aD. avaré Nuaber: DAMO27~02-1-0119 ‘ITL3: Sodium Todida symporter Gene Transfer for Imaging and ablation of Prostate Cancer PRINCIPAL INVESTIGATOR: Sissy M. Z:tang, Ph.D, CONTRACTING ORGENIZATION: The Ohio State University Research Poundazion, Cozumbus, Ohio 43219-1063 RPORT PAYY: ganvary 2004 WYER Ob HuvORY: Annual PREPARED FOR: U.S. Avny Medica! Research and Ma Fort Detrick, Maryland 21702 $012 hel Command DISTRIEUTION STATEYENT: Approved for Public Release; Distrizutie: Unlinited ‘he views, oginions and/or findings contained in this zepor: those of the author(s) and sioyid rot be construed as en off. Departnent of the Army rorition, policy cr decision anless so desiguated by other documentation "20040602 040 BEST AVANLABLE comy Form Approved REPORT DOCUMENTATION PAGE OME Yo. G74-0168 Tue oat BHT = REROAY HOPE ANG BATES COVERED dene sant! Annual (15 Doo 2002 ~ 14 sc 2003) En aio scare RINNE NERS Scdlum wedide Symorter Gene Trarefer for Imaging ard DeMDr7-02-1-0113 Sblazion of Frosvate Cancer Sissy %. dalarg, Pao. 7 FERFSRITS GRGARICATON HARES) AND ADRESSES, “EFERFDRT RCE She thio State Univereizy Research Foundation REPORT aun Conshue, One 43230-1063 eae fhiany. Goa ede F SPREGRIG ONTCRENE 8 BRESORNG MOTOR ‘Reeney nates) ato ADDRES) ‘Keeney Aeron? ALANOER sf, Army todical Roscarsk ané materie: Common Fort Detrick, Marylané 21702-5012 7 SORPTEMER TARY WPS Griginsl contains coler plates: #11 L0:¢ reproductions wil] be tn Black and white, Apgsaved for Publis Relesge; Dietetbut!an UrLinized 5 AASTARCT eas 200 Waste The sodicm ioside eyaporter (KIS} meiiates 1oaide uptake in thyrots tel! leylar o@lls and exovldas @ mecknnti fer offective rdiojodide srentront of residual, securren:, até Retastetic thyrois cancers, The obsestive of the pucpoaed research 1 Le Lest she hysothesis that exprece-on of exogenous MIIS ir prastatie kissin wilt, mable radioiogide to tocelize ané sblate rezicual prostate cnsor Following prostatescony, sich chet recusre:ce an2 metastasis of lle cigease can be prevented. Tow specifiy aims of this protect ere tor |1) seufian movastarse: provreneioa to distact Limp nodes and lunge Soliowize subcutanaous incculation of rata with MMILyin prostatia aéenseazcinoma colla Seouessioy INE, [21 snvervigate uhether redleledide Lleteyy will prevent. pataeraare Sod/ex prolong suxv-val in vars Uewting dohastancous “OUiyLi timbre that empress MNES) (3) Sctcunine the cepresaion lovol of RIE ragaired 20 e::eit selective rediolodide-rediaved Kilticg of Mamyiu-hi=s prostatic adeoncavcizoma cells in Glyo) ond, 14) vestrice AMIS cagrossion in prostatic tisaus urter transcriptional regulation of prostabe-sperific 74 SURDECT TERRE TE AER OF AOE wrostcte canser, sodiun ied-#e symporter, sere transfer, radioiodiae a TF SEDURTTY CEASSEICATON | 76. SECURITY CEAGEREATON | 18 GECUPY CLASGRATION —| Bi CHVTATON OF RESTRICT ornaroar ‘or as pace Onausreacr Tnetai fing Inevaneified Onedaveitied meLimived TEN ae s9oN a Seog Tan Bie 2 ‘8 M charg, 2098 annual report for OAMD7-02-1.0119 Table of Contents cover Pago 1 SF 298. Poge 2 TABLE OF GONTENTS.... Poge 3 INTRODUCTION. Page 4 BODY. Page 48 KEY RESEARCH ACCOMPLISHMENTS Page 6 REPORTABLE OUTCOMES. Pago 6 ONCLUSIONS. Page 6-7 REFERENCES. Page 7 APPENDIX 4 Page 8-15 APPENDIX 2s once Page 16-24 APPENDIX. Page 25-21 Pages r ‘Sef Jhiang, 2008 anual wp for DAWMDIT-02-1-0119 INTRODUCTION The sodium iod'de symporter (NIS} mediates iodids uptake in thyrold follicular calls and provides @ mechanism for effective radioiodide treatment of rosidual, recurrent, and Tetastatle thyrold cancers. The objective of the proposed research is to test the hypothesis lhl expression of exogenous hNIS in prastatic Hssue will enable radioiodide to localize and ablate residual prostate cancer following prostatectomy, such that Teourrence and metastasis of the disease can be prevented. The specific aims of this project are to: (1} confirm metastatle progression to distant lymph nadas and lungs following subcutaneous inooulation of rats with MATLyLU prostatic. adenocarcinama cols exprossing hNIS; (2) investigate whether radlofodide therapy will prevent metastases andior prolong survival in rats bearing subcutaneous MATLyLu tumors that express HNIS; (3) determine the expression level of hNIS required 10 elicit velectve radlo‘odide-medisted kiling of MATLyLU-ANIS. prostatic adenocarcinoma cells in vivo: and, (4) restrict HNIS expression in prostatic tissue Under transcriptional regulation of prostate-specific promoter. BODY Task 4. Confirm metastatic progression to distant iymph nodes and lungs following subcutanaous inoculation of rats wih MATLyLu prostatic adenocarcinoma cells expressing hNIS: Completed in Year 1 with one published paper KMD La Perle, D Shen, TLF Buckwaltar, 8 Wiliams, A Haynam, G Hinkle, R Pozderao, C Capen, and SM Jiang (2002) "i Vivo Expression and Funct on of the Sodium logide Symporter Following Gene Transfer in the MATLyLu Rat Model of Netastatle Prostata Cancar’, The Prostate, 80: 170-178. Task 2 Investigate whether radioiodide thorapy will prevent metastazes andiar prolong survival in rats beating subcutaneous MATLyLu tumors that express ANIS (Months 12- 24, completed) Appendix 1: KMD La Perle, EAG Blorime, CC Capen, and SM Jhang (2003) “Ettect of Exegenous Human Sodiurvlouide Symporter Express'on an Growth of MatLyLu Cells’, Thyroid 3433440 Appendix 2: DHY Shen, DK Marsee, J Schaap, W Yang, J-Y Cho, G Hinkle, HN Nagaraja, RT loos, RF Barth, end Sh Jhiang (2004) “Effec’s of dose, intervention time, and radior weld on sodium iodida symportar (NIS} targeted radionuclide Frerapy", Gene Therapy 112 161-169, ‘As reported in the previous annual report, we faund that NIS oxprosston inhibits MATLyLu cell growth (La Perle et al, 2063}, We also found (Rat the time course of tumor progression and spread of metastases vary significantly among animals even in the Same experimental group (La Perte et al, 2002, and La Per'e ot al, 2003}, Thus, we have chosan an alternate aniinal model fo investigate the theraneutc affectivaness of NIS- Page 4 ‘SM -hisng, 2008 anntal part for DARD17-C2-1-0119, maciated radioiodide therapy (Shon ot al, 2004). The effectiveness of NIG os a gene therapy agent ies in ifs ablity fo madiata the uptake of a variaty af radionuclides, such as 1, "|, and “Re, We have conducted a study comparing the therapeutic effectiveness of these diferent radioncclides In NIS-expressing gllomas, which have telaiively uniform survival tima among untreated animals. We show that: (1) the therapeutic effectivenass af "Vin prolanging the survival tima of rats hearing FS@/MNIS gliomas is dose- and treatment-ime-dapandent, (2) the number of remaining NIS expressing tumor cells decreased greatly in RGZ/NIS gllomas post "I treatment and ‘was inversely relaled o survivat tine; (2) 8 ani each of "lis as effective as 16 mCi "1 alone, despite @ smaller tumor absorbed dose; (4) "*ReO., a polent i erritir, is more efficlent than “| to enhance the survival of rats bearing F9B/hNIS gllomas. These studies demonstate the importance of radiopharmaceutlca” selection, dose, and tinlng of treatment (0 optimize the therapeutic effeciveness of NiS-largeted radionuclide thorapy. Task 3. Determine the expression levet of hNIS required to elicit sslective radiciodida- medlated killing of MatLyLU-hNIS prostatic adenocarcinoma cal's in vivo {months 18-36, In progress) We hypcthesize that radioiode uptake in vivo correlates with NIS expression levels within a certain range. We have been trying to establish MatLyLu clonal cells with doxyoycline inducible NIS expression so that we could determine the range of NIS expression levels required to aliit radioiodide-madiated killing effects. Unfortunately, wwe have failed ta acquire doxycyctine-induciblo MatLyLU-nNIS clones despite several attempts. However, se have been abla to establish doxyeyctine-inducib‘e hnNIS clones using other cell lings. We are nav trying to switch to other human prostate cancer cell lines to accomplish this task, Task 4. Restrict hNIS expression in prostatic tissue under transcriptioral regulation of prostate-spectf c promoters (Months 1-36, in pragress) Appendix 3: DK Marsee, DHY Shen, LR MacDonald, DD Vadysitisack, X Lin, G Hinkle, RT Koos, S14 Jhdang (2004) *Fnaging of Pulmnana-y Tumors Following RIS Gene Transfer Using ‘Single Photon Emission Computed Tomography’, Concer Gene Therapy in press, Since the most challenging issues of gene therapy remains vector delivery to the target tissue, we have beon trying fo use NIS as imaging renorter gene to optimize vector delivery efficiency, in addition to engineer adenovirus camying NIS under the contro! of prostate-spectfc pramoter. Due fo the high mortality assoctated with pulmonary tumor metastases, we have focused our MatLyLu studies on the use af NIS in gene therapy for lung tumor nodules (Marsee et al, 2004), In this study, we established NIS- expressing pulmonary tumois In nude mice by intravenous injection of tumor calls and investigated the minimal tumor sizo roquired for in vivo detection of pulmonary tumors by single photon emission computed tomography (SPECT) with pinhole collimation. to ander to define the axatumic location of NiS-expressing tumor nadules detactable by SPECT, we performed simultaneous, duslisotope imaging. We injected 1 mCi sonTo+ Pages StH Jhlang, 2008 annual sepor for DAMD17-02-1-0119 MAA via teil veln to Image pulmenary perfusion and injected 1 mCI Na" intrapertoneally to Image NIS-expressing tumors. Fused images showed that "Tc MAA perfusion defects correlated with NIS-mediated "uptake. Post-mortem analysis revealed that tumors 3 mm in diameter could ba dotected by SPECT with pintole collimation, These studies demonstrate the feasibilty of SPECT to datact pulmonary ‘umors expressing exogenous NIS in rice. We have also been exploring different methods to efficiently dellver adenoviaises to MalLyLu puknonery turnor nodules. fn vitro studies demonstrated that MatLyLu cells are highly susceptible to adenoviral infection. Inilally, we attempted ta deliver adenoviruses via intravenous delivery. Viral gere expression was found Ih the liver, but not in elther the lung parenchyma or pulmonary tumor nodules. We algo performed Intratracheal injection to celiver virusés to pulmonary wmors. Whereas the lung parenchyma showed strong viral gene exoression, the wmor nedules were not Infected. These results suggest that MatLyLu pulmonary tumors do not communicate with the alveotar alrspace, likely du to the localizatio of Maly. tumors on the surface of the lung, We are currently in the process of performing intrapleutal adenoviral delivery as a means to infect pulmonary metastases. KEY RESEARCH ACCOMPLISHMENTS. + Demonstrate the importance ef radiopharmaceutical selection, dase, and timing of treatment fo optimize the therapeullc effectiveness of NiS-targeted radionuclide therapy + Demonstrate the feasiiity of single photon emission computed tomography (SPECT) fo non-invasively detect pulmonary metastases of MatLyLU-NNIS tumors wit a dlameter of 3 mm in size in mie. REPORTABLE OUTCOMES Manuscript 1: KMD La Perle, EAG Blonme, CC Caper, and SM Jhiang (2003} "Effect of Exogenous Human Sodiumlodide Symporter Expression on Growth of MatLytu Cells", Thyroid 13133-4140 Manuscript 2: DHY Shen, OK Marsee, J Schaap, W Yang. J-Y Cho, G Hinkle, HN Nagaraja, RT Kloos, RF Barth, ark! SM Jhiang (2002) Efecis of dove, intervention line, and radionuciide on sodium iodide symportar (NIS) targeted radionuctide terony’, Gone Tharapy 14; 161-169. Manuscript 2: DK Marsee, DHY Shen, LR MacDonald, DD Vadysiisack, X Lin, G Hinkle, ST Klos, SM Jhiang (2004} Imaging of Pulmonary Tumors Following NIS Gere Transfer Using Single Photon Emission Computed Tomograptiy’, Cancor Gene THerany lh orass. CONCLUSIONS © Selecting the optimal raciopharmacauticals, as welt as the optimal dose and timing of treatment, could further optimize NIS-targeted radionuclide therapy for prostate cancer. Pages ‘SM Jnlang, 2003 annual reper for DAMIDI7-02-1.0119 + NIS can serve as an imaging reporter gane to optimize vector delivery for prostate cancar gene therapy. REFERENGES KMD Le Perle, D Shen, TLF Buckwaltor, B Willams, A Haynam, G Hinkle, R Pozderac, CC Capen, and 8M Jhiang (2002) “In’ Vivo Expression and Function of the Sodium lotiide Symporter Following Gere Transfer in the MATLyLu Rat Madel of Metastatic Prostate Cancer", The Prostate, 50: 170-178. KIND La Perle, EAG Blomme, CC Capen, and SM Jhiang (2093) "Effect of Exogenous Harman Sodiumifocide Symportse Exprescion on Grownh of MatLy.il Calle’, fhyrole 43134-4140 HY Shon, DK Matsoe, J Schaap, W Yang, J-Y Cha, G Hinkle, HN Nagaraja, RT Kloos, RF Barth, and SM Jhlang (2004) "Elfects of dose, intervention tims, and radionucide on sodium jodie symporter(NIS} targeted radionuclide therapy". Gene Therapy 11: 164-168. DK Marsee, DHY Shen, LR MacDonald, DD Vadysirisack, X Lin, G Hinkle, RT kloas, St uhiang (2004) "imaging of Pulinonery Tumo’s Folloulng NIS Gene Transfer Using Single Phaten Emission Computed Tomograpty", Cancer Gane Tharapy in press. APPENDICES ‘Three manusoripts are appended. Pane? Appendix 1 Effect of Exogenous Human Sodium lodide Symporter Expression on Growth of MATLyLu Cells Risa WD. Le Pte.) Die AG Rammras Crees @ Caren, awl Sis Wang! “Tha sodium iodide symporter (Nis) medias iodis wptake in Hye calls ena enable He fftve radii Gide tetmnent uf dngoid cancer, Ler ic mich intrest in fait edoinlide thenops i other cancers by IS ger tami, This study showed Tit exogennce DIS cxprseon decreased MATT yu al prosaic ae ‘octsaneme cell growth, Lumor gro ans uutnetatie progression Were sight any delayed in smugencic fete ingtel oni mized or lon papilations of SAIL YLAGNIS rolls eneapared ty rots with control famurs NAT TaN tumors in muck mite had a lose, albeit latistcaly sisfcant, ote rate tho antl Irmons The KET lebeling inn at Nlepoutve oss a3 Laver than io NiSumyalive ateas of tat mers le freed feom amined popwiilon af MALLyLu BIS cally Cacwtb of cong) populations of MAILyT w-NTS celts twas dslage providing InfRODUCTION Date Sam tere cunt tle ved ie soso Nai pucker 001 Tas Fete sysnqeecin ps Frt-an the Ssoliten! memnimanc of Dold Jo fenke {oklalcelle comple tre imnene acon of eo a" Geeeihonel sein se aioepances Guvesty 2° 78! dasoabe tepogphotne (4 Panel It. Conscquer yesh ienpeliar caagn OFT 029 fegher shen fat in pee 1 Ta spake oh fecal op ths pete he shyt wets, Wlodos ioe 2] ales yong 7. Tha {ilon Us sepect of moral Ujauidphsilogy "eon Cisceeetyenpied fe logan ge a obi re Exh rector ad sein aot sonra fo radimucide adauastaties The ard therapetc etmen for faa ener, whieh cembinay ageeOEY tolimea Ly tdalodite tape. tar oruled le coc! open seven vrs a he secuesce a8 ora fe wd ual de fc (2 batesul ofthe soveein ott hyroideaer ge bers oxy NS ae Dear atactv ir Be team ferret ol caer rece sr, Misi ave iva te nal apa ele FEE GNI or husnan USE! NS gene nance by ena Fedral cuales ete cel tl fe 8 nag 7 saline Letapy of eeel, Broa Papal Oy fro These results demotstvate fat SIS expression inlsbite MATLyLu eaD growth, hereby lita polanial senefi of US-madiated gene ther fe ener ‘eid, loose cone, aa aces xanga Unvostrsih, The wor of hoor ads ave sid Inunaéelet mies hing sbculaean oanan race eeopants A611 wl deer studi ose posnge [ermal (29 9 aly spate Be deeton oem Stuangons amar andelageesinipp tnedes yea aatigopry fllsainy ceo televia: gone tater of ENN a he MATLfL eigen ve madd of relate provintsconse ) Dchig te roure nh ody, se = Mire Gar sistas rumen ren rl oe (peal tet ate noi tan paradox veleters (Ded amore The theca sua tn ele this tnthe and estan eli 9° NT resin 3 the gers a Tybaclpoetaeedemocecrumes ls inocu Be Lae saemce nici ea Matai aret Methods samen NS eetoving Rect cel INA cana canning ther SSE CON or FLAN cDNA arate engnorer ng te Ferm wedor EXSN Gandy provided hy. Kale Tore tania he Ole Sate Crier, tambu O tv SN Th pen eared I rd san fe aence he Ie ST sa oc le [eraaiune Ts ‘cuneee pen feta, owes. Soubble CQUDNIS smb eangpize koa Cte of 10 10 the FLANIS. L ANOS SM enpmests MNTS cDNA nde the fate the 5 Mreney movie evens vr Tong ke Inbal ieee promi (0) ane ees a nev se lance gene (S) west ute ona se stpromote 5) (2) Relive geal wy preci Sb MATE ons ard syeganate Copernagen st aria! nook The MAC gla all foes me af sraral eines Cee ped feos x spuaturans pre adores {Copenhagea et 05). The SACI subtinn fe mpiy _Fevring vogae deperdsut, eaplti e t Figilp motte lo spllletel Tea wales ard forge afer saertamerneeon i apatite Cope? aye 216, ATI cots wene oblained Lion De. Inui K. Stcuiy Depermaent oF Peedi Drevaiion/ Ge atin, Universit of schigan, has Afb MO. Bape Callectan of Col Coltses (ECACC, sit, Salsbury. WME, United Kg, ard De Abert A. Cold (dsadanieh Zed ethats Viel sent Arverein, Te [Nethatiad}urd mans asinine n HIM 1643 sopple ‘nla lt 3 lye er. VE. sep sony IN) pga of naphunysin sane 2 eA ct > futon 1 m3 of oaon pra, exg/moF rain, Ss: of roneern, an? po shun en ie, Aloette mai an eget emp ae rom [GcoDHL Le Tecan ls (od ted, NY) we erent ells ere monetuced By inca Wah ImLof led. RStSH6 Sh FLPNIS EX, cr XEN so. ‘ns see i fo 24 fu Sibe-b ame ced ATLyLu cele wes peed with gral Cn otzin (G48) for 5 Ags fllowed Uy ewer in ME gfe CAIE Indie lea ell 3 ave 20 SomenfroncthentRIAT svcolsec expr aise Bren ovat inst sade, MR apres an raced {ths cine wont tiple neg ne way nord). Svar te Soothe rake Coperagen JG (dar Svagie Ley, ez IN) sere iaectsls eced using 2gnuge reel he ih Fan et 1 celle suspend it 3 ano Hans be anera ealegtvion (TES Rat were lise 3 a Sane wrath All eprint froceduna i Ue sty esses splint ea (onat Latemiory Animist Care and Use Ceacrithe of Ihe Di Sse Hay. 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Enagas weve proses) 32 as ‘hry re ren segracnied eng Seemerlaice (etal 5 Shchav ity ose taste tical nad erin to diainevsh mgt from pase tS EXPRESSION DECREASES MATLyLu GROWTH ject The setae prvi sl the rerio of Uinwcy tropes Their va asage analyte (Fe foe Boa FEA) genre he easraren of BEI Gh oe nue dor wera actly aalyae 5 de settee le este gra uve oer yl plats tae set wth 2210 parent oe teanednowe RIATLy La els for ve Each. cll [Eben iy typaiste Calle wae debicred with 25% Rapriiallyietirnine tae s ocd (UTA) aad Dunia wk vocyasiee et dags 1216 anal Inv sfaston niveasopy “Ssiscontloen peut es Tanatared MATESLU ct etched tov P73 est ah IS tpesJEDTA pled fy cnmapesin ed NE er 3 ote om pra Fine a espe Ds ue ase i ES ribs Used gaara, caylee, fal na eos aed ede deat pls (estes Petting, Rundi £4) UPrathin ctr nee Shaped wa ine SH kandi cles ase (Ga stew OF Sint ais ews cal data es cron a seers = shear ern ule gear EM. llc afeences bebe rane fe dtent dns wee rants th pha Trott sersan 30 (raped wales, Som Diego. CAL ing oaeseny melychs of tananee NOS} and TF [Kroner nid pe campus (al Satie penne sae hyp UU, ects eros gtd and slower ntastane pression of [MAT plsebls waors in Copsnnagen ais Sx dierent Se enpsimens eo cesta agrees fan De lee ren (Oats K 198 et noe, European Clas a Cel Gems {HACC Te Aen, Cal. Capeadager. ok were sub rusty ajecien with iar pant MATT lx cl, Nvtstc le cancer wih voles on {NSN} 26k thos, we MATEYLY cll tansdueed ooh romain es ry he rua ed term NFS ae lt Fn GAN Shc bMS Rtg wre fjted wh anit ule fap of A FLyLcADUS cell fn ur eetiaets hora: « ‘dhns, populalon re ected wt experimen all ‘Teor Eeteancs, Nie dised (oman cre il {ly anal ronda por of source Gike MET gene STS conernee ued REITER Sa. e7 whetve te baer sre derived oa IS “bet popriat of ear rete ere ae aed An Tanke Tl ae we jected eh = Detect ers a sons popolatan of ATLL PERE Although ts sage alae woe ml the tae MAT Ip LtSTEe rors mee Zid sate han he pores SEAT ya rare nascaey, Table 1, Because he Zh oom pods mph cade and mg ese eee ster nubcaangoe ron of tee Pores: KAT prone sehIATT pf cee olaeat for BEATE, Teor seksectens fen apne ne ene ied at parece) BST ols sale oe a mabe popslaon of Tay laeFLRNIS coll 4 TG LacFLANS tis ce tet0-ldenalle om povidone esperar TRCAGE: eRe 3 omer te ECAC cola bed (0 {reastsze conic afer abet coe Tres Ih node ae lang rtaatases cor deve etl ooh MATT yo on land fem De. ek ers WEkuancosehy ead ui Copetitogen mit oa ase cle Sat cae i ll nbn errors Hose MATELY fis wore mimtaecl Ni etsy expricg DSS FLAN, ond am wae ed it eter eed ar eons popslotion of Pwssliced cols WATIyLae ALANS rae Svc igneasy al Lia contd ATE SLAeLXSN (Deve fee Lsbe The Sten ar siz re Frock rne vals when 2 ecast poptlaie 8 Fld ‘Peall) of MATL yLa- FLAWS ele iee a [Bip U compazed i mea poplin 9 fle nelle. ote, ANTETuanerace ae Con Sats MALLU Toe WORMS we Cora Rats Ten Dug vats one diction 1 Perabl — Mecaalee NA Tee 2 uC 2 1 1 Nee) Mccmiy emt ROUSE 3 2 Paerml TeAaUt Sk Ts7 on 3 a 2 Rays Leae Mierd as 0ce 2 3 i Para ECACC xe 50 nae it} 2 3 Pins BAGC Nee O22 =uin it] i Ten" Goat Beat tans Tu a z i Fuleis Geto ket [7m a & z Len Golant Mowe ilar 18 a 4 5 figs atu Med 2a8 1 203 % i é LEN Catan Coot uae) 2 ry & Buss Cd Eon Bt AB 1 "BEM san ear