascular
`targeting strategy aimed at
`developing
`increasing the local efficacy and at enabling systemic
`administration of therapeutic doses We have shown
`recently that targeted delivery of TNF to tumor vessels
`can be achieved by coupling this protein with the
`CNGRC peptide an aminopeptidase N CD13 ligand
`the tumor neovasculature 14 In the pres
`that
`targets
`ent work we have investigated whether vascular tar
`geting with low doses of
`this conjugate called
`NGRTNF
`enhance
`of
`could
`the
`penetration
`drugs in tumors and improve their
`chemotherapeutic
`efficacy We show that systemic administration to mice
`of picogram doses of NGRmTNF 35 ngkg six
`orders of magnitude lower than the LD50 is sufficient
`to enhance the antitumor activity of melphalan and
`
`The Journal of Clinical Investigation
`
`August 2002
`
`I
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`I
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`Volume 110
`
`Number 4
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`I
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`475
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`Abraxis EX2046
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

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`doxorubicin with no evidence of increased toxicity In
`addition we provide evidence that vascular
`targeting
`with NGRTNF can reduce drug penetration barriers
`and increase the amount of doxorubicin that reaches
`cancer cells Finally we show that
`the delivery of
`minute amounts of NGRTNF to tumor vessels over
`comes another major problem associated with systemic
`administration of relatively high doses of TNF ie the
`induction of soluble TNF inhibitors
`
`Methods
`Tumor cell lines and reagents Mouse Bl6F1 melanoma and
`Thy 11 cDNAtransfected RMA cells RMAT lym
`phoma were cultured as described previously 14 15
`The mAb 6G1 rat anti p75 murine TNF mTNF
`receptor antagonist was produced and characterized
`as described previously 16 17 The mAb Vlq rat anti
`mTNF was kindly supplied by D Mannel University
`of Regensburg Regensburg Germany Melphalan
`Alkeran was obtained from Glaxo Wellcome Opera
`tions Dartford United Kingdom Doxorubicin
`from Pharmacia
`Adriblastina was purchased
`Upjohn SpA Milan Italy
`Preparation of human and murine TNF and NGRTNF
`Human and murine TNF and NGRTNF consisting of
`TNF fused with the Cterminus of CNGRCG were pre
`pared by recombinant DNA technology
`and purified
`from Escherichia coli cell extracts as described 14 All
`solutions used in the chromatographic steps were pre
`pared with sterile and endotoxinfree water SALF Lab
`oratorio Farmacologico SpA Bergamo Italy Protein
`concentration was measured using the BCA Protein
`Assay Reagent Pierce Chemical Co Rockford Illinois
`USA The in vitro cytolytic activity of human TNF
`hTNF estimated from a standard cytolytic assay with
`LM mouse fibroblasts 18 was 54 x 107 Umg where
`as that of purified NGRhTNF was 14 x 108 Umg The
`of mTNF was 76 x 107 Umg whereas
`that of NGRmTNF was 91 x 107 Umg The hydrody
`namic volumes of NGRmTNF NGRhTNF and
`mTNF were similar to those of hTNF a homotrimeric
`protein 19 by gel
`chromatography on a
`75 HR column Amersham Biosciences
`Superdex
`Europe GmbH Freiburg Germany Electrospray mass
`spectrometry of each product determined the follow
`ing molecular masses NGRhTNF 179376 ± 19 Da
`expected for CNGRCGhTNF1157 monomers 179394
`Da hTNF 17349
`± 13 expected
`for hTNF1157
`173507 NGRmTNF 1784116 ± 25 expected for
`CNGRCGmTNF1156 178442 mTNF 173849 ± 2
`expected for MetmTNFiis 6 173867 The endotoxin
`content of each product measured using the quantita
`limulus amoebocyte lysate LAL test
`tive chromogenic
`Inc Walkersville Maryland USA
`BioWhittaker
`was NGRhTNF 0079 Uug hTNF 0117 Uµg
`NGRmTNF 0082 Upg mTNF 161 Upg
`on animal models were
`In vivo studies Studies
`approved by the Ethical Committee of the San Raffaele
`H Scientific Institute and performed according to the
`
`cytolytic activity
`
`filtration
`
`prescribed guidelines C57BL6 mice Charles River
`Laboratories Calco Italy weighing 1618 g were chal
`lenged with subcutaneous
`flank of
`injection in the left
`5 x 104 RMAT or B16F1 living cells 412 days later
`the mice were treated with TNF or NGRTNF solutions
`100 fl
`followed 2 hours later by administration of
`melphalan or doxorubicin solution 100 µ1 Unless
`specified all drugs were administered intraperitoneal
`ly All drugs were diluted with 09 sodium chloride
`containing 100 1igm1 endotoxinfree HSA Farma
`Biagini SpA Lucca
`for doxorubicin
`Italy except
`which was diluted with 09 sodium chloride alone
`Tumor growth was monitored daily by measuring the
`tumors with calipers as previously described 20 Ani
`mals were sacrificed before the tumors reached 1015
`cm in diameter Tumor sizes are shown as mean ± SE
`five animals per group Statistical analysis was per
`formed by two tailed t test Differences between groups
`were considered significant when P was less than 005
`Soluble TNF receptor assays Soluble p55TNF receptor
`sTNFR1 and soluble p75TNF receptor sTNFR2 in
`animal sera were measured using the Quantikine M kit
`RD Systems Inc Minneapolis Minnesota USA
`Detection of doxorubicin in tumors C57BL6 mice bear
`ing B16F1 or RMAT tumors diameter 051 cm were
`treated with NGRTNF 01 ng diluted in 09 sodium
`chloride containing 100 pgm1 HSA intraperitoneal
`ly or with diluent alone followed 2 hours later by dox
`orubicin 320 ig intraperitoneally After 2 hours the
`animals were sacrificed and the tumors were excised
`Each tumor was weighed disaggregated
`in cold PBS and filtered through 70 pm filters The
`cells were resuspended in cold PBS 50 ml centrifuged
`460g 10 minutes 4°C resuspended in cold PBS 25
`mlg of tumor tissue and mixed with freshly prepared
`PBS containing 8 formaldehyde 25 mlg of tissue
`The cells were stored in the dark at 4°C overnight and
`then analyzed by FACS The FACScan BD Biosciences
`Erembodegen Belgium was calibrated with cells recov
`ered from untreated tumors Each sample was then
`analyzed using the FL3 filter and CellQuest BD Bio
`sciences software
`
`resuspended
`
`Results
`Dose response curves ofNGRmTNF and mTNF in murine
`lymphoma and melanoma models The antitumor
`activity
`of NGRmTNF and mTNF was first characterized
`in
`the absence of chemotherapeutic drugs To compare
`the dose response curves of NGRmTNF and mTNF
`we performed several experiments based on single or
`repeated intraperitoneal administration of various
`doses of NGRmTNF and mTNF from 001 to 10000
`ng to RMAT lymphoma or B16F1 melanoma bear
`ing mice Murine TNF delayed tumor growth when
`adminis tered ai high doses 10000 ng Figure la no
`effects were induced by doses lower than 100 ng either
`with single Figure la or with repeated administra
`tions Figure lb NGRmTNF was markedly more
`In this case we observed antitumor effects even
`potent
`
`476
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`The Journal of Clinical Investigation
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`Figure 1
`Effect of mTNF and NGRmTNF on tumor growth and body weight of
`animals bearing RMAT lymphomas Animals bearing RMAT tumors
`five mice per group were treated intraperitoneally with NGRmTNF
`or mTNF at day 12 after tumor implantation a or at days 1011 and
`12 b in two separate experiments Exp 1 and Exp 2 Tumor vol
`umes in Exp 1a and Exp 2b and animal body weight in Exp 1c
`14 days after treatment are shown The arrowheads inc indicate the
`time of treatment
`
`hypothesized that the lower efficacy of 10 ng of NGR
`mTNF was related to induction of sTNFR1 andor
`sTNFR2 and consequently to neutralization of its
`interaction with membrane receptors
`To test
`this hypothesis we measured the levels of
`sTNFR1 and sTNFR2 in the serum of tumorbearing
`mice collected 1 hour after administration of various
`doses of mTNF and NGRmTNF As expected both
`products induced sTNFR2 shedding but not sTNFR1
`shedding at doses greater than 4 ng Figure 2a
`To assess whether sTNFR2 shedding regulates the
`activity of NGRmTNF we
`coadministered this
`cytokine with mAb 6G1 an antagonist antisTNFR2
`antibody that prevents the binding of mTNF to solu
`ble and membrane murine TNFR2 16 The antitu
`mor activity of 10 ng of NGRmTNF was potentiated
`by mAb 6G1 Figure 2b in line with the hypothesis
`that sTNFR2 plays a role in inhibiting the antitumor
`effects of NGRmTNF
`To further support this hypothesis we compared the
`in vivo dose response curve of NGRmTNF with that of
`NGRhTNF taking advantage of the fact
`human cytokine cannot bind murine sTNFR2 22 We
`found that the doseresponse curve of NGRhTNF was
`not bell shaped and that 10 ng of NGRhTNF is as
`active as 1 ng Figure 2c It is also remarkable that 1 ng
`was sufficient to induce the maximum antitumor effect
`This may suggest that receptor binding on vessels can
`be achieved with very low blood levels of NGRhTNF
`
`that
`
`the
`
`a
`
`NGRmTNF
`ornTNF
`
`Exp 2
`
`co 500
`
`co
`
`300
`
`100
`
`0 5
`0
`
`0
`
`Ts
`a
`
`E > E I
`
`c7
`
`4r11
`
`o oo o
`o o
`
`1
`
`00
`
`Exp 1
`
`700
`
`a
`
`Co
`
`JI
`
`cl 300
`
`loo
`
`0
`
`Dose ng
`
`Dose ng
`
`NGFmTNF
`
`14
`
`15
`
`16
`12
`Time day
`
`100 0 1
`10000 10
`
`I
`
`13
`
`7
`14
`
`15
`
`I
`
`I
`
`160 0
`
`Exp1
`20 mTNF
`
`111 Is
`
`is
`
`18
`
`17
`
`16
`
`Animalweightg
`
`12
`
`13
`
`Dose ng
`
`o mTNF
`
`NGRmTNF
`
`600 7
`
`P< 005
`
`NS
`
`P< 005
`
`mAb 6G1
`El+ mAb 6G1
`
`g E 400
`T
`
`7
`E cz 200
`z
`I
`
`Ed
`
`01
`0
`10
`NGRmTNF ng
`
`<17 400
`E
`
`IT
`
`NGRhTNF
`TOhTNF
`Tnil
`
`> 200
`
`0
`
`liE
`
`ac
`
`2 6
`
`S
`
`0 001 01
`10 100 1000
`Dose ng
`
`1
`
`0
`
`1
`
`10 100 1000
`Dose ng
`
`with doses as low as 001 ng Figure 1 a and b How
`the dose response curve was more complex For
`ever
`instance the effect of 10 ng was surprisingly lower than
`that of 00101 ng and 100010000 ng A bell shaped
`dose response curve was observed in several other
`experiments conducted in the RMAT model as well as
`in the B 16F1 melanoma model not shown These
`that the efficacy of low doses of NGR
`results suggest
`mTNF is markedly higher than that of mTNF and that
`doses of NGRmTNF greater than 110 ng activate
`negative feedback mechanisms that inhibit its poten
`tial antitumor activity
`Nanogram but not picogram doses ofNGRTNF induce sol
`uble TNF receptor shedding The protective mechanisms
`responsible for the bell shaped dose response curve of
`NGRmTNF were then investigated Since exogenous
`ly administered TNF can induce shedding of soluble
`TNF receptors sTNFRs shedding in vivo 21 we
`
`treatment with
`
`Figure 2
`Circulating levels of sTNFR2 and their role in regulating the activity
`of NGRmTNF and NGRhTNF a Serum levels of sTNFR1
`and
`sTNFR2 in B1 6F1 tumor bearing mice 1 hour after
`various doses of NGRmTNF or mINF Animals three mice per
`group were treated at day 6 b Effect of the antisTNFR2 mAb
`6G1 on the antitumor activity of NGRmTNF The mAb 6G1 100
`11g was administered to animals bearing B16F1 tumors at day 5 and
`8 Each animal was treated 1 hour later with NGRmINF at the indi
`cated doses and 2 hours later with melphalan 90 lug five mice
`group c Effect of NGRhINF and hTNF on the growth of RMAT
`tumors Mice were treated with various doses of each cytokine at day
`11 NS not significant t test
`
`per
`
`15
`
`=125
`
`sify
`
`10
`
`2
`
`75
`
`5
`
`25
`
`15
`
`=125
`
`Ii
`
`10
`
`ccc
`
`LL2
`
`75
`
`5
`
`25
`05
`
`The Journal of Clinical Investigation
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`August 2002
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`I Number 4
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`of NGRmTNF induced stronger antitumor effects than
`did the single agents indicating a synergistic effect Fig
`ure 3c Remarkably the combination of melphalan with
`01 ng of NGRmTNF was more effective than the com
`bination with 5000 ng of mTNF Figure 3 c and d We
`observed this synergism even when NGRmTNF 01 ng
`was injected intravenously not shown
`Two similar experiments were conducted with dox
`orubicin in the B16F1 model Animals were treated 5
`days after tumor implantation with NGRTNF diluted
`in 09 sodium chloride containing 100 µgm1HSA or
`with diluent alone and 2 hours later with various doses
`of doxorubicin 20320 µg intraperitoneally In both
`experiments the effect of doxorubicin plus NGRmTNF
`was stronger than that of doxorubicin alone Figure 4
`a b and e indicating that NGRmTNF markedly
`improves the efficacy of this drug For example the
`effect of doxorubicin 40 µg plus NGRmTNF 01 ng
`was stronger than that of 320 µg of doxorubicin alone
`Figure 4b while the effect of doxorubicin 20 µg plus
`NGRmTNF was weaker Figure 4a From these results
`we estimate that the activity of doxorubicin is potenti
`ated eight to tenfold by NGRmTNF in this model
`In another series of experiments we measured the
`effect of NGRmTNF in combination with melphalan
`or doxorubicin administered 1012 days after tumor
`implantation ie when the tumors were well estab
`lished Synergism between low doses of NGRmTNF
`and chemotherapy was observed in both B16F1 and
`RMAT models Figure 5 Of note a single treatment
`with NGRTNF plus melphalan cured three out of five
`RMAT tumorbearing mice In contrast no cure was
`observed with B16F1 tumors treated with NGRmTNF
`combined with either doxorubicin or melphalan Like
`the stronger immunogenicity of the
`ly this reflects
`Raucher virus induced lymphoma RMAT compared
`with the spontaneous B16F1 melanoma
`In conclusion these results suggest
`that picogram
`doses of NGRTNF
`are sufficient
`to improve the
`response of tumors to melphalan and doxorubicin
`Low doses of NGRmTNF are not toxic and do not increase
`the toxicti of melphalan To estimate the efficacytoxici
`ty ratio of each treatment we monitored animal body
`weight daily and animal survival after treatment While
`therapeutic doses of mTNF 10000 ng induced
`marked loss ofbody weight in RMATbearing animals
`therapeutic doses of NGRmTNF
`Figure lc left
`0011 ng did not cause loss of body weight nor ani
`mal death Figure lc right The toxicity of NGR
`mTNF in combination with melphalan was then exam
`ined Three out of ten mice bearing the RMAT tumor
`died 3 days after treatment with 200 µg of melphalan
`alone Neither NGRmTNF nor mTNF 1 ng each
`increased the lethality of melphalan 200 µg as in both
`cases only two out of Len animals died
`therapeutic doses of NGR
`In the B 16F1 model
`mTNF 01 ng did not cause loss of body weight even
`when combined with melphalan Figure 3e In con
`trast melphalan combined with therapeutic doses of
`
`Melphalan alone
`
`NGRmTNF or
`mTNF alone
`
`NGRmTNF
`01 ng
`a mTNF
`01 ng
`o None
`11
`
`1
`
`Melphalan jig
`
`I
`
`0 9
`
`0
`
`4
`
`8
`
`12
`
`Melphalan 90 g
`+ NGRmTNF
`
`4
`
`16d
`
`8
`
`12
`
`16
`
`Melphalan 90 g
`
`+ mTNF
`
`NGRmTNF ng
`00
`01
`400
`2000
`
`I
`
`1
`
`mTNF ng
`00
`a 01
`v1000
`0 5000
`II
`
`I
`
`12
`
`4
`16
`Time day
`Melphalan 90 hg f
`+ NGRmTNF
`
`1
`
`12
`
`16
`
`Melphalan 90 hg
`+ mTNF
`
`I
`
`I
`
`a
`
`900
`
`0 600
`
`300
`
`7>
`
`900
`
`600
`
`300
`
`7>
`
`63
`
`00
`
`05 1
`
`m>
`
`Ta 0
`E o
`
`E 5
`
`0c0
`
`E2
`
`15
`
`1
`
`0I1
`
`4002000
`NGRmTNF ng
`
`0
`
`011
`
`10100
`
`mTNF ng
`
`5000
`
`Figure 3
`Effect of melphalan alone a or in combination with NGRmTNF
`c or mTNFd on the tumor growth ad and body weight e and
`f of mice bearing B1 6F1 melanoma The animals were treated
`intraperitoneally with the drugs and the doses indicated in each panel
`five animals per group at days 47 and 9 after tumor implantation
`indicated by arrows
`
`Taken together
`the results of these experiments
`strongly suggest that NGRmTNF and mTNF at doses
`greater than 4 ng induce shedding of sTNFR2 in
`amounts sufficient
`to inhibit their antitumor activity
`Picogram doses ofNGRmTNF are sufficient to enhance the
`effect of melphalan and doxorubicin We then
`therapeutic
`investigated whether targeted delivery of low doses of
`NGRmTNF to tumor vessels could enhance
`the antitu
`mor activity
`drugs These experi
`of chemotherapeutic
`ments were conducted using the B16F1 model a spon
`taneous mouse melanoma characterized
`by scarce
`immunogenicity and low sensitivity to melphalan and
`using the RMAT model Melphalan 90 µg was unable
`the growth of B 16F1 tumors when injected
`alone Figure 3a Similarly mTNF 01 ng alone
`intraperitoneally was virtually inactive while the same
`dose of NGRmTNF modestly delayed the tumor growth
`Figure 3b The combination of melphalan with 01 ng
`
`Lo affect
`
`478
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`The Journal of Clinical Investigation
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`I Number 4
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`Figure 4
`Effect of various doses of doxorubicin alone white bars or in com
`bination with NGRmTNF black bars on the tumor growth a and
`b body weight c and d and survival e of mice bearing B16F1
`
`melanomas The drugs were administered to the animals five mice
`per group intraperitoneally 5 days after tumor implantation
`
`Fvn
`
`ii
`
`L
`
`L
`
`900
`
`600 1
`
`300 I
`
`0
`
`0
`
`40
`
`80 160 320
`
`iNursm iNr
`ng
`01
`
`1110
`
`a
`
`Exp
`
`1
`
`800
`
`§ E 600
`
`71
`
`400
`
`7>
`
`1 6 200
`
`01it
`
`0
`
`20
`
`80
`
`L
`
`I
`320d
`
`3 1Exp 1
`
`2 Exp 2
`
`0 1
`
`80 160 320
`
`Doxorubicin
`
`lig
`0
`80
`80
`320
`320
`
`40
`
`320
`0
`Doxorubicin jtg
`NGRmTNF
`ng
`A0
`01
`
`O 0
`
`O 0
`
`1
`
`D
`
`2 1
`
`1 2 3
`
`cs
`
`c°
`
`o
`E 0
`
`C C
`
`o >
`5
`
`E
`0 o
`
`0
`
`20
`
`80
`
`Exp 1 + Exp 2
`n= 10
`
`E
`
`V r
`
`f
`
`E
`
`c
`
`Ta
`
`The role of TNFR1 and TNFR2 was then studied
`To this end we evaluated the effect of melphalan in
`combination with 001 ng or 01 ng of NGRhTNF a
`agonist 22 The effect of melphalan
`TNFR1specific
`in the B16F1 model was potentiated by NGRhTNF
`Figure 6b suggesting that TNFR1 activation is suf
`ficient for the synergism
`The synerg between NGRmTNF and chemotherapy is not
`To assess whether
`the
`dependent on tumor cell cytotoxict
`synergism depends directly on cytotoxicity against
`tumor cells we measured the effect of each compound
`alone or in combination on cultured B16F1 cells Nei
`ther melphalan nor NGRmTNF alone or in combina
`tion killed these cells in a 48 hour in vitro assay not
`shown Similarly NGRmTNF did not enhance the
`cytotoxic activity of doxorubicin in vitro not shown
`These results suggest
`that the synergism observed in
`on cytotoxic effects
`vivo is not directly dependent
`against tumor cells and point to an indirect role of a
`component of the tumor stroma eg the endothelial
`lining of tumor vessels
`NGRmTNF increases the penetration of doxorubicin in
`murine melanomas and lymphomas We then investigat
`ed whether NGRmTNF could increase the penetra
`tion of chemotherapeutic drugs in tumors To this
`aim we measured the amount of doxorubicin that
`had penetrated B16F1 and RMAT tumors 2 hours
`after administration taking advantage of the fluo
`this drug 23 Preliminary
`rescent properties of
`
`RMAT
`
`05
`
`15
`
`NGRmTNF Melphalan
`ng
`jig
`0
`0
`
`O
`
`a
`
`0
`
`01
`
`50
`
`50
`
`35
`
`10
`
`15
`
`20
`
`25
`
`30
`
`B16F1
`
`05
`
`NGRmTNF Doxorubicin
`ng
`lig
`
`O 0
`
`01
`
`0
`
`01
`
`0
`
`0
`
`80
`
`80
`
`1000
`
`750
`
`500
`
`250
`
`0
`
`QE
`
`5 1800
`
`1200
`
`600
`
`01
`10
`
`12
`
`16
`14
`Time day
`
`18
`
`20
`
`E
`
`E<
`
`C5
`
`10
`
`15 20
`
`25
`
`30
`10
`Time day
`
`15
`
`20 25
`
`30
`
`mTNF 5 fig induced marked loss of body weight Fig
`ure 30 In addition NGRmTNF 01 ng did not
`increase the loss of body weight caused by high doses
`of doxorubicin Figure 4 c and d
`that picogram doses of NGR
`These results suggest
`mTNF increase the response of tumors to melphalan
`and doxorubicin with no evidence of increased toxicity
`TNT RI activation is necessary and sufficient for the syner
`gism between NGRTNF and chemotherapeutic
`drugs The
`mechanisms of the synergism between low doses of
`NGRmTNF and chemotherapy were then investigated
`these mechanisms rely on TNFRs
`To assess whether
`activation we tested the effect of mAb Vlq a neutraliz
`ing antimTNF antibody on the antitumor
`of
`NGRmTNF 01 ng in combination with melphalan 90
`fig Vlq inhibited at least partially the antitumor activ
`ity of these drugs in the B16F1 model Figure 6a This
`suggests that the interaction between the TNF moiety
`and TNFRs is critical
`for the activity of the conjugate
`
`activity
`
`Figure 5
`Effect of melphalan or doxorubicin alone or in combination with
`NGRmTNF on well established RMAT and B16F1
`tumors Each
`animal was treated with the drugs and the doses indicated in each
`per group at time points indicated by the arrows
`panel five animals
`The numbers on each curve indicate the animals that were tumor
`free at day 43
`
`The Journal of Clinical Investigation
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`August 2002
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`Volume 110
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`I Number 4
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`Figure 6
`Role ofTNF receptors in the synergistic activity of NGRmTNF and
`melphalan a Effect of mAb V1q an antimTNF neutralizing anti
`
`body on the antitumor activity of melphalan in combination with
`NGRmTNF in the B16F1 model The drugs were administered at day
`5 V1q and NGRmTNF were premixed and incubated
`for 1 hour
`before injection into animals b Effect of melphalan in combina
`tion with NGRhTNF at the indicated doses
`
`Melphalan 90 jtg
`+ NGRhTNF
`
`NGRhTNF ng
`
`0 0
`
`01
`01
`
`600
`
`400
`
`200
`
`Melphalan 90 ug
`+ NGRmTNF
`NGRmTNF ng
`
`tg
`
`0 0
`
`1
`01
`+ V1g 7
`00
`
`Melphalan
`
`ib
`
`12
`
`0
`
`14
`Time day
`
`6
`
`6
`
`Co
`
`Figure 7 ch This suggests
`
`that NGRmTNF
`increased the number of cells that were reached by dox
`orubicin as well as the intracellular amount of drug
`
`a
`
`300
`
`I 200
`
`>
`
`100
`
`1
`
`0
`
`the nuclei of B16F1 cells
`experiments showed that
`become fluorescent after these cells are exposed to
`doxorubicin in vitro Figure 7a The fluorescence
`and stable for at least 24
`is dose dependent
`signal
`hours when the cells are fixed with formaldehyde
`and kept at 4°C as measured by FACS Figure 7b
`Thus the fluorescence intensity of tumor cells recov
`ered from animals after treatment
`is an indication of
`the amount of doxorubicin
`that has penetrated
`that 01 ng of NGRmTNF
`tumors We observed
`administered 2 hours before doxorubicin increased
`intensity and the percentage of pos
`the fluorescence
`itive cells recovered from both B16F1 and RMAT
`two to fivefold
`tumors 2 hours after treatment
`
`Discussion
`Alteration of vascular permeability and interstitial pres
`sure endothelial cell damage and fibrin deposition are
`important mechanisms for the antitumor activity of
`TNF either alone or in combination with chemother
`apeutic drugs After infusion in animals or patients
`TNF can also induce negative feedback mechanisms
`that neutralize most of these effects For example TNF
`even at moderate doses can induce the release of solu
`ble OS and p75 TNF receptors that may prevent
`its
`interaction with membrane receptors 21 24
`the body
`Although these soluble inhibitors may protect
`from the harmful effects of this cytokine they may also
`its antitumor activity and could explain in
`prevent
`
`B16F1
`n= 10
`
`e
`
`80
`
`60
`
`rid
`
`40 I
`
`71
`
`5 200
`
`B16F1
`n = 10
`
`20
`
`10
`
`Meanfluorescence
`
`B16F1
`
`None
`
`Doxorubicin
`NGRmTNF +
`doxorubicin
`
`200
`
`160
`
`120
`
`80
`
`40
`
`0
`
`a
`
`Doxorubicin ugm1
`loo
`0
`
`0
`
`80
`
`h
`
`e 60
`
`713 40
`
`15 20
`
`RMAT
`n = 6
`
`0
`
`g50
`
`40
`
`30
`
`20
`
`10
`
`Meanfluorescence
`
`10°
`
`10
`
`10
`
`103
`
`10
`
`RMAT
`
`None
`
`Doxorubicin
`
`NGRmTNF +
`doxorubicin
`
`10
`101
`103
`Fluorescence
`
`104
`
`0 JLmlMIM
`01
`0
`NGRmTNF ng
`
`0
`
`01
`0
`NGRmTNF ng
`
`0
`
`200
`
`160
`
`120
`
`80
`
`40
`
`0
`
`z 0
`
`0
`
`1
`
`10
`Doxorubicin ugrni
`
`100
`
`400
`
`c9
`
`63Q
`
`6 200
`
`4=
`
`a2
`
`0
`
`Figure 7
`
`Effect of NGRmINF on the penetration of doxorubicin in B16F1 and RMAT tumors a Bright field upper panels and fluorescence lower
`panels microscopy of B16F1 cells incubated in vitro with 100µgm1doxorubicin 30 minutes 37°C Inset Merge of bright field and flu
`images b Stability of the B16F1 fluorescence signal after in vitro treatment with doxorubicin B16F1 cells were incubated with
`orescence
`in culture medium 30 minutes 37°C washed with 09 sodium chloride and fixed with 4 formaldehyde
`various doses of doxorubicin
`The cells were then incubated for 0 hours or 24 hours in culture medium at 4°C washed again and analyzed by FACS c and f Represen
`tative FACS analysis of cells recovered from B16F1 c or RMAT f tumors 2 hours after in vivo administration of doxorubicin
`alone 320
`1tg or in combination with NGRmTNF 01 ng Dashed lines indicate the fluorescence interval considered positive d and g Mean ± SE
`fluorescence of B16F1 d or RMAT g cells recovered from tumors e and h Mean ± SE of positive cells recovered from B16F1 e or
`RMAT h tumors P< 005 statistical analysis by two tailed t test
`
`480
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`The Journal of Clinical Investigation
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`August 2002
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`Volume 110
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`I Number 4
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`Downloaded from httpwwwjciorg on May 31 2017
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`that
`
`Normal vessels
`a Low dose 00101 ng
`NGR
`
`Blood flow
`
`NGRTNF
`
`Tumor vessels
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`TNFRs
`b Moderate dose 10 ng
`
`II
`
`io
`
`II
`
`U
`
`MIIII
`
`UN
`sTNFRs ill
`
`11
`
`II
`
`CD13
`
`II
`
`ii
`
`C High dose >1000 ng
`
`I
`
`I
`
`I
`
`Figure 8
`Schematic representation ofthe hypothetical
`
`interactions of low a
`moderate b and high c doses of NGRTNF with soluble and
`
`membrane
`in normal vessels CD13negative
`and in
`receptors
`tumor associated vessels CD13positive Black arrows indicate TN F
`receptor signaling or extracellular domain shedding
`
`part the need of high doses of TNF for effective thera
`py In this work we postulated that homing low doses
`of TNF to tumor vessels represents a new strategy to
`avoid toxic reactions as well as negative feedback mech
`anisms while preserving its synergism with chemother
`apy To verify this hypothesis we have investigated the
`low doses of
`antitumor activity of high and
`NGRmTNF and mTNF ranging from picogram to
`microgram quantities in two murine models based on
`RMAT lymphoma
`subcutaneous
`and
`B16F1
`melanoma tumors The study was carried out using
`these cytokines alone or in combination with melpha
`lan or doxorubicin While mTNF was virtually inactive
`in these models at doses lower than 1001000 ng we
`found Lila NGRnaTNF even alone could induce anti
`tumor effects with doses as low as 00101 ng Since
`the LIDso values of mTNF and NGRmTNF are similar
`and correspond to about 50000 ng 25 mgkg in
`RMAT tumorbearing mice 14 these results indicate
`
`ratio of NGRmTNF is
`the efficacytoxicity
`104105 times greater than that of mTNF
`Administration of minute amounts of NGRmTNF
`00101 ngmouse 055 ngkg about
`106 fold lower
`than the LD50 to tumorbearing animals potentiated
`the antitumor activity of melphalan and doxorubicin
`with no evidence of increased toxicity as judged by
`tumor mass reduction animal survival and weight
`loss
`that NGRmTNF
`treatment This suggests
`after
`improves the therapeutic index of these drugs It
`noteworthy that 5 x 104 fold greater doses of mTNF
`eg 5000 ngmouse in the B16F1 model were neces
`sary to enhance the effect of melphalan to comparable
`levels causing marked loss of body weight
`The fact that both melphalan and doxorubicin at
`doses virtually inactive in the B16F1 model reduced
`tumor growth when combined with NGRmTNF
`indicates that these drugs act synergistically Studies
`on the mechanism of action showed that the syner
`gism relies on the interaction of NGRmTNF with
`TNFR1 on stromal cells most
`likely endothelial
`cells and much less on tumor cells In addition we
`targeting with NGRmTNF
`found that vascular
`improves cytotoxic drug penetration in tumors It
`noteworthy that NGRmTNF increased both the per
`centage of cancer cells that can be reached by dox
`orubicin in 2 hours and the intracellular amount of
`drug suggesting that NGRTNF can alter drug pen
`etration barriers Previous studies showed that TNF
`can rapidly increase endothelial permeability 25 26
`fluid pressure 8 both
`and can decrease interstitial
`believed to be critical for drug penetration in tumors
`1 Possibly these mechanisms increase convective
`
`is
`
`is
`
`transport of drugs through tumor vessel wall and
`interstitium finally resulting in increased
`drug
`uptake by tumor cells The timing of administration
`for these mechanisms as TNF can
`is likely critical
`also induce intravascular coagulation 27 leading to
`vessel occlusion and reduction of tumor perfusion
`In keeping with this view we observed that the effect
`of melphalan was higher when this drug was admin
`istered 2 hours after NGRTNF than when it was
`administered after 6 hours data not shown Besides
`these mechanisms other known effects of TNF on
`endothelial cells could contribute to its overall anti
`tumor activity including the induction of endothe
`leukocyte adhesion molecules
`inflammatory
`lial
`cytokines chemokines class II molecules and pro
`factors 11 12 These mechanisms
`coagulant
`together with improved chemotherapeutic drug pen
`etration could contribute to activating inflammato
`ry and immune responses
`The hypothesis that vascular
`targeting could avoid
`negative feedback mechanisms usually associated with
`TNF therapy is supported by the observa Lion tha
`picogram doses of NGRmTNF do not induce soluble
`receptor shedding while both NGRmTNF and mTNF
`rapidly induce the release of sTNFR2 into the circula
`tion at doses greater than 410 ng These levels of
`
`The Journal of Clinical Investigation
`
`I
`
`August 2002
`
`Volume 110
`
`I
`
`I Number 4
`
`481
`
`

`

`Downloaded from httpwwwjciorg on May 31 2017
`
`httpsdoiorg101172JC115223
`
`sTNFR2 inhibited most of the antitumor activity of 10
`ng of NGRmTNF and may explain the paradoxical
`observation that long is less active than 01 ng Likely
`a large proportion of injected molecules were rapidly
`complexed by sTNFRs and their activity was blocked
`The molecular mechanisms underlying the selective
`interaction of low doses of NGRmTNF with tumor
`blood vessels have been partially elucidated We have
`shown recently that different CD13 isoforms are
`expressed in tumorassociated vessels in e