Antitumor Effects of Chimeric Receptor Engineered Human T Cells Directed to Tumor Stroma

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1 The Amerin Soiety of Gene & Cell Therpy originl rtile Antitumor Effets of Chimeri Reeptor Engineered Humn T Cells Direted to Tumor Strom Sunith Kkrl 1,2,3, Kevin KH Chow 1,2,3, Melind Mt 1,2,4, Donld R Shffer 1,2,3, Xio-Tong Song 1,4, Meng-Fen Wu 5, Ho Liu 5, Lis L Wng 2,6, Dvid R Rowley 7, Klus Pfizenmier 8 nd Stephen Gottshlk 1,2,3,4,6 1 Center for Cell nd Gene Therpy, Texs Children s Hospitl, The Methodist Hospitl, Bylor College of Mediine, Houston, Texs, USA; 2 Texs Children s Cner Center, Texs Children s Hospitl, Bylor College of Mediine, Houston, Texs, USA; 3 Interdeprtmentl Progrm in Trnsltionl Biology nd Moleulr Mediine, Bylor College of Mediine, Houston, Texs, USA; 4 Deprtment of Pthology nd Immunology, Bylor College of Mediine, Houston, Texs, USA; 5 Biosttistis Shred Resoure Dn L Dunn Center, Bylor College of Mediine, Houston, Texs, USA; 6 Deprtment of Peditris, Bylor College of Mediine, Houston, Texs, USA; 7 Deprtment of Moleulr nd Cellulr Biology, Bylor College of Mediine, Houston, Texs, USA; 8 Institute of Cell Biology nd Immunology, University of Stuttgrt, Stuttgrt, Germny Cner-ssoited firolsts (CAFs), the priniple omponent of the tumor-ssoited strom, form highly protumorigeni nd immunosuppressive miroenvironment tht medites therpeuti resistne. Co-trgeting CAFs in ddition to ner ells my therefore ugment the ntitumor response. Firolst tivtion protein-α (FAP), type 2 dipeptidyl peptidse, is expressed on CAFs in mjority of solid tumors mking it n ttrtive immunotherpeuti trget. To trget FAP-positive CAFs in the tumor-ssoited strom, we genetilly modified T ells to express FAP-speifi himeri ntigen reeptor (CAR). The resulting FAP-speifi T ells reognized nd killed FAP-positive trget ells s determined y proinflmmtory ytokine relese nd trget ell lysis. In n estlished A549 lung ner model, doptive trnsfer of FAP-speifi T ells signifintly redued FAP-positive stroml ells, with onomitnt derese in tumor growth. Comining these FAP-speifi T ells with T ells tht trgeted the EphA2 ntigen on the A549 ner ells themselves signifintly enhned overll ntitumor tivity nd onferred survivl dvntge ompred to either lone. Our study undersores the vlue of o-trgeting oth CAFs nd ner ells to inrese the enefits of T-ell immunotherpy for solid tumors. Reeived 8 Novemer 212; epted 1 April 213; dvne online pulition 4 June 213. doi:1.138/mt INTRODUCTION The tumor-ssoited strom hs grnered inresing ttention for its role in inititing nd sustining tumor growth. Oupying up to 9% of the tumor mss, 1 the strom is omposed of heterogeneous ell types, of whih ner-ssoited firolsts (CAFs) re prepondernt. 2 CAFs support tumor progression diretly through prrine seretion of ytokines, growth ftors nd so on, 3 nd indiretly y mediting resistne to hemotherpy, rdiotherpy, nd immunotherpy. 4,5 Additionlly, therpies direted to ner ells often fil to erdite CAFs, whih n reinstte tumorigeni milieu nd fvor reurrene. 6,7 It is now evident tht CAFs express mrkers tht distinguish them from their norml ounterprts, 8 llowing them to e seletively trgeted. One suh mrker is firolst tivtion protein-α (FAP), type 2 dipeptidyl peptidse originlly isolted from CAFs in humn sroms. 9 FAP expression ws susequently deteted on ner-ssoited firolsts in over 9% of ommon epithelil ners inluding oloretl, rest, pnreti, skin, nd lung 1 tumors, nd is often orrelted with poor prognosis. 11 Seletive ltion of FAP-positive stroml ells in trnsgeni mouse model permitted immunologil ontrol of tumor growth, inditing their signifint immunosuppressive funtion in the miroenvironment. 12 Trgeting FAP-positive ner-ssoited firolsts therefore presents n ttrtive strtegy to ugment urrent immunotherpies. While severl groups hve evluted the use of FAP-trgeted vines, 13 no study so fr hs determined whether the doptive trnsfer of FAP-speifi T ells enhnes urrent T-ell therpy pprohes for solid tumors. Here we report the development of FAP-speifi himeri ntigen reeptor (CAR) to rediret T ells to FAP-positive nerssoited firolsts. These T ells reognize nd kill FAP-positive trgets in vitro nd suppress tumor growth in oth loo-regionl nd systemi tumor models. When omined with CAR-T ells trgeting tumor-ssoited ntigen, they signifintly enhned ntitumor effets in omprison to nimls treted with FAP- or tumor-speifi T ells lone. RESULTS Genertion of FAP-speifi CAR modified T ells We generted seond genertion CAR speifi for oth murine nd humn FAP (mhfap) using the single hin vrile Correspondene: Stephen Gottshlk, Center for Cell nd Gene Therpy, Bylor College of Mediine, 112 Btes Street, Suite 177, Houston, TX 773, USA. E-mil: smgotts@txh.org Moleulr Therpy vol. 21 no. 8, ug

2 FAP-speifi T Cells for Tumor Strom Trgeting The Amerin Soiety of Gene & Cell Therpy frgment sfv MO36 (mhfap-car; Figure 1). 14,15 T ells were trnsdued with retrovirl vetor enoding the mhfap-car to generte FAP-speifi T ells. Five dys fter trnsdution, CAR expression ws mesured y flow ytometry using CH2CH3 ntiody. Over 75% of the T ells were CAR positive (n = 5; rnge %; Figure 1) nd ontined oth CD4-positive nd CD8-positive T-ell popultions. FAP-speifi T ells reognize nd kill oth humn nd murine FAP-positive trgets To investigte the funtionlity of FAP-speifi T ells, we used qrt-pcr mplifition nd/or FACS nlysis to mesure the expression of humn FAP y pnel of ell lines, inluding the metstti lung firolst ell line (Hs894), prostte ner-ssoited firolst ell line HPS-19I, 16 melnom (SENMA), nsophryngel rinom (C666.1), gliolstom (U87), pnreti dutl rinom (PL45), lung ner (A549) nd lympholstoid ells (LCLs). All lines expressed FAP exept for A549 nd LCLs (Figure 2,). To demonstrte FAP-speifi reognition of trget ells, we first trnsdued FAP-negtive A549 ells with lentivirl vetor enoding either murine or humn FAP (A549. mfap or A549.hFAP; Figure 2,). We o-ultured tumor ells with FAP-speifi T ells or nontrnsdued (NT) T ells for 24 hours nd mesured proinflmmtory ytokines in the ell ulture superntnts y Multiplex nlysis. FAP-speifi T ells reognized oth murine (A549.mFAP) nd humn (A549.hFAP) ell lines s evidened y the relese of proinflmmtory ytokines suh s IFNγ nd TNFα with no relese on exposure to FAP-negtive A549 trget ells (P <.5). While FAP-speifi T ells lso sereted IL-6 nd IL-13, they did not serete signifint mounts of GM-CSF, IL-2, IL-4, IL-5, IL-7, or IL-8. Similrly, CD4 Counts FAP sfv 5 LTR CH2CH3 TM CD28 ζ-hin 3 LTR (mhfap) 47.6% CD8 FAP.CAR 44.% Counts 1.4% CD4 + FAP.CAR 9.5% CD % 95.6% Figure 1 Genertion of FAP-speifi T ells. () Shemti of the FAPspeifi CAR retrovirl vetor. () Representtive dt from one donor showing CAR expression nd T-ell susets. produed little to no proinflmmtory ytokines in response to either FAP-positive or FAP-negtive trget ells (Figure 2, Supplementry Figure S1). In stndrd 51 Cr relese ssy, FAP-speifi T ells hd signifint ytotoxi tivity ginst A549.mFAP nd A549.hFAP, wheres did not (P =.5; Figure 2d). To provide dditionl evidene tht the expression of FAP-CARs is ritil for the oserved effet, we performed ytotoxiity ssy using T ells expressing prostte stem ell ntigen (PSCA)-speifi CAR. 17 While PSCA-speifi T ells killed PSCA-trnsdued K562 ells, PSCA-speifi T ells did not kill prentl A549 ells or A549 ells-trnsdued with murine or humn FAP (Figure 2e). Hene, T-ell reognition depends on the expression of FAP-speifi CARs y T ells nd the presene of murine or humn FAP on trget ells. We onfirmed the ove findings y using rnge of FAPpositive nd FAP-negtive tumor ell lines. FAP-speifi T ells sereted signifintly more IFNγ (P <.5) nd TNFα (P <.5) thn in o-ulture ssys with FAP-positive ells (SEMNA, U87, Hs894, nd HPS-19I) ut not with FAPnegtive ells (LCL nd A549; Figure 3,). FAP-speifi T ells lso killed FAP-positive (SENMA, U87, Hs894, nd HPS-19I; P <.5) ut not FAP-negtive (LCL, A549) trget ells in stndrd 51 Cr relese ssys (Figure 3). showed little to no ytolyti tivity. Thus FAP-speifi T ells reognize nd kill trgets tht nturlly express FAP. FAP-speifi T ells hve ntitumor tivity in loo-regionl tumor model To ssess the effets of trgeting FAP-positive ner-ssoited firolsts in the tumor-ssoited strom, we first used looregionl tumor model with FAP-negtive LCLs. We trked tumor growth in vivo y seril ioluminesene imging of modified LCLs expressing GFP firefly luiferse fusion protein (LCL.eGFP. fflu). 18 To determine if LCL.eGFP.ffLu indued FAP-positive retive strom in vivo, we injeted severe omined immunodefiieny (SCID) mie suutneously (s..) with LCL.eGFP.ffLu ells, euthnized them t different time points post tumor injetion, nd quntified murine FAP in the tumors y qrt-pcr mplifition. FAP expression ws deteted when mesured three dys post tumor ell injetion (Figure 4). The presene of retive strom ws onfirmed y immunohistohemistry for smooth musle α-tin nd immunofluoresene stining for FAP (Supplementry Figure S2). To investigte the effets of trgeting FAP-positive retive strom on tumor growth, we injeted suutneously n dmixture of LCL.eGFP.ffLu ells nd FAP-speifi T ells or nontrnsdued T ells (n = 1). Bioluminesene imging showed tht FAP-speifi T ells signifintly deresed tumor growth ompred to mie reeiving (P =.3 on dy 4; P =.3 on dy 8; P =.2 on dy 11, nd P =.1 on dy 14; Figure 4,). To exlude in vivo indution of FAP expression on LCL tumor ells themselves, we generted seond FAP-speifi CAR tht only reognizes humn FAP (hfap-car, Supplementry Figure S3) nd would therefore not e retive ginst the mfap present on the murine strom. hfap-speifi T ells hd no signifint ntitumor tivity when injeted with LCL.eGFP.ffLu ells t ny time point (P =.43 on dy 4; P =.64 on dy 8; P =.35 on dy 11, nd P =.17 on dy 14; Supplementry Figure S4,). Hene, indution vol. 21 no. 8 ug. 213

3 The Amerin Soiety of Gene & Cell Therpy FAP-speifi T Cells for Tumor Strom Trgeting Hs894 HPS-19I LCL A549 A549.mFAP A549.hFAP SENMA 5 C666.1 C t sore PL45 U U87 SENMA Hs894 PL45 1 A LCL A549.hFAP A549.mFAP Cell ounts FAP 25, 2, 25, 2, IFNγ (pg/ml) 15, 1, TNFα (pg/ml) 15, 1, 5, 5, A549 A549.mFAP A549.hFAP A549 A549.mFAP A549.hFAP d % Lysis (1:1 E:T rtio) e % Lysis (1:1 E:T rtio) PSCA-T ells A549 A549.mFAP A549.hFAP A549 A549.mFAP A549.hFAP K562.PSCA Figure 2 FAP-speifi T ells reognize nd kill humn or murine FAP-positive ells. () qrt-pcr. () FACS nlysis on pnel of ner ell lines nd ner-ssoited firolst ell lines using humn FAP primers. () NT- or were o-ultured with FAP + trget ells (A549.mFAP, A549.hFAP) nd FAP - trgets (A549) t 2:1 effetor: trget (E:T) rtio. The prodution of proinflmmtory ytokines IFN-γ nd TNF-α y T ells ws determined y Milliplex MAP fter 24 hr (n = 4 independent replites; mens + SD). (d,e) FAP-, PSCA-, nd were tested in stndrd 6-hr hromium relese ssys t 1:1 E:T rtio (n = 5; men + SD). P <.5; P <.5. of humn FAP on tumor ells nnot explin the enefit of FAPspeifi T ells in this xenogrft model nd the oserved ntitumor tivity depends insted on the presene of T ells tht reognize nd trget murine FAP. FAP-speifi T ells hve ntitumor tivity in systemi tumor model We next mesured the effets of strom trgeting with FAP-speifi T ells in systemi rther thn loo-regionl tumor model. We used n A549 lung ner model sine ner-ssoited firolsts ply n importnt role in lung ner development 19 nd A549 ells re negtive for FAP expression. We generted n A549. egfp.fflu ell line to llow noninvsive ioluminesene imging. To ssess if A549 ells indue FAP-positive strom in vivo, we injeted A549.eGFP.ffLu ells into the til vein of SCID- Beige (SCID-Bg) mie. Mie were then euthnized t different time points nd mfap in the lungs quntified y qrt-pcr nlysis. mfap expression ws deteted when mesured on dy 4-post Moleulr Therpy vol. 21 no. 8 ug

4 FAP-speifi T Cells for Tumor Strom Trgeting The Amerin Soiety of Gene & Cell Therpy IFNγ (pg/ml) TNFα (pg/ml) 2, 15, 1, 5, LCL A549 U87 SENMA HPS-19I Hs894 2, 15, 1, 5, Murine FAP fold expression % Lysis (1:1 E:T rtio) LCL A549 U87 SENMA HPS-19I Hs894 Figure 3 FAP-speifi T ells reognize nd kill ells expressing FAP from its endogenous promoter. () NT- or were o-ultured with FAP + trget ells (U87, SENMA, HPS-19I, nd Hs894) nd FAP - (LCL nd A549) t 2:1 effetor: trget (E:T) rtio. () The prodution of IFN-γ nd TNF-α y T ells ws determined y Milliplex MAP fter 24 hr (n = 3 independent replites; men ± SD). () FAP- nd were tested in stndrd 6-hr hromium relese ssys t 1:1 E:T rtio (n = 3 independent replites; men ± SD). P <.5. tumor ell injetion (Figure 5). Immunohistohemil nlysis of lungs exised on dy 11 post tumor ell injetion showed tumors with FAP-positive strom. No suh strom ws present in ontrol mie (Supplementry Figure S5). To evlute the ntitumor effets of trgeting FAP-positive ells, A549.eGFP.ffLu ells were injeted intrvenously into SCID-Bg mie on dy. On dy 4, FAP-speifi T ells or were dministered intrvenously in onjuntion with n LCL A549 U87 SENMA HPS-19I Hs Figure 4 FAP-speifi T ells suppress LCL tumor growth. () Indution of murine FAP expression in vivo. Reltive fold expression (2^-ΔΔC t ) ws quntified in omprison to norml skin using murine FAP primers. Eh dot represents single tumor smple from different mie t eh time point. () An dmixture of LCL.eGFP.FFLu ells nd NT-T ells. () were injeted suutneously long with 1,5 U of IL-2 dministered i.p. (n = 1/group) on dy. Bioluminesene signl (rdine = photons/se/m 2 /sr) ws monitored over time. P <.5; P <.5; P <.5. intrperitonel injetion of rhil-2 (1,5 U). A totl of three doses of T ells nd IL-2 were dministered t weekly intervls. Untreted mie served s ontrols. Quntifition of ioluminesent imging showed exponentil tumor growth in ontrol mie reeiving NT-T ells, while mie reeiving FAP-speifi T ells hd signifint derese in tumor growth (FAP vs. Untreted P <.1, FAP vs. NT P =.12 on dy 2; Figure 5 e). Moreover, FAP-speifi T ells signifintly prolonged survivl of mie ompred with the vol. 21 no. 8 ug. 213

5 The Amerin Soiety of Gene & Cell Therpy FAP-speifi T Cells for Tumor Strom Trgeting Murine FAP fold expression 1 1 Pretretment Dy 5 Dy 15 Dy 25 Untreted 1 9 Untreted d e f Perent survivl NT (n = 9) FAP (n = 8) 8 Untreted (n = 9) Figure 5 FAP-speifi T ells derese tumor growth nd improve survivl in n A549 lung ner model. () A549.eGFP.FFLu ells were injeted i.v. Lungs were exised nd qrt-pcr ws used to determine reltive levels of murine FAP expression in the lungs using murine FAP primers. Reltive fold expression (2^-ΔΔCt) ws quntified in omprison to norml lungs. Eh dot represents single lung smple from different mie t eh time point. () A549.eGFP.FFLu ells were injeted i.v. into mie on dy nd treted i.v. with nd 1, 5 U IL-2 dministered i.p. on dy 4. Tretment ws repeted two more times week prt. Untreted mie nd mie treted with served s ontrols. Tumor progression ws followed y in vivo ioluminesene imging. Imges of representtive nimls re shown. ( e) Solid lines represent eh individul mouse. (f) Kpln Meier survivl urve of untreted mie (n = 9) or those treted with FAP (n = 8) or (n = 9). untreted ohort or those treted with (P <.1; Figure 5f). Tretment with hfap-speifi T ells hd no effets similr to the NT-ontrol T ells (Supplementry Figure S6), inditing tht the ntitumor effet is result of trgeting mfap-positive strom. To provide diret evidene tht FAP-speifi T ells kill mfap-positive ner-ssoited firolsts we injeted mie intrvenously with A549.eGFP.ffLu ells followed y FAP-speifi or on dy 4. mfap expression in the lungs ws determined 48 hours lter. FAP-speifi T ells signifintly deresed mfap expression in omprison to (P =.43; Figure 6). We then injeted mie intrvenously with A549.eGFP.ffLu ells, nd on dy 4 we exised the lungs nd dissoited them to single ell suspensions. Cells were susequently o-ultured with FAP-speifi or for 72 hours efore FACS nlysis for GFP-positive ner ells nd GFPnegtive/FAP-positive ner-ssoited firolsts. FAP-speifi T ells eliminted FAP-positive ner-ssoited firolsts, while did not (P <.5; Figure 6,). While mfap-speifi T ells inhiited tumor growth, the tumors eventully progressed. To disover if this espe is due to limited T-ell persistene, A549 were injeted intrvenously into 15 mie on dy. On dy 4, egfp.ffluexpressing FAP-speifi T ells (or) egfp.fflu-expressing hfap-speifi T ells (or) egfp.fflu-expressing NT-T ells were injeted intrvenously into mie (5 nimls per group; Supplementry Figure S7). Additionlly, five nontumor ering mie reeived egfp.fflu-expressing FAP-speifi T ells s ontrols. All nimls reeived n intrperitonel injetion of rhil-2 (1,5 U). in vivo imging system ws used to follow T-ell persistene. FAP-speifi T ells homed to lungs, where they expnded nd persisted for one week. In ontrst, h nd disppered within 48 hours fter dministrtion s did FAP-speifi T ells tht were injeted into nontumor ering mie (Supplementry Figure S7). Thus tumor progression is most likely due to limited T-ell persistene in vivo. The in vivo experiments onduted so fr demonstrted the ntistroml tivity of FAP-speifi T ells in the sene of overt toxi effets. To provide dditionl sfety dt, tumor-ering mie were injeted on dy 4 with egfp.fflu-expressing FAP-speifi T ells. Forty-eight hours post injetion mie were euthnized nd lungs, one mrrow, spleen, liver, nd kidneys were sujeted to pthologil nlysis. Untreted mie (n = 5) served s ontrols. Pthologil nlysis reveled mixed inflmmtory ells omposed predominntly of neutrophils, infiltrting the perivsulr nd perironhil sites of mie injeted with FAP-speifi T ells tht were sent in untreted ontrols. No histologil differenes were seen in other tissues (Supplementry Figure S8). Similrly, skin wound heling ssy performed one dy prior to T-ell injetion of tumor-ering mie showed no differene etween FAPspeifi T ells nd (n = 5; Supplementry Figure S8,). Moleulr Therpy vol. 21 no. 8 ug

6 FAP-speifi T Cells for Tumor Strom Trgeting The Amerin Soiety of Gene & Cell Therpy Murine FAP fold expression FAP Isotype 15 1 % FAP + ells 5 versus tumor GFP Norml Tumor versus tumor Figure 6 FAP-speifi T ells reognize nd kill murine FAP-positive stroml ells. () A549.eGFP.FFLu were injeted i.v. on dy. On dy 4 mie were treted with NT- or dministered i.v. long with 1,5 U IL-2 dministered i.p. Lungs were exised 48 hr post tretment nd reltive fold expression (2^-ΔΔCt) ws quntified in omprison to norml lungs y qrt-pcr using murine FAP primers. Dt re mens otined from six lung speimens in eh tretment group. Four norml lung speimens were used s ontrol P <.5. () A549.eGFP. FFLu ells were injeted i.v. into SCID-Beige mie on dy. Lungs were exised on dy 4, dissoited to form single ell suspensions nd oultured with NT- or t 2:1 E:T rtio or left untreted. 72 hr lter the presene of FAP-positive ells nd GFP-positive tumor ells ws determined y FACS nlysis. Representtive dt for one suh single ell suspension is shown. () Perentge FAP+ ells were determined for 25,-gted events. Eh symol represents one lung speimen. P <.5. FAP FAP Untreted Tumor Differentil gene expression is indued in lungs of mie treted with FAP-speifi T ells ompred to To explore the hnges in the lung miroenvironment ssoited with the ntitumor response, we exised lungs from mie reeiving FAP-speifi or 48 hours post tretment. After ffymetrix nlysis (n = 6 replites for eh group) using mouse genome 1. ST rrys, we seleted differentilly expressed genes tht were oth 1.5-fold hnge from seline nd signifintly different (P <.5) etween the experimentl (FAP-speifi T ells) nd ontrol group (). Aout 277 genes were upregulted nd 75 genes downregulted in mie treted with FAP-speifi T ells ompred to those reeiving. Pthwy nlysis of the differentilly expressed genes reveled enrihment of genes involved in hemokine nd ytokine ytokine reeptor intertion (P = ), toll-like reeptor pthwy (P = ), nturl killer ell medited ytotoxiity (P =.1), Jk-STAT pthwy (P =.44), B-ell reeptor signling (P =.266), ntigen proessing nd presenttion pthwy (P =.329), omplement nd ogultion sdes (P =.238), RIG-1-like signling pthwy (P =.238), pthwys in ner (P =.382dy), type 2 interferon signling (P = ), IL-2 (P =.382) nd IL-3 reeptor signling (P =.9), nd EGFR1 signling pthwy (P =.366) s speified y Kyoto Enylopedi of Genes nd Genomes (KEGG) nd wikipthwys dtse (Supplementry Tle S1). Co-trgeting ner ells nd CAFs results in enhned ntitumor effets We next determined if the ntitumor effets oserved with FAPspeifi T ells ould e further enhned y dding T ells diretly trgeted to the tumor ells themselves. We trgeted the A549 tumorssoited ntigen erythropoietin-produing heptoellulr rinom A2 (EphA2), whih is lso overexpressed y primry lung tumor ells. 2 T ells expressing n EphA2-speifi CAR were generted s previously desried. 21 EphA2-speifi CAR ws expressed y medin of 36.6% of vetor-treted T ells (rnge %; n = 5; Supplementry Figure S9). In funtionl ssys, EphA2-speifi T ells reognized nd killed EphA2 positive trgets (NCI-H1299, A549, nd K562. hepha2) ut not EphA2-negtive K562 ells (Supplementry Figure S9,). We injeted A549.eGFP.ffLu ells intrvenously into SCID-Bg mie, nd then on dy 7 we injeted these nimls intrvenously with FAP-speifi T ells (n = 5), EphA2- speifi T ells (n = 5) or n dmixture of FAP-speifi T ells nd EphA2-speifi T ells (n = 5) long with one intrperitonel injetion of rhil-2 (1,5 U). A totl of three doses of T ells nd IL-2 were dministered t weekly intervls. We monitored tumor growth y ioluminesene imging, nd untreted mie served s the ontrol. After four weeks, FAP- nd EphA2-speifi T ells hd signifint ntitumor tivity ( vs. untreted P =.4; EphA2-T ells vs. untreted P =.267). However, the omintion of FAP- nd EphA2-speifi T ells hd the gretest ntitumor tivity ( + EphA2-T ells vs. P =.2; + EphA2-T ells vs. EphA2-T ells P =.163 on dy 29; Figure 7 e), suffiient to signifintly inrese the survivl of mie reeiving oth FAP-speifi nd EphA2-speifi T ells (P <.1, Figure 7f). These mie did however eventully suum to the disese. To evlute if reurrent tumors onsisted of ntigen loss vrints, the tumors were exised vol. 21 no. 8 ug. 213

7 The Amerin Soiety of Gene & Cell Therpy FAP-speifi T Cells for Tumor Strom Trgeting Pretretment Untreted EphA2-T ells + EphA2-T ells 1 9 Untreted Dy Dy d Dy EphA2-T ells e EphA2-T ells f Untreted (n = 4) FAP (n = 5) EphA2 (n = 5) Perent survivl FAP + EphA2 (n = 5) Figure 7 Co-trgeting CAFs nd ner ells results in n enhned ntitumor response nd improved survivl. () A549.eGFP.FFLu ells were injeted i.v. on dy. On dy 7, mie reeived n i.v. dministrtion of (or) EphA2-T ells, (or) nd EphA2-T ells. All groups reeived n i.p. injetion of 1,5 U IL-2. Tretment ws repeted two more times week prt. Untreted mie served s ontrols. Tumor progression ws followed y in vivo ioluminesene imging. Imges of representtive nimls re shown. ( e) Solid lines represent eh individul mouse (f) Kpln Meier survivl urve of untreted mie (n = 4) or those treted with (n = 5) or EphA2-T ells (n = 5), or FAP nd EphA2-T ells (n = 5). nd nlyzed for the expression of EphA2. All tested tumors (n = 6) were EphA2 positive, exluding ntigen loss s potentil explntion (Supplementry Figure S1). DISCUSSION Here we desrie the development nd hrteriztion of novel CAR speifi for FAP, nd show tht T ells expressing this CAR n effetively trget humn nd murine FAP-positive ells. These FAP-speifi T ells produe immunostimultory ytokines nd indue tumor ytolysis when o-ultured with FAP-positive trgets. The FAP-speifi T ells lso trgeted murine FAP-positive ner-ssoited firolsts in humn-tumor xenogrft model diminishing the growth of FAP-negtive tumors. Finlly, the omined trgeting of FAP-positive ner-ssoited firolsts nd the tumor-ssoited ntigen EphA2 on the tumor ells themselves enhnes the ntitumor tivity of either omponent lone. The tumor miroenvironment is omplex milieu with heterogeneous omponents nd is no longer onsidered n innoent ystnder in ner development. 22 Insted, it plys n essentil role in tumor initition, progression, 23 nd medites therpeuti resistne. 24 Trgeting these uxiliry ells in ddition to the ner ells my therefore inrese the effetiveness of nertrgeted therpies. 25 FAP-positive ner-ssoited firolsts re the entrl ellulr omponent of the tumor strom nd mke ritil ontriution to immunosupression, 12 stromgenesis, vsulriztion, nd extrellulr mtrix remodeling. 26 These effets hve een vlidted in prelinil studies, in whih trgeting of FAP with monolonl FAP ntiody onjugted to mytnsinoid, disrupted firolsti nd vsulr strutures with onomitnt derese in tumor growth nd miniml side-effets. 27 Unfortuntely, however, infusion of FAP-speifi monolonl ntiody in phse 2 linil tril showed no ojetive tumor response. 28 Similrly, phse 2 linil trils with smll moleule inhiitors suh s PT-1 tht trget ll memers of the prolylpeptidse fmily inluding FAP filed to show linil enefit. 29 Immunotherpy using vines ginst FAP hs een omined with hemo- or other tumor direted therpies, 13,3,31 ut this pproh hs yet to e evluted in the lini, nd there is onern out the low ojetive response rte of ner vines in generl. 32 Thus, new immunotherpy pprohes re needed to trget FAP-positive ner-ssoited firolsts. The doptive trnsfer of T ells engineered to express CARs hs shown enourging ntitumor tivity, espeilly for treting CD19-positive hemtologil mlignnies While CAR-T ells hve een less widely used to tret solid tumors, reent linil trils provide evidene for effiy, with sustined remissions otined in ptients with tive disese. 37,38 The doptive trnsfer of FAP-speifi CAR-T ells is therefore promising pproh to trget FAP-positive ner-ssoited firolsts in the tumor miroenvironment. Moreover, the FAP-speifi T ells, we used, Moleulr Therpy vol. 21 no. 8 ug

8 FAP-speifi T Cells for Tumor Strom Trgeting The Amerin Soiety of Gene & Cell Therpy n reognize nd kill oth humn nd murine FAP-positive trgets with similr ffinity, due to the ross-retivity of the FAP-speifi sfv CAR. 39 This llowed us to evlute the effets of trgeting the (murine) tumor strom within nd round the (humn) tumor xenogrft in n immunodefiient mouse model. We used this FAP-CAR in two different tumor models: the loo-regionl B-ell lymphom model ws used to test the role of FAP-positive strom during tumor initition, while the nonsmll ell lung rinom xenogrft model ws used to explore the systemi effets of FAP-speifi T ells on estlished tumor. In the first model, (humn) LCLs effiiently indued retive (murine) FAP-positive strom suutneously. If LCLs were o-injeted with FAP-speifi T ells, tumor growth ws signifintly deresed when ompred to ontrol T ells. This effet ws result of trgeting murine FAP in the tumor strom, sine T ells restrited in speifiity for humn FAP hd no ntitumor tivity. In the systemi model, FAP-negtive A549 ells indued FAP-positive strom in the lungs of injeted mie, n effet tht ws diminished y dministrtion of FAP-speifi T ells with resultnt derese in tumor growth nd improved survivl, with no toxiity or negtive effets on wound heling. Reent evidene indites tht FAP expression my not e unique to tumor strom nd my e present on mlignnt ells themselves Although we onfirmed these findings in severl other ner ell lines y using qrt-pcr nd FACS nlysis, the sme ssys filed to detet FAP expression in ny of the lines used in the in vivo models we desrie, nd so the ntitumor effets of FAP-speifi T ells nnot e ttriuted to diret effet on ner ells. Insted, gene expression nlysis of the lungs of tumor-ering mie treted with FAP-speifi T ells showed upregultion of murine genes involved in innte immune pthwys (e.g. hemokine nd tolllike reeptor signling pthwys). Additionlly, downregultion of genes suh s IL-6 suggests tht trgeting FAP-positive strom ould potentilly stunh the supply of solule ftors required for tumor growth. 19 Hene, destrution of FAP-positive strom my llevite the immunosuppressive miroenvironment nd llow infiltrtion of immune ells, whih indiretly medite ntitumor effets. Further mehnisti nlysis wits the development of suitle immunoompetent mouse model. More interestingly, our results lso show tht o-trgeting the tumor diretly (tumor ells) nd indiretly (FAP-positive strom) enhnes the overll ntitumor tivity. To trget A549 ner ells, we used EphA2-speifi T ells tht reognize EphA2 expressed on A549 ells. While expression of EphA2 hs een oserved on the tumor vsulture of severl tumors, 43 qrt-pcr mplifition reveled no indution of murine EphA2 in the A549 model used (Supplementry Figure S11). Injetion of either EphA2- or FAP-speifi T ells on dy 7 post tumor injetion improved survivl over ontrol nimls (inditing tht strom trgeting lone n e s effetive s trgeting tumor ntigen) ut o-injetion of FAP-speifi nd EphA2-speifi T ells ws etter still. Using murine systems expressing model ntigens, investigtors hve shown tht onsistent erdition of solid tumors requires the destrution of oth strom nd ner ells. 44 The urrent dt extend these findings to humn tumors expressing linilly relevnt humn tumor nd stroml ntigens. Long-term follow-up of the nimls showed tumor reurrene, even though the regrowing tumors ontinued to express the trgeted ntigens. FAP-speifi T ells only persisted for up to one week post infusion nd it is likely tht this limited T-ell persistene oupled with the highly ggressive tumor model ounted for the reurrenes oserved. In onlusion, our findings provide prelinil evidene for the therpeuti potentil of FAP-speifi T ells. These T ells destroy FAP-positive strom nd hve ntitumor tivity in vivo. When o-dministered with tumor-speifi T ells, FAP-speifi T ells enhne their ntitumor tivity in vivo. Thus, trgeting FAP with FAP-speifi T ells either lone or s n djuvnt therpy hs the potentil to improve urrent immunotherpeuti pprohes for ner. MATERIALS AND METHODS Tumor ell lines. The non-smll ell lung ner ell lines NCI-H1299 nd A549, hroni myelogenous leukemi ell line K562, metstti lung firolst line Hs894, gliolstom line U87, nd pnreti ner ell line PL45 were purhsed from the Amerin Type Culture Colletion (ATCC; Mnsss, Virgini). HPS-19I ws provided y Dr. Dvid Rowley, outhor. C666.1 ws provided y Dr. Nny R-Tru, University of North Crolin, Chpel Hill, NC. LCLs were generted s previously desried. 45 The Chrterized Cell Line Core Fility t MD Anderson Cner Center, Houston, Texs, performed ell line vlidtion. For the proedures on the genertion of genetilly modified ell lines, see Supplementry Mterils nd Methods. Genertion of n FAP-speifi CAR. The mhfap -ross retive single hin vrile frgment (sfv MO36) ws previously generted y phge disply from n immunized FAP/ knok-out mouse. 39 This sfv (designted mhfap) ws suloned into n SFG retrovirl vetor ontining the humn IgG1-CH2CH3 domin, CD28 trnsmemrne domin, nd ostimultory domins derived from CD28 nd the CD3 ζ-hin. 46 The loning of the FAP-speifi CAR ws verified y sequening (Seqwright, Houston, Texs). The PSCA-speifi CAR ws kindly provided y Dr. Jun Ver, Center for Cell nd Gene Therpy, Bylor College of Mediine, Houston, Texs. Retrovirus prodution nd trnsdution of T ells. CAR expressing T ells were generted s previously desried. 21 Briefly, PBMCs, otined from helthy donors in ordne to protools pproved y the Institutionl Review Bord of Bylor College of Mediine, were stimulted on OKT3 (Ortho Bioteh, Bridgewter, New Jersey) nd CD28 (Beton Dikinson, Mountin View, Cliforni) ntiodies-oted non-tissue ulture treted 24-well pltes. Humn interleukin-2 (IL-2; Proleukin; Chiron, Emeryville, Cliforni) ws dded to ultures on dy 2, nd on dy 3 T ells were trnsdued with the retrovirl prtiles on RetroNetin (Clonteh, Mountin View, Cliforni) oted pltes in the presene IL-2. T ells were susequently expnded with IL-2., used s ontrols, were tivted with OKT3/CD28 nd expnded in prllel with IL-2. Flow ytometry. For immunophenotyping, ells were stined with fluoresein-onjugted monolonl ntiodies (Beton Dikinson, Sn Jose, Cliforni) direted ginst CD4, CD8, nd CD56 surfe proteins. Isotype ontrols were immunogloulin G1 fluoresein isothioynte (IgG1-FITC; BD), IgG1 phyoerythrin (IgG1-PE; BD), IgG1 peridinin hlorophyll protein (IgG1-PerCP; BD), nd isotype Cy5 (Jkson ImmunoReserh Lortories, West Grove, Phildelphi). The surfe expression of CAR on T ells ws nlyzed using CH2CH3 Cy5 ntiody (Jkson ImmunoReserh Lortories). For eh smple, 1, ells were nlyzed y FACSCliur instrument (BD, Beton Dikinson, Mountin View, Cliforni) with the Cell Quest Softwre (Beton Dikinson) or with FCS Express softwre (De Novo Softwre, Los Angeles, Cliforni). FAP-positive ells were stined using the sheep polylonl vol. 21 no. 8 ug. 213

9 The Amerin Soiety of Gene & Cell Therpy FAP-speifi T Cells for Tumor Strom Trgeting nti-fap (R&D Systems, In., Minnepolis, Minnesot) for 1 hour followed y donkey nti-sheep IgG (H+L)-PerCP for 3 minutes (R&D Systems) for tumor smples, nd Northern Lights Anti-Sheep IgG-NL 557 for 1 hour (R&D Systems) for ell lines. Ex vivo funtionl nlysis of T ells. NT- nd CAR-T ells were plted t 2:1 rtio with tumor ells. Cytokine relese fter 24 hours of ulture ws mesured using the 13-plex Milliplex MAP (EMD Millipore, Billeri, Msshusetts). Stndrd hromium ( 51 Cr) relese ssys were performed s previously desried. 47 Loo-regionl LCL tumor model. All niml experiments followed protool pproved y the Bylor College of Mediine Institutionl Animl Cre nd Use Committee. Institute for Cner Reserh (ICR)-SCID mie were purhsed from Toni (IrT:ICR-Prkdsid; Fox Chse C.B-17 SCID ICR; Toni, Hudson, New York). Mle 8- to 12-week-old mie were sulethlly irrdited (23 Gy) 48 hours prior to tumor hllenge. An dmixture of LCL.eGFP.ffLu ells with either NTontrol T ells or FAP-speifi T ells in PBS were dministered suutneously in to the flnk region. rhil-2 (1,5 U) ws dministered intrperitonelly. Isofluorne nesthetized nimls were imged using the IVIS system (IVIS, Xenogen Corp., Almed, Cliforni) 1 to 15 minutes fter 15 mg/kg D-luiferin (Xenogen) ws injeted per mouse intrperitonelly. The photons emitted from the luiferse-expressing tumor ells were quntified using Living Imge softwre (Cliper Life Sienes, Hopkinton, Msshusetts). A onstnt region-of-interest ws drwn over the tumor region nd the intensity of the signl mesured s totl photon/ seond/m 2 /sterdin (p/s/m 2 /sr). Animls were initilly imged every two dys, nd one week therefter. Mie were euthnized when tumors eme neroti or when they met euthnsi riteri (weight loss, signs of distress) in ordne with the Center for Comprtive Mediine t Bylor College of Mediine. Systemi A549 tumor model. All niml experiments followed protool pproved y the Bylor College of Mediine Institutionl Animl Cre nd Use Committee. Around 9- to 12-week-old SCID Beige mie were purhsed from Chrles River (CB17.Cg-PrkdsidLystg/Crl; Fox Chse SCID Beige mouse; Chrles River Lortories Interntionl, In., Wilmington, Msshusetts) A549.eGFP.ffLu ells in PBS were injeted intrvenously on dy. To determine the effiy of trgeting FAP-positive murine strom, mie were treted with n intrvenously dministrtion of either or FAP-speifi T ells on dy 4-post tumor hllenge. Untreted nimls served s ontrols. rhil-2 (1,5 U) ws dministered intrperitonelly in ll the groups. To determine the effiy of o-trgeting ner-ssoited firolsts nd ner ells, mie were treted on dy 7 post tumor hllenge with either FAP-speifi T ells or EphA2-speifi T ells or FAP-speifi T ells nd EphA2-speifi T ells. Untreted nimls served s ontrols. rhil-2 (1,5 U) ws dministered intrperitonelly in ll the groups. Animls were imged s desried. Quntittive rel time PCR. RNA ws extrted from ell lines using the RNesy Mini Kit (Qigen, Vleni, Cliforni). The RNesy Lipid Tissue Mini Kit (Qigen) ws used to extrt RNA from exised tumors. Reltive quntifition of FAP mrna expression ws done using TqMn One- Step RT-PCR Mster Mix Regents (Applied Biosystems, Brnhurg, New Jersey) using the primer/proes for murine FAP, humn FAP, murine et tin, nd humn et tin (ll from Applied Biosystems). Gene expression rry nlysis. Aout A549.eGFP.FFLu ells were injeted intrvenously into SCID-Beige mie on dy. On dy 4 mie were treted intrvenously with NT- or long with 1,5 U IL-2 dministered intrperitonelly. Lungs were exised 48 hours post T-ell injetion nd totl RNA ws isolted using RNesy Lipid Tissue Mini Kit (Qigen). Trnsriptionl profiling ws performed using GeneChip Mouse Gene 1. ST Arry (Affymetrix, Snt Clr, Cliforni.). Arrys of tumors treted with NT- or were performed s triplites using six lung smples in eh group. Dt preproessing nd summriztion were performed using Roust Multihip Averge. Sttistil tests of differentil expression were onduted using the moderted pired t-test. The Benjmini Hoherg multiple testing djustment ws pplied in order to ontrol the flse disovery rte. Genes present t signifintly different (P <.5; fold hnge 1.5) levels in tumor ering lungs treted FAP-speifi or were sumitted to IPA softwre (Ingenuity Systems, Redwood, Cliforni) nd nlyzed to unover gene pthwys tht were overrepresented in the dtset. Both trnsriptionl profiling nd nlysis of mirorry dt were performed y Genome explortions USA (Memphis, Tennessee). Sttistil nlysis. All in vitro experiments were performed either in triplite, qudruplite, or in pentuplite. GrphPd Prism 5 softwre (GrphPd softwre, In., L Joll, Cliforni) ws used for sttistil nlysis. Mesurement dt were presented s men ± stndrd devition (SD). The differenes etween mens were tested y nonprmetri Mnn Whitney U test. The signifine level used ws P <.5. For the mouse experiments, tumor rdine dt were log-trnsformed nd summrized using men ± SD t seline nd multiple susequent time points for eh group of mie. Fold hnges in tumor rdine from seline t eh time point were lulted nd ompred etween groups using one-wy Anov. Survivl determined from the time of tumor ell injetion ws nlyzed y the Kpln Meier method nd y the log-rnk test. SUPPLEMENTARY MATERIAL Figure S1. FAP-speifi T ells serete vrile mounts of proinflmmtory ytokines. Figure S2. Retive strom is indued in LCL xenogrfts. Figure S3. T ells speifi for humn FAP (hfap) reognize nd kill hfap-positive trgets. Figure S4. hfap-speifi T ells hve no ntitumor tivity in vivo. Figure S5. mfap is indued in A549 lung ner xenogrfts. Figure S6. hfap-speifi T ells hve no ntitumor tivity in A549 systemi model. Figure S7. FAP-speifi T ells expnd nd persist only in the presene of tumor. Figure S8. Tretment with FAP-speifi T ells does not indue ute orgn dmge or ffet wound heling. Figure S9. EphA2-speifi T ells reognize nd kill humn FAPpositive trgets. Figure S1. Reurrent tumors ontinue to express trget ntigen. Figure S11. Murine EphA2 expression is not indued in A549 lung ner xenogrfts. Tle S1. Differentilly expressed genes in lungs of tumor-ering mie treted with FAP-speifi or. Mterils nd Methods. ACKNOWLEDGMENTS We thnk Clion M. Rooney for helpful disussions nd dvie. We lso thnk LTerri Willims, Troung Dng nd Steven Ressler for their help. K.K.H.C. is Melnik sholr nd ws supported y NIH grnts 5T32HL92332 nd 5T32GM733. This work ws supported y NIH grnts 1R1CA A1 nd P1CA94237, nd y the Adrienne Helis Mlvin Medil Reserh Foundtion through its diret enggement in the ontinuous tive ondut of medil reserh in onjuntion with Bylor College of Mediine nd the Adoptive T-ell Therpy for Lung Cner Progrm. The Chrterized Cell Line Core Fility t MD Anderson Cner Center is funded y NCI # CA The Center for Cell nd Gene Therpy hs reserh ollortion with Celgene nd lueird io. S.K., K.K.H.C., D.R.S., X.T.S., K.P., nd S.G. hve ptent pplitions in the field of T-ell nd gene-modified T-ell therpy for ner. Moleulr Therpy vol. 21 no. 8 ug

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