Redirected Antitumor Activity of Primary Human Lymphocytes Transduced With a Fully Human Anti-mesothelin Chimeric Receptor

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1 originl rticle Redirected Antitumor Activity of Primry Humn Lymphocytes Trnsduced With Fully Humn Anti-mesothelin Chimeric Receptor Evripidis Lnitis 1,2, Mthilde Poussin 1, In S Hgemnn 3, George Coukos 1, Rphel Sndltzopoulos 2, Nthlie Scholler 1 nd Dniel J Powell Jr 1,3 1 Deprtment of Ostetrics nd Gynecology, Ovrin Cncer Reserch Center, University of Pennsylvni, Phildelphi, Pennsylvni, USA; 2 Deprtment of Moleculr Biology nd Genetics, Democritus University of Thrce, Alexndroupolis, Greece; 3 Deprtment of Pthology nd Lortory Medicine, Armson Cncer Center, University of Pennsylvni, Phildelphi, Pennsylvni, USA Cncer regression y gene-modified T cells ering chimeric ntigen receptor (CAR) exodomin of mouse origin cn e limited y the induction of trnsgene immunogenicity resulting in poor persistence nd function in vivo. The development of functionlly-ctive CAR of humn origin cn ddress this issue. Here, we constructed nd evluted fully humn nti-mesothelin CARs comprised of humn mesothelin-specific singlechin ntiody vrile frgment (P4 scfv) coupled to T cell signling domins. Primry humn T cells expressing P4 CAR specificlly produced proinflmmtory cytokines, degrnulted nd exerted potent cytolytic functions when cultured with mesothelin-expressing tumors in vitro. P4 CAR T cells lso medited ystnder killing of mesothelin-negtive cncer cells during coculture. CAR rectivity ws not rogted y solule tumor-secreted or recominnt mesothelin protein even t suprphysiologicl levels. Importntly, doptive trnsfer of P4 CARexpressing T cells medited the regression of lrge, estlished tumor in the presence of solule mesothelin in xenogenic model of humn ovrin cncer. Thus, primry humn T cells expressing fully humn ntimesothelin CAR efficiently kill mesothelin-expressing tumors in vitro nd in vivo nd hve the potentil to overcome the issue of trnsgene immunogenicity tht my limit CAR T cell trils tht utilize scfvs of mouse origin. Received 31 My 211; ccepted 28 Octoer 211; dvnce online puliction 29 Novemer 211. doi:1.138/mt Introduction Successful T cell immunotherpeutic strtegies re limited y the tolernce to self-ntigens, rendering the identifiction nd expnsion of tumor-rective T cells with high vidity for tumorssocited ntigens difficult. 1 Further, solid tumors often downregulte mjor histocomptiility complex (MHC) clss I nd/or other molecules relted with the ntigen processing mchinery s mechnism for evding immune response. 2 To ovite these ostcles, tumor ntigen-specific T cells hve een engineered to express chimeric ntigen receptors (CAR) or T odies comprised of n ntigen-specific single-chin ntiody vrile frgment (scfv) fused to intrcellulr signling domins derived from receptors involved in lymphocyte ctivtion. 3 CARs cn functionlly redirect T cells with high specificity to vrious surfce ntigens on tumor cells independent of MHC restriction nd ntigen processing, nd therefore ypss mjor mechnisms y which tumors escpe immune recognition. CARs trgeting vrious tumor-ssocited ntigens hve een developed, chrcterized, nd tested. 4 Despite encourging preclinicl results, CAR therpy hs hd limited success in the clinic primrily due to poor long-term persistence of the engineered T cells following infusion to ptients. This my e ttriuted in prt to the frequent use of scfvs of mouse origin which renders these constructs susceptile to host immune recognition nd responses ginst xenogeneic regions of the molecule. Xenogeneic responses hve een oserved in clinicl trils of CAR therpy. For exmple, ptients who received utologous T cells trnsduced to express CAR of mouse origin mounted humorl immune responses ginst the trnsgeneering cells, which my hve limited their persistence in vivo nd their ility to respond ginst ntigen-expressing tumor cells. 5,6 Mesothelin is glycosylphosphtidyl inositol-linked memrne glycoprotein overexpressed on the cell surfce of mesotheliom, ovrin cncer s well s cncers of the pncres, stomch, nd lung. 7 9 Mesothelin lso exists s solule form nd is serum iomrker for lung, mesotheliom, nd ovrin cncer The iologicl function of mesothelin is still uncler; however mesothelin inds to CA125, plsm glycoprotein on tumor cells, suggesting tht mesothelin my contriute to peritonel nd pleurl metstsis. 13,14 Mesothelin expression is ssocited with chemoresistnce, shorter disese-free survivl, nd worse overll survivl of ptients with epithelil ovrin cncer. 15 Accordingly, mesothelin represents n ttrctive trget for immune-sed therpies. While vccintion with grnulocyte mcrophge-colony stimulting fctor-trnsduced pncretic cncer lines cn induce in vivo mesothelin-specific CD8 + T cells with the cpcity to kill mesothelin-expressing cncer cells in n MHC clss I-restricted fshion, 16 more recent work hs shown tht humn T cells ering n nti-humn mesothelin CAR of mouse origin (referred to s SS1) exhiit MHC-independent effector functions Correspondence: Dniel J Powell, University of Pennsylvni, Rm BRB II/III, 421 Curie Blvd., Phildelphi, Pennsylvni 1914, USA. E-mil: pod@mil.med.upenn.edu Moleculr Therpy 1

2 Humn Mesothelin-specific CAR Therpy of Cncer The Americn Society of Gene & Cell Therpy in vitro nd induce the regression of humn mesotheliom xenogrfts in vivo in immunodeficient mice. 17 Here, we ddress the potentil issue of CAR trnsgene immunogenicity nd report tht primry humn T cells cn e efficiently trnsduced to express fully humn nti-mesothelin-specific CAR using lentivirl vectors, nd tht fully humn CAR-trnsduced T cells demonstrte specific proinflmmtory cytokine secretion nd potent cytolytic ctivity in response to humn cncer cells expressing mesothelin, resist solule mesothelin inhiition, medite ystnder killing, nd medite regression of estlished humn tumor in xenogeneic mouse model of dvnced ovrin cncer. Results CAR construction The humn nti-humn mesothelin-specific P4 scfv ws selected for CAR construction sed upon its high inding ffinity nd specificity for mesothelin ( /mol/l). 18 P4 CAR constructs comprised the P4 scfv linked to CD8α hinge nd trnsmemrne region, followed y CD3ζ signling moiety lone (P4-z) or in tndem with the CD28 intrcellulr signling motif were generted (P4-28z; Figure 1). An nti-cd19 CAR contining CD3ζ lone or with CD28 signling motifs in tndem (CD19-28z) ws used s n ntigen-specificity control. 19 Primry humn T cells were efficiently trnsduced with CAR lentivirl vectors with trnsduction efficiencies reproducily ove 9% (Figure 1), nd equilirted to 8% y dding untrnsduced T cells for ll functionl ssys. Primry humn P4 CAR T cells exert ntigen-specific function in vitro We first tested whether solule or immoilized mesothelin protein stimultion induces P4-z CAR-trnsduced T cell ctivtion in vitro. Solule mesothelin protein did not induce ctivtion of P4 CAR T cells, even t high protein concentrtions (5 μg/ml). However, cross-linking of the CAR y immoilized mesothelin protein resulted in roust T cell ctivtion nd secretion of high levels of interferon-γ (IFN-γ) (Figure 2). To evlute the ility of P4-z CAR T cells to respond to mesothelin expressed on the cell surfce, AE17, mouse mlignnt mesotheliom cell line, ws modified to express humn mesothelin (AE17M; Figure 2). 2 P4-z CAR T cells secreted IFN-γ in response to AE17M cells, ut not to prentl AE17 cells (Figure 2c). Since ovrin cncers frequently express mesothelin, 21 pnel of estlished humn ovrin cncer cell lines of disprte humn leukocyte ntigen-hplotype tht expressed surfce mesothelin t vrying levels (, OVCAR-3, SKOV-3, OVCAR-5) or not t ll (C3 nd OVCAR-2) ws ssemled for functionl ssys (Figure 2). P4-z CAR-trnsduced T cells secreted IFN-γ, mcrophge inflmmtory protein-1α (MIP-1α), tumor necrosis fctor-α nd interleukin-2 (IL-2) in response to mesothelin + tumor lines stimultion, ut not when stimulted with mesothelin neg lines (Figure 2c). Low levels of IL-4 (<25 pg/ml) nd higher levels of IL-1 (<7 pg/ml) were secreted y P4 CAR T cells in response to mesothelin-expressing cncer cells (dt not shown). P4-z CAR T cells recognized nd responded to stimultion y ll humn ovrin cncer cells expressing detectle mesothelin on their surfce (Figure 2d). The mount of IFN-γ secreted correlted with the level of surfce protein expressed y tumor cells (Figure 2e). Control T cells trnsduced to express green fluorescent protein (GFP) or the CD19-z CAR did not produce cytokines fter mesothelin + tumor stimultion, demonstrting the need for ntigen-specificity. Primry humn P4 CAR T cells exhiit potent cytolytic function Degrnultion is quntittive indictor of lytic function y T cells. 22 P4 CAR CD8 + T cells degrnulted in response to ovrin cncer z Anti-mesothelin P4 scfv VH L VL Anti-CD19 scfv CD3ζ VH L VL CD3ζ :CD8 leder :CD8 hinge :CD8 TM :CD28 TM 28z VH L VL CD28 CD3ζ VH L VL CD28 CD3ζ Intrcellulr domins Anti-mesothelin CAR Intrcellulr domins Anti-CD19 CAR P4-z P4-28z CD19-z CD19-28z (22,41) 1 (8,248) 1 (54,272) 1 (12,184) 96% 8 87% 8 96% 8 91% scfv expression Figure 1 Genertion nd specific immune recognition y P4 nti-mesothelin CAR-trnsduced humn T cells in vitro. () Schemtic representtion of P4 sed chimeric ntigen receptor (CAR) constructs contining the CD3ζ cytosolic domin lone (P4-z) or in comintion with the CD28 costimultory module (P4-28z). The nti-cd19-z nd nti-cd19-28z CARs re shown. () P4 CAR expression (filled lck histogrms) ws detected on humn CD3-gted cells vi recominnt mesothelin protein stining fter trnsduction with lentivirus compred to untrnsduced T cells (open histogrms). Anti-CD19 trnsduced T cells were detected using protein L followed y SA-PE. Trnsduction efficiencies re indicted with the percentge of CAR expression nd the men fluorescence intensity of the trnsduced popultions in prentheses. L, linker; P4, ntimesothelin scfv; VH, vrile H chin; VL, vrile L chin; SA-PE, streptvidin-phycoerythrin; scfv, single-chin ntiody vrile frgment; TM, trnsmemrne region. 2

3 Humn Mesothelin-specific CAR Therpy of Cncer Humn ovrin cncer cell lines Mouse mesotheliom cell line IFN-γ concentrtion (pg/ml) c IFN-γ concentrtion (pg/ml) 35, 28, 21, 14, 7, 12, TNF-α concentrtion (pg/ml) 9, 6, 3, None GFP CD19-z P4-z Solule mesothelin IFN-γ TNF-α Immoilized mesothelin None AE17 AE17M C3 K562-CD19 MIP-1α concentrtion (pg/ml) IL-2 concentrtion (pg/ml) P4-z CD19-z GFP 5, 4, 3, 2, 1, Stimultor cell lines OVCAR-3 SKOV-3 6, OVCAR-5 C3 OVCAR Humn mesothelin expression None AE17 MIP-1α IL-2 AE17M C3 K562-CD19 IFN-γ concentrtion (pg/ml) IFN-γ concentrtion (pg/ml) , 2, 15, 1, 5, 25, 2, 15, 1, 5, None AE AE1M 21, OVCAR SKOV-3 R 2 =.8518 OVCAR-5 C3 2, 4, 6, 8, Mesothelin MFI Figure 2 Mesothelin redirected T cells secrete Th1 proinflmmtory cytokines in response to plte-ound mesothelin or tumor cell surfcessocited mesothelin. () Trnsduced T cells respond ginst immoilized ut not solule mesothelin. P4-z T cells or control CD19-z nd GFP T cells (1 5 cells/well) were incuted with either 5 μg/ml of solule or plte-immoilized mesothelin. After overnight incution, culture superntnts were nlyzed for humn Th1/Th2 cytokines using cytometric ed rry technology. Concentrtion of IFN-γ ws expressed in pg/ml. () Surfce mesothelin expression (solid lck histogrms) y vrious humn ovrin cncer cell lines ws evluted y flow cytometry. The ntive mouse mlignnt mesotheliom cell line AE17 which does not express humn mesothelin ws engineered to express high surfce levels of humn mesothelin (AE17M) s shown y flow cytometry; isotype ntiody control (open gry histogrms). (c) Primry humn T cells trnsduced with P4-z preferentilly produce Th1 cytokines fter stimultion with mesothelin + cncer cell lines. Trnsduced T cells (1 5 CAR + T cells) were cultured lone (none) or stimulted overnight with n equl numer of humn mesothelin + AE17M nd or ntigen-negtive AE17 nd C3 cncer cell lines. Cell-free superntnt from three independent cultures ws hrvested nd pooled fter ~2 hours of incution nd the indicted humn Th1/Th2 cytokines were quntified using cytometric ed rry technology. Vlues represent cytokine concentrtion (pg/ml). (d) Antigen-stimulted IFN-γ secretion y P4-z ut not CD19-z or GFP T cells following overnight incution with ovrin cncer cell lines expressing different levels of surfce mesothelin. Men IFN-γ concentrtion ± SEM (pg/ml) from triplicte cultures is shown. (e) Correltion of mesothelin expression (men fluorescence intensity; MFI) on mesothelin-expressing tumor cells ws plotted versus the production of IFN-γ y P4-z CAR-trnsduced T cells cocultured with these tumor cells. CAR, chimeric ntigen receptor; GFP, green fluorescent protein; IFN, interferon; IL, interleukin; MIP, mcrophge inflmmtory protein; Th1, T helper 1; Th2, T helper 2; TNF, tumor necrosis fctor. d e cell lines expressing mesothelin (, OVCAR-3, SKOV-3) or AE17M, with upregulted surfce coexpression of moilized CD17 (lysosoml-ssocited memrne protein 1) nd the ctivtionssocited mrker CD69, ut not when stimulted with mesothelin neg lines (AE17 nd OVCAR-2; Figure 3). AE17M stimultion reproducily resulted in moderte CD17 upregultion nd sustntil poptosis of CAR T cells (dt not shown). CD17 expression ws restricted to T cells expressing the P4 chimeric receptors (dt not shown). Mock-T cells nd nti-cd19 CAR T cells did not degrnulte in response to mesothelin + cells (dt not shown). In 4 hours chromium relese ssys, P4-z CAR T cells specificlly lysed AE17M cells ut not the prentl AE17 line (Figure 3). P4-z CAR T cells lso directly nd efficiently lysed the mesothelin + humn ovrin cncer cell line, ut not mesothelin neg C3 cells (Figure 3). CD19-z CAR or GFP trnsduced T cells showed no cytotoxic ctivity ginst the sme trget cells, excluding llorectivity or nonspecific lysis. These results were reproducile in extended 18-hour ssys, with incresed specific lysis from P4 Moleculr Therpy 3

4 Humn Mesothelin-specific CAR Therpy of Cncer The Americn Society of Gene & Cell Therpy Isotype None AE17 AE17M Q4 Q4 Q Q OVCAR-3 SKOV-3 OVCAR CD Q Q Q Q CD17 Mesothelin + trgets Mesothelin trgets 1 8 AE17M 1 8 AE17 GFP CD19-z P4-z Specific lysis (%) C E/T rtio Figure 3 Cytolytic ctivity of nti-mesothelin lentivirl vector-engineered T cells. () P4 CAR T cells degrnulte nd express T cell ctivtion mrkers in response to mesothelin-specific stimultion. P4 CAR T cells were cultured without trget cells (none) or with the indicted mesothelinnegtive or positive tumor cell trgets for 5 hours, while eing stined y n nti-cd17, ntiody conjugted with FITC. After the incution period, T cells were stined for CD8 nd CD69 nd nlyzed y flow cytometry. () Antigen-specific killing of mesothelin + tumor cells y P4 CAR T cells. Primry humn T cells trnsduced to express either P4-z or CD19-z CARs or GFP were cocultured with Cr 51 -leled ntive AE17, AE17M,, nd C3 cell lines for 4 hours t the indicted effector to trget rtio. Percent specific trget cell lysis ws clculted s (experimentl spontneous relese) (mximl spontneous relese) 1. Error rs indicte stndrd devition. CAR, chimeric ntigen receptor; E/T rtio, effector cell to trget cell rtio; GFP, green fluorescent protein; FITC, fluorescein isothiocynte. CAR T cells (Supplementry Figure S1). Anti-CD19 CAR T cells did however lyse CD19 + K562 cells, demonstrting their cpcity to respond (Supplementry Figure S2). Anti-mesothelin CAR T cells medite ystnder killing of mesothelin-negtive tumor cells Since estlished cell lines my not ccurtely represent the heterogeneity of complex solid tumor smples, mesothelin expression ws evluted using immunohistochemicl nlysis in 18 cses of high-grde ovrin serous crcinoms in tissue microrry. All cses contined t lest one primry lesion nd one metsttic site. Mesothelin ws expressed t vrious levels mong the tumor sites, rnging from undetectle (score ) to strong stining (score 3+; Figure 4). Mesothelin ws expressed in t lest one primry site in ll (18/18) cses (Figure 4), nd 94% (17/18) of cses showed mesothelin expression t ll primry nd metsttic sites. Of ll 4

5 Humn Mesothelin-specific CAR Therpy of Cncer Cses (n = 18) Sites Primry Metsttic Legend c Specific lysis (%) hours coculture 18 hours coculture AE17cr AE17cr + AE17M AE17Mcr AE17 + AE17Mcr hours coculture 18 hours coculture C3cr C3cr+ cr C3 + cr E/T rtio Figure 4 Bystnder killing induced y P4-redirected T cells. () Mesothelin immunostining showing regionl diversity of mesothelin expression in high-grde ppillry serous ovrin denocrcinom. Originl mgnifiction ws 2. () Hetmp illustrtion of mesothelin expression level in primry nd metsttic ovrin crcinom cses. (c) P4-z CAR T cells were cocultured with ntigen-negtive Cr 51 -leled AE17 (upper) or C3 tumor cells (lower) in the presence or sence of unlelled ntigen-positive AE17M (upper) or cells (lower), respectively, for 4 hours (left pnels) nd 18 hours (right pnels) t the indicted effector to trget rtio. As positive controls, P4-z CAR T cells were cocultured with ntigen-positive Cr 51 - leled AE17M (upper) or tumor cells (lower) in the presence or sence of unlelled ntigen-negtive AE17 (upper) or C3 cncer cells (lower), respectively. Percent specific trget cell lysis ws clculted s (experimentl spontneous relese) (mximl spontneous relese) 1. Error rs indicte stndrd devition. CAR, chimeric ntigen receptor; E/T rtio, effector cell to trget cell rtio. sites, 93% (63/68) expressed mesothelin t some level, nd mesothelin scores were generlly similr cross metsttic nd primry sites (Supplementry Figure S3). Heterogeneity in the expression levels of mesothelin protein mong the different sites ws present in 56% (1/18) of cses. Consistent with existing dt, 23 regionl diversity in mesothelin expression level ws oserved, with some res of tumor eing devoid of detectle mesothelin expression, suggesting tht cncer cells in these regions my e resistnt to the direct cytotoxic effects of P4 CAR T cells. We therefore leled ntigen-negtive tumor cells (C3 nd AE17) with chromium nd cocultured them with P4 CAR T cells in the presence or sence of mesothelin + tumor cells ( nd AE17M) to evlute whether P4 CAR T cells cn elicit ystnder killing of mesothelin neg tumor cells. No killing of mesothelin-negtive cells ws detected fter 4 hours in either the presence or sence of mesothelin + tumor cells (Figure 4c). However, fter 18 hours of coculture, ystnder killing of the mesothelin neg tumor cells y P4 CAR T cells ws detected ut only in the presence of mesothelin + tumor trgets (Figure 4c). In humn tumor cell cultures, ystnder killing ws T cell dose-dependent. Importntly, the level of chromium relesed y ystnder killing sustntilly exceeded the level of spontneous relese in control cultures of tumor lone, excluding the likelihood of chromium reuptke y mesothelin + cncer cells. Direct killing of mesothelin + trgets y P4 CAR T cells ws lso oserved nd ws not inhiited y the presence of mesothelin neg trgets. Lstly, the ystnder effects oserved were not due to tumorsecreted mesothelin trnsfer from ntigen-positive to ntigennegtive cells (Supplementry Figure S4). Tumor-stimulted response y P4 CAR T cells is not inhiited y tumor-secreted mesothelin Ovrin cncers secrete high levels of solule mesothelin protein which is frequently found in the serum nd scites fluid of ptients, nd represents iomrker of disese. 1,12,24 Consistent with these results, we found high levels of solule mesothelin (~1 μg/ml) in scites fluid collected from ptients with epithelil ovrin cncer (Figure 5). High levels of solule mesothelin were lso secreted y cncer cell lines expressing surfce mesothelin ( nd AE17M), ut not mesothelin neg cells (C3 nd AE17), in overnight culture (Figure 5). To determine whether solule mesothelin locks P4 CAR T cell ctivity, cocultures were estlished in the presence or sence of tumor-secreted mesothelin from cell-free tumor superntnts (~25 ng/ml finl concentrtion). First (P4-z) or second genertion (P4-28z) CAR T cells cocultured with mesothelin + cells retined their IFN-γ response in the presence of tumor-derived solule mesothelin (Figure 5). Similr results were oserved from cocultures of P4 CAR T cells nd OVCAR-5 s trget cells, which re mesothelin low Moleculr Therpy 5

6 Humn Mesothelin-specific CAR Therpy of Cncer The Americn Society of Gene & Cell Therpy Mesothelin concentrtion (pg/ml) 1,, 1, 1, 1, 1 c P4 CAR expression C3 AE17 AE17M RMPI-1 Untrnsduced.6% P4-z % IFN-γ concentrtion (pg/ml) IFN-γ concentrtion (pg/ml) 1, 16, 12, 1, 1 8, 4, 1 1 None (meso +++ ) OVCAR-5 (meso + ) CD19-K562 (meso ) R-1 medium medium 5, 4, 3, 2, 1, R-1 medium medium 25, 2, 15, 1, 5, R-1 medium medium P4-z P4-28z P4-z P4-28z P4-z P4-28z CD19-28z rmesothelin Control protein AE17M d Normlized MFI of retined mesothelin (%) Mesothelin ound T cells (%) 4 ºC 1 37 ºC Hours Figure 5 P4 CAR T cells exhiit effector functions ginst tumor cells in the presence of recominnt or tumor-derived mesothelin. () Solule mesothelin is present in the superntnt of mesothelin expressing tumor cells nd in the ovrin cncer-derived scites fluid. Tumor-free cell superntnts or scites fluid were nlyzed for the presence of solule mesothelin using ELISA ssy s descried in Mterils nd Methods. () Anti-mesothelin T cells were incuted with mesothelin + tumor cells in the presence of mesothelin rich medium or RPMI 1% FBS medium. After overnight incution, superntnts were ssyed for IFN-γ y ELISA (results depict the men ± SEM of triplicte wells). (c) T cells were incuted overnight with AE17M nd tumor cells in the presence or sence of solule mesothelin or control. The mount of IFN-γ in culture superntnts ws determined using ELISA (results depict the men ± SEM of triplicte wells). (d) Mesothelin dissocition ssy. P4-z CAR T cells were leled with recominnt iotinylted mesothelin nd then incuted t 37 C or 4 C in time course in the presence of tenfold excess of noniotinylted mesothelin. Antigen retention on the cell surfce ws ssessed y flow cytometry y dding SA-PE fter the end of ech culture period. (Upper) Percent retined mesothelin (y-xis) ws normlized nd scored s men fluorescence intensity (MFI) postincution preincution MFI 1. (Lower) The percentge of preound CD3 + T cells with positive mesothelin inding compred to similrly treted untrnsduced T cells from the sme donor s control ws plotted on the y-xis. ELISA, enzyme-linked immunosorent ssy; FBS, fetl ovine serum; IFN, interferon; SA-PE, streptvidin-phycoerythrin. (Figure 2). CD19-redirected T cells lso retined their ctivity ginst CD19 + K562 trget cells in the presence of solule mesothelin protein. Additionl coculture ssys of P4-z CAR T cells with mesothelin + cncer cells ( nd AE17M) were performed in the presence of recominnt mesothelin protein t suprphysiologicl levels (5 μg/ml); fivefold higher concentrtions thn tht mesured in ptient-derived scites fluid (Figure 5). To more rigorously ssess locking cpcity, P4 CAR T cells were lso diluted tenfold with untrnsduced T cells efore coculture to reduce their frequency to 1% of totl T cells (Figure 5c). Agin, no locking of P4 CAR T cell tumor recognition nd IFN-γ secretion y recominnt mesothelin protein ws detected (Figure 5c). Control protein t identicl concentrtion lso did not lock T cell responses. We next longitudinlly mesured y flow cytometry the dissocition of iotinylted-mesothelin protein tht ws preound to P4-z CAR T cells in the presence of tenfold higher concentrtion of uniotinylted protein nd incuted t either 37 C or 4 C, s surrogte indictor of CAR ffinity. A modest dissocition of iotinylted mesothelin (3%) ws detected over 6 hours t 4 C sed upon men fluorescence intensity, lthough ll CAR T cells remined ound t some level (Figure 5d). At 37 C progressive, sustntil loss of detectle surfce protein ws evident within 1 minutes nd fully displced to ckground levels (9%) fter 6 hours, showing the trnsient nture of P4 CAR inding to solule mesothelin protein under stndrd culture conditions tht my provide the opportunity for CAR re-enggement with surfce ound ntigen. Antitumor ctivity of primry humn T cells expressing P4 nti-mesothelin CAR in vivo To evlute the cpcity of P4 CAR T cells to induce regression of lrge, estlished humn tumors in vivo, second genertion P4-28z CAR T cells were used, since it hs een previously shown tht costimulted CAR T cells exhiit enhnced effector functions in vivo P4-28z CAR T cells used for infusion showed specific nd enhnced IFN-γ secretion s well s IL-2 nd tumor necrosis fctor-α in response to mesothelin + tumor stimultion in vitro, compred with P4-z CAR T cells, confirming their 6

7 Humn Mesothelin-specific CAR Therpy of Cncer Luc ctivity (photons/sec) Tumor volume (mm 3 ) Peripherl T count (cells/µl) c 1,2 Sline GFP 1, CD19-28z P4-z 8 P4-28z e 1, 1, Dys post-tumor injection CD19-28z P4-28z CD4 CD8 Dys post-tumor injection P428-z CD19-28z f CAR expression (%) d Sline 1 GFP CD19-28Z P4-28z dy 12 dy 29 CD19-28z dy 12 dy 29 P4-Z P4-28Z Gted on humn CD3 + T cells P428-z CD19-28-z Imge Min = 59 Mx = 72,33 6, 5, 4, 3, 2, 1, Counts Color r Min = 3,617 Mx = 61,989 Imge Min = 5 Mx = 7, Counts Color r Min = 2, Mx = 42, 4, 3, 2, 1, Figure 6 Mesothelin re-directed T cells exert potent effector functions in vivo. () Rpid regression of lrge pre-estlished tumors in vivo y P4-28z CAR T cells: effect of the CD28 costimultory signling domin. NSG mice ering estlished sucutneous tumor were treted with intrtumorl injections of P4-z nd P4-28z CAR + T cells or control CD19-28z nd GFP T cells or sline on dy 45 nd 55. Tumor growth ws ssessed y cliper mesurement. Tumors treted with P4-28z CAR-trnsduced T cells (~75% CAR expression) rpidly regressed (rrows indicte dys of T cell infusion); tumors treted with sline, GFP or CD19-28z CAR-trnsduced T cells did not regress 3 weeks post-first T cell dose (P <.5). Equl doses of P4-z CAR-trnsduced T cells (~75% CAR expression) only slowed the tumor growth (P =.5). Results re expressed s men tumor volume (mm 3 ± SEM) with n = 5 for ll groups. () fluc + ioluminescence signl ws decresed in P4-z nd P4-28z CAR-treted mice compred with the CD19-z nd the control tretment groups 3 weeks fter the first T cell dose. (c) P4-28z T cells inhiit tumor outgrowth nd scites formtion in murine model of peritonel crcinomtosis. NSG mice received intrperitonel injection of fluc + tumor cells nd were rndomized into two groups efore eginning therpy with T cells expressing P4-28z or CD19-28z vi intrvenous infusion on dy 14 nd 19 fter tumor inocultion. Tumor growth ws monitored y ioluminescence imging (dys 12 nd 29 shown). Photon emission from fluc + tumor cells ws quntified nd the men ± SEM ioluminescence signl determined with n = 5 for oth groups. (d) fluc + ioluminescence signl ws rpidly decresed nd reched the ckground luminescence level in P4-28z CAR-treted mice compred with the CD19-28z 2 weeks fter the first T cell dose. (e) Stle persistence of P4 CAR-engineered humn T cells in vivo. Peripherl lood ws collected 3 weeks fter the first T cell infusion nd quntified for the solute numer of humn CD4 + nd CD8 + T cells/µl of lood. Men cell count ± SEM is shown with n = 5 for ll groups. (f) Surfce CAR expression on persisting P4 + nd CD19-specific humn CD3 + T cells derived from the lood of treted mice mesured y flow cytometry. Men CAR + expression frequency per CD3 + T cell ± SEM per group is shown with n = 5 for ll groups. CAR, chimeric ntigen receptor; GFP, green fluorescent protein; NSG, NOD/SCID/γ-chin /. functionl rectivity (Supplementry Figure S5,). NOD/ SCID/γ-chin / (NSG) mice with estlished sucutneous tumors ( 15 mm 3 ) received intrtumorl injections of CAR T cells on dys 47 nd 57 post-tumor inocultion. Tumor growth ws modestly inhiited in mice receiving P4-z CAR T cells (P =.7), compred to sline, CD19-28z CAR T cells or GFP T cell control groups 3 weeks fter first T cell dose (Figure 6). In contrst, mice receiving P4-28z T cells experienced rpid tumor regression which ws significntly etter thn P4-z T cells (P <.5), indicting tht incorportion of CD28 signls enhnces net ntitumor ctivity in vivo (Figure 6,). Furthermore, xenogeneic model of dvnced intrperitonel metsttic cncer ws estlished to evlute the functionl ctivity of P4 CAR T cells ginst tumor in more physiologiclly relevnt comprtment. NSG mice were inoculted intrperitonelly with fluc + cells. Two weeks postinocultion, two intrvenous injections of P4-28z T cells resulted in rpid tumor regression in ll mice (Figure 6c,d). Disese progression occurred in ll mice receiving CD19-28z T cells. Mice treted with P4-28z CAR did not develop distended domens or scites, nd exhiited profound enhncement in tumor-relted survivl (P <.5) with no cses of tumor-relted mortlity. In the CD19-28z control group, ll mice hd to e euthnized due to disese progression dys post first T cell infusion (dt not shown). Three weeks fter the first T cell dose, peripherl lood CD8 + T nd CD4 + T cell counts from mice injected with P4-28z T cells were significntly Moleculr Therpy 7

8 Humn Mesothelin-specific CAR Therpy of Cncer The Americn Society of Gene & Cell Therpy higher thn in the CD19-28z group (P <.5; Figure 6e). Mice tht received P4-28z CAR T cell trnsfer lso hd incresed persistence of humn T cells ering surfce scfv (57%), compred to CD19-28z CAR T cell-treted mice (.82%; Figure 6f). Similr differences in lood counts nd CAR expression were oserved in mice with sucutneous tumors (Supplementry Figure S5c,d). Discussion To dte, only one other mesothelin-specific CAR hs een reported, 17 the SS1 CAR, whose specificity is derived from the mouse ntihumn mesothelin scfv SS1. 28 SS1 CAR T cells exert potent effector functions ginst cncer cell lines expressing mesothelin in vitro nd erdicte estlished mesothelioms in preclinicl studies. 17 Two phse I studies 29,3 of SS1 coupled to Pseudomons exotoxin A (PE38) hve shown ntitumor ctivity in sujects with mesothelin + tumors nd chimeric ntiody-sed on SS1 scfv (MORA-9) remins under investigtion in phse II study for mesotheliom nd pncretic cncer ( Still, humn nti-mouse ntiody responses hve not een ssessed in these SS1-treted sujects. However, the mouse origin of the SS1 scfv predicts its propensity for inducing xenogeneic responses upon trnsfer to humn sujects. The immunogenicity of mouse trnsgenes is noted in numerous trils of doptive immunotherpy using utologous T cells modified to express mouse-derived scfvs or tumor ntigen-specific T cell receptors (TCRs). 5,31 33 Trnsfer of T cells outfitted with xenogeneic CARs to immunocompetent sujects induces trnsgene-specific immune responses, which my limit the persistence nd function of the gene-modified cells. 5,31,32 Humorl responses re often noted. Induction of oth humorl nd cellulr immune responses hve een reported in ptients treted with ex vivo-engineered nti-caix CAR T cells. 33 Such responses were directed ginst the xenogeneic complementrity-determining nd frmework regions of the CAR vrile domins. Although lymphodepleting chemotherpy cn trnsiently disle endogenous T nd B cell responses, trnsfer of utologous T cells ering murine nti-cea TCR to ptients with metsttic colorectl cncer fter lymphodepleting preconditioning still induced ntimouse TCR-specific IgG ntiodies tht ws cple of impiring the functionlity of crcinoemryonic ntigen (CEA)-specific TCR T cells in vitro. 34 Although significnt correltion etween trnsgene immunogenicity nd efficcy hs not een estlished, the ove mentioned clinicl experiences rtionlize the construction of receptors from humn derivtives to minimize the immunogenic potentil of CARs nd optimize therpy. Our CAR construct utilizes the humn nti-mesothelin P4 scfv, originlly developed y selective enrichment of yest-disply humn scfv lirry, 35 which demonstrtes high specificity with inding of Meso-Ig protein detectle in the rnge of ng/ml. 18 An importnt issue concerning ntiody-sed therpies directed ginst mesothelin is the stimultory or possile inhiitory effect of solule ntigen on the ility to trget memrneound mesothelin, prticulrly in ovrin cncer ptients where high levels of solule mesothelin is present in serum or scites fluid. 1,12,24 Immoilized mesothelin protein ws le to ctivte P4 CAR T cells, feture not detectle when solule mesothelin ws used. Until now, resistnce of nti-mesothelin CAR T cells hs not een tested. P4 CAR T cells chllenged with ovrin cncer cells expressing high or low levels of mesothelin-resisted functionl inhiition y solule mesothelin protein, even t suprphysiologicl levels. Resistnce ws confirmed y their cpcity to medite the regression of estlished tumors in vivo in the presence of high levels of cncer-secreted mesothelin in the serum of treted mice (59 ng/ml ± 2.87 ng/ml, n = 3, dt not shown). Our findings expnd upon prior reports, nd suggest CAR s ility to resist solule ntigen lockde lies in its ility to disengge solule ntigen t physiologicl temperture, nd the need for cross-linking of criticl mss of CAR receptors on the T cell surfce which is provided y immoilized, ut not solule, mesothelin protein. The P4 nti-mesothelin CAR llows for the direct cytolytic destruction of ntigen-positive tumor cells, however, tumor ntigens re often heterogeneously expressed mong prticulr cncer histologies. Our immunohistochemicl nlysis of multiple serous denocrcinom tumor sections revels disprte regionl mesothelin expression with little to no detectle mesothelin expression in certin tumor res. We find tht ntigen-ctivted P4 CAR T cells my exert effector functions cple of inducing ystnder killing of cncer cells not expressing the trget ntigen. Solule fctors relesed y CAR-ctivted effector cells re indeed le to inhiit the growth of tumor cells lcking cognte ntigen. 39 The mechnism ccounting for CAR-medited ystnder cytotoxicity in our study is unknown, however, our results suggest tht ntigen-less tumor cells within heterogeneous field of mesothelinexpressing tumor my e susceptile to indirect destruction y mesothelin-redirected T cells tht hs previously rected ginst djcent ntigen-positive cncer cells. However, ystnder ctivity my cuse dmge to the surrounding norml tissue. Thus, finetuning of CAR specificity nd ctivity is criticl. P4 CAR T cells control lrge, well-estlished tumors in immunodeficient mice. Consistent with multiple reports, 19,4,41 first genertion CAR possessing only CD3z signling hd modest impct on tumor outgrowth in vivo, while second genertion CAR comprising CD3z fused to CD28 costimultory domin medited tumor regression in vivo. 27 T cells modified to express n irrelevnt nti-cd19 CAR or GFP were unle to lter tumor growth demonstrting the high specificity of the CAR system nd ruling out the possiility of xenogeneity s the source of the tumor response. Persistence of humn T cells ws highest in mice treted with P4 CARs compred with control CD19-28z T cells demonstrting tht ntigen exposure is sufficient to support preferentil CAR-trnsduced T cell engrftment nd persistence in vivo. However, the persistence of P4 CAR T cells ws independent of the ddition of the CD28 costimultory domin, s noted previously. 4 Our results show tht primry humn T-cells expressing fully humn CAR-trgeting mesothelin re highly effective in response controlling lrge, well-estlished tumors. Although the sfety nd efficcy of mesothelin-directed CAR therpy hs yet to e tested in the clinic, the fully humn P4 CAR descried here is well positioned to resist solule protein inhiition, elude trnsgene immunogenicity nd mximize ntitumor efficcy of doptively trnsferred T cells in vivo. 8

9 Humn Mesothelin-specific CAR Therpy of Cncer Mterils nd Methods Anti-mesothelin CAR construction. The ptor2 plsmid contining the nti-mesothelin scfv P4 18,42 ws used s templte for PCR mplifiction of 795-p P4 frgment using the following primers: 5 -GCGAGATCTCAG GTACAGCTGCAGCAGTC-3 (BglII is underlined) nd 5 -CGCGCTAGC GGAGAGGACGGTCAGTTGGG-3 (NheI is underlined). The resulting PCR product contining BglII site on the 5 - end nd NheI site on the 3 - end ws digested with the relevnt enzymes. Third genertion self-inctivting lentivirl expression vectors pelns previously descried were digested with BmHI nd NheI to crete comptile cohesive ends nd gel purified. The digested PCR products were then inserted into the pelns vector contining CD3z or CD28-CD3z T cell signling domins in which trnsgene expression is driven y the elongtion fctor-1α (EF-1α) promoter. The resulting construct ws designted pelns-p4-z/cd28-z. Recominnt lentivirus production. High-titer repliction-defective lentivirl vectors were produced nd concentrted s previously descried. 293T humn emryonic kidney cells were seeded t per T-15 tissue culture flsk 24 hours efore trnsfection. All plsmid DNA were purified using the QIAGEN Endo-free Mxi prep kit (Qigen, Vlenci, CA). Cells were trnsfected with 7 μg pvsv-g (VSV glycoprotein expression plsmid), 18 μg of μg prsv.rev (Rev expression plsmid), 18 μg of pmdlg/p.rre (Gg/ Pol expression plsmid), nd 15 μg of pelns trnsfer plsmid using Express Inn (Open Biosytems, Huntsville, AL). The virl superntnt ws hrvested t 24 nd 48 hours post-trnsfection. Virl prticles were concentrted nd resuspended in.4 ml y ultrcentrifugtion for 3 hours t 25, rpm with Beckmn SW28 rotor (Beckmn Coulter, Fullerton, CA). Humn T cell trnsduction. Primry humn T cells, which were purchsed from the Humn Immunology Core t University of Pennsylvni, were isolted from helthy volunteer donors following leukpheresis y negtive selection. All specimens were collected under University Institutionl Review Bord-pproved protocol, nd written informed consent ws otined from ech donor. T cells were cultured in complete medi (RPMI 164 supplemented with 1% het-inctivted fetl ovine serum, 1 U/ml penicillin, 1 μg/ml streptomycin sulfte, 1-mmol/l HEPES), nd stimulted with nti-cd3 nd nti-cd28 mas coted eds (Invitrogen, Crlsd, CA) s descried. 43 Twelve to twenty-four hours fter ctivtion, T cells were trnsduced with lentivirl vectors t multiplicity of infection of ~5 1. CD4 + nd CD8 + T cells used for in vivo experiments were mixed t 1:1 rtio, ctivted, nd trnsduced. Humn recominnt interleukin-2 (Novrtis, St Louis, MO) ws dded every other dy to 5 IU/ml finl concentrtion. Cell density of cells/ml ws mintined. Rested engineered T cells were djusted for identicl trnsgene expression efore functionl ssys. Cell lines. Lentivirus pckging ws performed in the immortlized norml fetl renl 293T cell line purchsed from Americn Type Culture Collection (Mnsss, VA). Humn cell lines used in immune-sed ssys include the estlished humn ovrin cncer cell lines SKOV3,, OVCAR3, OVCAR5, OVCAR-2, nd C3. For ioluminescence ssys, trget cncer cell lines were trnsfected to express firefly luciferse (fluc), enriched y ntiiotic selection positive expression y ioluminescence imging. For specificity controls, the mouse mlignnt mesotheliom cell line, AE17, ws trnsduced with lentivirus to express humn mesothelin (AE17-M). K562, humn erythroleukemic cell line, CD19-expressing K562 (CD19 + K562) cells, nd mesothelin/cd19-expressing K562 (Meso + CD19 + K562) cells were kindly provided y Crl June (University of Pennsylvni). 293T cells nd tumor cell lines were mintined in RPMI-164 (Invitrogen) supplemented with 1% (vol/vol) het-inctivted FBS, 2 mmol/l L-glutmine, nd 1 μg/ml penicillin, nd 1 U/ml streptomycin. All cell lines were routinely tested for mycoplsm contmintion. Cytokine relese ssys. Cytokine relese ssys were performed y coculture of T cells with either solule or immoilized yest-derived recominnt mesothelin or trget cells per well in triplicte in 96-well round ottom pltes in finl volume of 2 μl of T cell medi. After 2 24 hours, coculture superntnts were ssyed for presence of IFN-γ using n ELISA Kit, ccording to mnufcturer s instructions (Biolegend, Sn diego, CA). Vlues represent the men of triplicte wells. IL-2, IL-4, IL-1, tumor necrosis fctor-α nd MIP-1α cytokines were mesured y flow cytometry using Cytokine Bed Arry, ccording to mnufcturer s instructions (BD Biosciences, Sn Jose, CA). Cytotoxicity ssys. 51 Cr relese ssys were performed s descried. 44 Trget cells were leled with 1 μci 51 Cr t 37 μc for 1.5 hours. Trget cells were wshed three times in phosphte-uffered sline (PBS), resuspended in complete medi t 1 5 vile cells/ml nd 1 μl dded per well of 96-well V-ottom plte. Effector cells were wshed twice in complete medi nd dded to wells t the given rtios. Pltes were quickly centrifuged to settle cells, nd incuted t 37 C in 5% CO 2 incutor for 4 or 18 hours fter which time the superntnts were hrvested, trnsferred to LumPlte nd counted using 145 Microet Liquid Scintilltion Counter (Perkin-Elmer, Wlthm, MA). For the ystnder cytotoxicity ssys, 51 Cr leled mesothelin-negtive trget cells were mixed with unleled mesothelin-positive trgets cells t rtio 1:1 for finl concentrtion of 1 5 vile cells/ml efore eing incuted with the effector T cells t the given rtios. Spontneous 51 Cr relese ws evluted in trget cells incuted with medium lone. Mximl 51 Cr relese ws mesured in trget cells incuted with sodium dodecyl sulfte t finl concentrtion of 2% (vol/vol). Percent specific lysis ws clculted s (experimentl spontneous lysis/mximl spontneous lysis) 1. Mesurement of solule secreted mesothelin. Cell culture superntnts, scites fluids, nd NSG mice-derived serum were nlyzed for their mesothelin concentrtion using the Humn Mesothelin DuoSet Kit (R&D Systems, Minnepolis, MN) ccording to the mnufcturer s instructions. Blocking ssys. Mesothelin-positive tumor cells were seeded t wells of 96-well U-ottom plte t cell/2 μl nd cultured overnight. The next dy the medi ws removed nd cells were wshed once with PBS or left untreted. P4 CAR T cell were then dded to the tumor cells left untreted or to whom fresh medium ws dded nd the percentge of inhiition of tumor recognition ws clculted. To clculte the ckground inhiition possily derived from immunoinhiitory cytokines nti-cd19- BBZ T cells were resuspended in either conditioned medi prior their ddition to dhered nd wshed K562-CD19 cells. For the locking experiment using recominnt mesothelin P4 CARs were incuted with the or AE17M cells in the presence of 5 μg/ml recominnt mesothelin nd the IFN-γ secretion ws determined fter n overnight coculture. Immunohistochemistry. Institutionl review ord pprovl ws otined. We retrieved records from 18 consecutive ptients with metsttic ppillry serous ovrin cncer (FIGO stge IIB nd ove) undergoing primry resection t our institution etween 25 nd 28. Slides were reviewed nd nnotted nd prffin-emedded tissue locks were selected to construct tissue microrry of primry nd metsttic tumors. Including primry sites nd metstses, totl of 72 tumor deposits were represented on the rry. A men of 3.7 sites were included per ptient. The most common metsttic sites included omentum, peritoneum (e.g., cul-de-sc), uterine seros, nd owel wll. For ech lock, triplicte.6 mm cores of tumor were plced on tissue microrry. Prffin sections of the rry (5 µm) were stined with nti-mesothelin (1:1, ct #MS-132; NeoMrkers, Fremont, CA) ccording to stndrd protocols in our lortory. Mesothelin expression in ech core ws scored y light microscopy t 2 mgnifiction using semiquntittive scle rnging from to 3. Antigen dissocition ssy. P4 CAR or untrnsduced T cell were hrvested, wshed once with fluorescent-ctivted cell sorting uffer nd stined with.5 μg/ml yest-derived iotinylted-mesothelin protein 18 for 3 minutes t 4 C. Then the cells were wshed two times efore the ddition of 5 μg/ml Moleculr Therpy 9

10 Humn Mesothelin-specific CAR Therpy of Cncer The Americn Society of Gene & Cell Therpy of noniotinylted mesothelin competitor nd incution t 4 C or 37 C for different time points ( < t > 6 hours). At indicted time points cells were removed from 4 C or 37 C, wshed gin, leled with iotinylted streptvidin, wshed nd nlyzed for percent mesothelin-positive nd men fluorescence intensity y flow cytometry. Xenogrft model of ovrin cncer. All nimls were otined from the Stem Cell nd Xenogrft Core of the Armson Cncer Center, University of Pennsylvni. Six to 12-week-old NSG mice were red, treted, nd mintined under pthogen-free conditions in-house under University of Pennsylvni IACUC pproved protocols. For n estlished ovrin cncer model, 6- to 12-week-old femle NSG mice were inoculted sucutneously with fluc + cells on the flnk on dy. After tumors ecome plple t out 6 weeks, humn primry T cells were ctivted, nd trnsduced s descried ove. After 2 weeks of T cell expnsion, when the tumor urden ws ~15 25 mm 3, mice were injected intrtumorlly with T cells. Tumor dimensions were mesured with clipers, nd tumor volumes clculted using the formul V = 1/2(length width 2 ), where length is the gretest longitudinl dimeter nd width is the gretest trnsverse dimeter. Animls were imged efore T cell trnsfer nd out every week therefter to evlute tumor growth. Photon emission from fluc + cells ws quntified using the Living Imge softwre (Xenogen, Almed, CA) for ll in vivo experiments. For the intrperitonel model of ovrin cncer, 8 to 12-week-old NSG mice were injected intrperitonelly with fluc + cells. Two weeks fter peritonel inocultion, mice ering estlished tumors received intrvenously dministered T cells. Mice were scrificed when they ecme distressed nd moriund. To monitor the extent of tumor progression, the mice were imged weekly. In ll models, five mice were rndomized per group efore tretment. Bioluminescence imging. Tumor growth ws lso monitored y Bioluminescent imging. Bioluminescent imging ws performed using Xenogen IVIS imging system nd the photons emitted from fluc- expressing cells within the niml ody were quntified using Living Imge softwre (Xenogen). Briefly, mice ering fluc + tumor cells were injected intrperitonelly with D-luciferin (15 mg/kg stock, 1 μl of D-luciferin per 1 g of mouse ody weight) suspended in PBS nd imged under isoflurne nesthesi fter 5~1 minutes. A pseudocolor imge representing light intensity (lue, lest intense; red, most intense) ws generted using Living Imge. Bioluminescent imging findings were confirmed t necropsy. Flow cytometric nlysis. The following monoclonl ntiodies were used for phenotypic nlysis: PE mouse nti-humn CD3; FITC ntihumn CD4; APC nti-humn CD8; PE-nti-humn CD45; APC-Cy7 nti-humn CD69; FITC nti-humn CD17; nd FITC nti-humn CD17. 7-Aminoctinomycin D (7-AAD) ws used for viility stining. All monoclonl ntiodies were purchsed from BD Biosciences. In T cell trnsfer experiments, peripherl lood ws otined vi retro-oritl leeding nd stined for the presence of humn CD45, CD4, nd CD8 T cells. After gting on the humn CD45 + popultion, the CD4 + nd CD8 + susets were quntified using TruCount tues (BD Biosciences) with known numers of fluorescent eds s descried in the mnufcturer s instructions. Tumor cell surfce expression of mesothelin ws performed using solule P4 nti-mesothelin scfv followed y PE-leled streptvidin. T cell surfce expression of the P4 or nti-cd19 CAR ws evluted using V5-tgged recominnt mesothelin followed y Alex-647 conjugted nti-v5 monoclonl ntiody (AD Serotec, Rleigh, NC) or iotinylted protein L (GenScript, Pisctwy, NJ) followed y PE-leled streptvidin respectively. Acquisition nd nlysis ws performed using BD FACS CANTO II with DIVA softwre (BD Biosciences). Degrnultion ssy. The degrnultion ssy ws performed s erlier descried 22 with minor modifictions. Trget cells (1 1 5 ) were cocultured with n equl numer of effector cells in.1 ml per well in 96-well plte in triplicte. Control wells contined either T cells lone. Anti-CD17 nd A Anti-CD17 (1 μl per well) or IgG1 conjugted to FITC (BD Biosciences) were dded in ddition to 1 μl/smple of monensin (BD Biosciences) nd incuted for 4 5 hours t 37 C. Cells were wshed two times with PBS, stined for expression of the P4 CAR, CD8, nd CD69 nd nlyzed on FACS DIVA II. Sttisticl nlysis. Sttisticl nlysis ws performed using two-wy repeted mesures nlysis of vrince for the tumor urden (tumor volume, photon counts). Student s t-test ws used to evlute differences in solute numers of trnsferred T cells, cytokine secretion, nd specific cytolysis. Kpln Meier survivl curves were compred using the log-rnk test. GrphPd Prism 4. (GrphPd Softwre, L Joll, CA) ws used for the sttisticl clcultions. P <.5 ws considered significnt. SUPPLEMENTARY MATERIAL Figure S1. Antigen-specific killing of mesothelin positive tumor cells y P4 CAR T cells in 18-hour 51 Cr-relese ssy. Figure S2. Antigen-specific killing of CD19 + trgets y nti-cd19 CAR T cells 4 nd 18 hours post co-culture. Figure S3. Expression levels of mesothelin protein mong the different serous ovrin denocrcinom sites in individul cses. Figure S4. Mesothelin is not trnsferred from ntigen-positive to ntigen-negtive cells. Figure S5. In vitro cytokine secretion nd in vivo persistence of first nd second genertion P4 CAR T cells. ACKNOWLEDGMENTS The uthors grtefully cknowledge Drs Crl H. June, Michel Klos, Gwenn Dnet-Desnoyers, Steven M. Aleld, Edmund K. Moon, Michel C. Milone, nd De-Gng Song, s well s Denrd Dngj, for helpful suggestions, discussions nd regents. This work ws supported y grnts from the W.W. Smith Chritle Trust, the Sndy Rollmn Ovrin Cncer Foundtion nd the Joint Fox Chse Cncer Center, nd University of Pennsylvni Ovrin Cncer SPORE (P5 CA83638). REFERENCES 1. 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