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An Agonist of Toll-Like Receptor 5 Has Radioprotective Activity In

An Agonist of Toll-Like Receptor 5 Has Radioprotective Activity In

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DOI: 10.1126/science.1154986, 226 (2008);
320
Science 
 
et al.
Lyudmila G. Burdelya,
ModelsRadioprotective Activity in Mouse and PrimateAn Agonist of Toll-Like Receptor 5 Has
 
www.sciencemag.org (this information is current as of December 10, 2009 ): The following resources related to this article are available online at 
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Science 
2008 by the American Association for the Advancement of Science; all rights reserved. The titleCopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the
Science 
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evaluated species distribution models and Zonationsolutions for their taxa. A.R. conducted the geographicinformation system (GIS) analyses to produce the SAPMpriority map (Fig. 2B, black outlines). C.K. and A.C. wrotethe initial draft of the manuscript; all authors commentedon subsequent drafts.
Supporting Online Material
www.sciencemag.org/cgi/content/full/320/5873/222/DC1MethodsSOM TextFigs. S1 to S11Table S1ReferencesExtended AcknowledgmentsAppendix S1GIS data of Fig. 214 January 2008; accepted 7 March 200810.1126/science.1155193
An Agonist of Toll-Like Receptor 5 HasRadioprotective Activity in Mouseand Primate Models
Lyudmila G. Burdelya,
1
*
Vadim I. Krivokrysenko,
2
*
Thomas C. Tallant,
3
Evguenia Strom,
2
Anatoly S. Gleiberman,
2
Damodar Gupta,
1
Oleg V. Kurnasov,
4
Farrel L. Fort,
2
Andrei L. Osterman,
4
Joseph A. DiDonato,
3
Elena Feinstein,
2
Andrei V. Gudkov
1,2
The toxicity of ionizing radiation is associated with massive apoptosis in radiosensitive organs.Here, we investigate whether a drug that activates a signaling mechanism used by tumor cellsto suppress apoptosis can protect healthy cells from the harmful effects of radiation. We studiedCBLB502, a polypeptide drug derived from
Salmonella
flagellin that binds to Toll-like receptor5 (TLR5) and activates nuclear factor
k
B signaling. A single injection of CBLB502 before lethaltotal-body irradiation protected mice from both gastrointestinal and hematopoietic acute radiationsyndromes and resulted in improved survival. CBLB502 injected after irradiation also enhancedsurvival, but at lower radiation doses. It is noteworthy that the drug did not decrease tumorradiosensitivity in mouse models. CBLB502 also showed radioprotective activity in lethallyirradiated rhesus monkeys. Thus, TLR5 agonists could potentially improve the therapeuticindex of cancer radiotherapy and serve as biological protectants in radiation emergencies.
T
he toxicity of high-dose ionizing radiation(IR) is associated with induction of acuteradiationsyndromes(
1
)involvingthehem-atopoietic system (HP) and gastrointestinal tract (GI). The extreme sensitivity of HP and GI cellstogenotoxicstresslargelydeterminestheadverseside effects of anticancer radiation therapy andchemotherapy (
2
). Development of radioprotect-ants for medical and biodefense applications has primarily focused on antioxidants that protect tis-sues(
3
)andcytokinesthatstimulatetissueregen-eration (
4
).Here, we have explored whether radiopro-tection can be achieved through suppressionof apoptosis, the major mechanism underlyingmassive cell loss in radiosensitive tissues (
5
 – 
).Specifically, we have attempted to pharmaco-logically mimic an antiapoptotic mechanism fre-quently acquired by tumor cells, i.e., constitutiveactivation of the nuclear factor 
 – 
k
B (NF-
k
B) pathway (
8
). NF-
k
B is a transcription factor that  plays a key role in cellular and organismal re-sponse to infectious agents as a mediator of innate and adaptive immune reactions. The link  between NF-
k
B and the mammalian response toIR has been established by previous work show-ing that GI radiosensitivity is enhanced in micewith a genetic defect in NF-
k
B signaling (
9
).Activation of NF-
k
B induces multiple factorsthat contribute to cell protection and promotetissue regeneration, including apoptosis inhibitors,reactive oxygen species scavengers, and cyto-kines. Finally, NF-
k
B activation is among themechanisms by which tumors inhibit function of the p53 tumor suppressor pathway (
10
), one of the major determinants of radiosensitivity (
11
).In order to activate NF-
k
B in GI cells with-out inducing acute inflammatory responses, westudied factors produced by benign microorga-nisms in the human gut that activate NF-
k
B by binding to Toll-like receptors (TLRs) expressed by host cells (
12
). Stimulation of TLR signaling by commensal microflora plays a protective rolein the GI tract (
13
). In particular, we focused onTLR5, which is expressed on enterocytes, den-dritic cells (
14
), and endothelial cells of the smallintestine lamina propria (
15
). Endothelial cellapoptosis has been identified as an important contributor to the pathogenesis of GI acute ra-diation syndrome (
16 
). The only known ligandand agonist of TLR5 is the bacterial protein fla-gellin (
17 
).To investigate whether flagellin has in vivoradioprotective activity, we injected flagellin pu-rified from
Salmonella enterica
serovar Dublin(
18
) into NIH-Swiss mice 30 min before total- body
g
irradiation(TBI).Treatmentwith0.2mg/kgof body weight of flagellin protected mice fromlethaldosesof10and13Gythatinducemortalityfrom HP and GI acute radiation syndromes, re-spectively (Fig. 1A). Flagellin did not rescue micefrom 17 Gy TBI but prolonged their median sur-vival from 7 to 12 days. The dose-modifying fac-tor (DMF, the fold change in irradiation doselethal for 50% of animals) of CBLB502 in NIH-Swiss mice was 1.6, exceeding that of other ra-dioprotective compounds, such as cytokines or amifostine, used at nontoxic doses (
3
).Toreducetheimmunogenicityandtoxicityof flagellin,wetookadvantageofstudiesthatmappedthe TLR5-activating domains of flagellin to itsevolutionarilyconservedNandCtermini(Fig.1B)(
19
). We tested a series of engineered flagellinderivatives for NF-
k
B activation in vitro (Fig.1B and fig. S1). The most potent NF-
k
B acti-vator, designated CBLB502, included thecomplete N- and C-terminal domains of flagellinseparated by a flexible linker (fig. S1). CBLB502 produced in
Escherichia coli
as a recombinant  protein retains entirely the NF-
k
B
 – 
inducing acti-vity and exceptional stability of flagellin (
18
), yet is substantially less immunogenic (fig. S2). It isalso less toxic than flagellin, with a maximumtolerated dose (MTD) in mice of 25 mg/kg ascompared with the 12 mg/kg MTD of flagellin(
20
). Flagellin derivatives that failed to activate NF-
k
B in vitro did not provide radioprotection invivo (one example is shown in Fig. 1C), whichsuggested that activation of TLR5 signaling isnecessary for radioprotection.To test whether CBLB502 retained the radio- protectiveefficacyofflagellin,weadministeredsingle injection of the compound (0.2 mg/kg) to NIH-Swiss mice 30 min before 13 Gy TBI. Thetreatment (
18
) rescued more than 87% of micefrom radiation-induced death (Fig. 1C). At thisradiation dose, the most powerful previously de-scribed radioprotectants provided about 54% pro-tection[amifostine(
21
)]orhadnoprotectiveeffect atall[5-androstenediol(5-AED)orNeumune(
22
)](Fig.1C).Notably,the moderateprotectiveeffect observed with amifostine against 13 Gy TBI re-quiredinjectionofadose(150mg/kg)closetoitsMTD(200mg/kginNIH-Swissmice).CBLB502showed a significantly stronger protective effect (
 P 
<0.05)whenitwasinjectedatlessthan1%of its MTD.To address the practicality of CBLB502 as anantiradiationdrug,weinvestigatedthetimeframefor effective administration of the compound at differentradiationdoses.CBLB502protectedmiceagainst the very high doses of radiation that in-ducelethalHPorcombinedHPandGIsyndromes(10 Gy and 13 Gy, respectively) only when in- jected 15 to 60 min before TBI (Fig. 1D). Thecompound provided no survival benefit if in- jectedbeforethistimeintervalorafterirradiation.
1
Department of Cell Stress Biology, Roswell Park CancerInstitute, Buffalo, NY 14263, USA.
2
Cleveland BioLabs, Inc.(CBLI), Buffalo, NY 14203, USA.
3
Department of Cell Biology,Lerner Research Institute, Cleveland Clinic, Cleveland, OH44195,USA.
4
BurnhamInstituteforMedicalResearch,LaJolla,CA 92037, USA.*These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail:andrei.gudkov@roswellpark.org; efeinstein@cbiolabs.com
11 APRIL 2008 VOL 320
SCIENCE
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226
REPORTS
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However, at a radiation dose of 9 Gy, whichkilled>90%ofcontrol[phosphate-bufferedsaline(PBS)
 – 
injected]mice,CBLB502providedradio- protective benefits when administered as early as24 hours before, or up to 1 hour after, irradiation.WithinjectionofCBLB5021hourpostirradiation,40% of the CBLB502-treated mice survived ascompared with 7% of the control mice (Fig. 1D).Thus, CBLB502 is effective as both protectsagainst and mitigates radiation-induced injury.We next evaluated the effect of CBLB502 onradiationdamagetotheGItractandHPsystemat the tissue and cellular levels. Treatment of NIH-Swiss mice with 0.2 mg/kg CBLB502 30 min before 15 Gy TBI dramatically reduced the pro- portion of apoptotic cells in the lamina propria of the small intestine of irradiated mice, includingvascular endothelial cells (Fig. 2A and fig. S3A)(
18
). This observation is consistent with the postulated role of endothelial apoptosis in theGItoxicityofradiation(
16 
).Inaddition,wefoundthat the radiation-induced reduction in small in-testine crypt size and cell density that was ob-servedincontrolNIH-Swissmicewasameliorated byCBLB502pretreatment(0.2mg/kggiven1hou before15GyTBI)(Fig.2Bandfig.S3B).CBLB502treatment did not preserve the morphology of the small intestine in irradiated MOLF/Ei mice(Fig. 2B), a strain with defective TLR5 response because of a germline mutation (
23
), which sug-gests that radioprotection by CBLB502 is indeedTLR5-dependent.CBLB502-mediatedprotectionof the GI tract was also illustrated by preservationofcryptstemcellsinthesmallintestine.5-Bromo-2
-deoxyuridine (BrdU) was used to label prolif-eratingcryptcellsinirradiated(13Gy)NIH-SwissmicepretreatedwithPBSorCBLB502(
18
).PBS-injected mice showed a near-complete loss of crypt stem cells,whereas CBLB502-treated mice
Fig. 1.
Flagellin-mediated radioprotection of mice (
18
). (
A
) Groups of 20NIH-Swiss mice were injected with flagellin or PBS 30 min before TBI. Rep-resentative resultsfromone of three independent experimentsare shown. Thedifference in survival between control and experimental groups was sta-tisticallysignificant(
P
<0.05bytwo-tailedFisher
stest)ondays15to30,9to30, and 8 to 13 after 10, 13 and 17 Gy TBI, respectively. (
B
) Generation of anoptimized flagellin derivative. (Bottom) Schematic of the domain compositionof flagellin. Flagellin derivatives were composed from sequences in theconservedN-andC-terminaldomainsrequiredforTLR5activation(A,B,andCand A
and B
, respectively). Amino acid numbering is from flagellin of
 Salmonella enterica
serovar Dublin (GenBank accessionno. AAA27081). (Top)Assessment of NF-
k
B activation by flagellin (FliC) derivatives using elec-trophoretic mobility shift assay. Flagellin itself and variants AA
, AB
, and BA
induced NF-
k
B, whereas other combinations of N and C termini (BB
, CA
, andCB
); isolated N termini (A, B, and C); glutathione-
 S 
-transferase (GST)-fusionsofCtermini(GST-A
,GST-B
);andmixturesofisolatedNandCtermini(A+A
,A+B
) were not active. Positive control: TNF
a
; negative controls: untreatedcells (Un) and cells incubated with GST alone (GST). Flagellin variant AA
(redarrow) was renamed CBLB502. (
C
) NIH-Swiss mice were treated with CBLB502(0.2 mg/kg), flagellin derivative CB
(0.2 mg/kg), amifostine (150 mg/kg),5-AED (30 mg/kg), or PBS before receiving 13 Gy TBI. *
P
< 0.03 by two-tailedFisher
s test for comparison of survival in CBLB502- and amifostine-treatedgroups. (
D
) ICR mice (15 per group) were injected with CBLB502 at the in-dicated timesrelative to 9, 10, or 13 Gy TBI (red arrow). Controlmice receivedPBS 30 min before TBI. The percentage of mice surviving at day 30 post-irradiation is plotted.
www.sciencemag.org
SCIENCE
VOL 320 11 APRIL 2008
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