Characterization and Functional Analysis of scfv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma

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1 originl rticle The Americn Society of Gene & Cell Therpy Chrcteriztion nd Functionl Anlysis of scfv-sed Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Gliom Giedre Krenciute 1 3, Simone Kres 1 3, Dvid Torres 1 3, Meng-Fen Wu 4, Ho Liu 4, Ginpietro Dotti 5, Xio-Nn Li 2,3, Mciej S Lesnik 6, Irin V Blysnikov 6 nd Stephen Gottschlk 1,2,3,7 1 Center for Cell nd Gene Therpy, Texs Children s Hospitl, Houston Methodist, Bylor College of Medicine, Houston, Texs, USA; 2 Texs Children s Cncer Center, Texs Children s Hospitl, Bylor College of Medicine, Houston, Texs, USA; 3 Deprtment of Peditrics, Bylor College of Medicine, Houston, Texs, USA; 4 Deprtment of Biosttistics, Shred Resource Dn L Duncn Cncer Center, Bylor College of Medicine, Houston, Texs, USA; 5 Deprtment of Microiology nd Immunology, University of North Crolin, Chpell Hill, North Crolin, USA; 6 Deprtment of Surgery, The Brin Tumor Center, University of Chicgo, Chicgo, Illinois, USA; 7 Deprtment of Pthology nd Immunology, Bylor College of Medicine, Houston, Texs, USA Immunotherpy with T cells expressing chimeric ntigen receptors (CARs) is n ttrctive pproch to improve outcomes for ptients with gliolstom (GBM). IL13Rα2 is expressed t high frequency in GBM ut not in norml rin, mking it promising CAR T-cell therpy trget. IL13Rα2-specific CARs generted up to dte contin mutted forms of IL13 s n ntigen-inding domin. While these CARs trget IL13Rα2, they lso recognize IL13Rα1, which is rodly expressed. To overcome this limittion, we constructed pnel of IL13Rα2-specific CARs tht contin the IL13Rα2-specific single-chin vrile frgment (scfv) 47 s n ntigen inding domin, short or long spcer regions, trnsmemrne domin, nd endodomins derived from costimultory molecules nd CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive trget cells in coculture nd cytotoxicity ssys with no cross-rectivity to IL13Rα1. However, only IL13Rα2-CAR T cells with short spcer region produced IL2 in n ntigen-dependent fshion. In vivo, T cells expressing IL13Rα2-CARs with short spcer regions nd CD28.ζ, 41BB.ζ, nd CD28.OX4.ζ endodomins hd potent nti-gliom ctivity conferring significnt survivl dvntge in comprison to mice tht received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2- trgeted immunotherpy pproches for GBM nd other IL13Rα2-positive mlignncies. Received 8 August 215; ccepted 23 Octoer 215; dvnce online puliction 1 Decemer 215. doi:1.138/mt IRODUCTION The outcome for gliolstom (GBM) remins poor nd immunotherpy with vccines or GBM-specific T cells is one ttrctive strtegy to improve survivl, since it does not rely on the cytotoxic mechnisms employed y conventionl therpies such s chemotherpy nd rdition. 1 3 Genetic modifiction with chimeric ntigen receptors (CARs) llows for the rpid genertion of tumor-specific T cells. For GBM-trgeted CAR T-cell therpies, severl ntigens re ctively eing pursued including interleukin 13 receptor α2 (IL13Rα2), humn epiderml growth fctor receptor 2 (HER2), epiderml growth fctor vrint III (EGFRvIII), nd erythropoietin-producing heptocellulr crcinom A2 (EphA2) IL13Rα2, cncer testis ntigen, is n idel trget for CAR T-cell therpy for GBM since it is expressed in 5 8% of GBM cells nd gliom-inititing cells, which re resistnt to conventionl therpies, ut not in norml rin IL13Rα2 expression is ssocited with poor prognosis, 15 nd while initil studies indicted tht IL13Rα2 is decoy receptor, more recent studies hve demonstrted tht IL13Rα2 prevents poptosis nd induces TGF-β secretion CARs consist of n ectodomin, which is most commonly derived from single-chin vrile frgment (scfv), spcer region, trnsmemrne domin, nd n endomin Current IL13Rα2-specific CARs do not contin scfvs, ut n IL13-mutein with one or two mino cid sustitutions to preferentilly redirect T cells to IL13Rα While one study showed tht T cells expressing IL13-mutein CARs did not recognize IL13Rα1-positive cells, 22 other studies including ours demonstrted tht IL13-mutein CAR T cells redily recognize IL13Rα1-positive trgets rising concerns of on trget/off cncer toxicity. 7,8 To selectively trget IL13Rα2, we previously hd generted monoclonl ntiody (MA) nd scfv tht specificlly recognized IL13Rα2 (clone 47; ) with high ffinity nd no cross-rectivity to IL13Rα1. 23,24 Here, we report the development of IL13Rα2-specific CARs with -sed ntigeninding domin (IL13Rα2-CARs). We show tht IL13Rα2-CARs require short spcer region for optiml functionlity, nd tht IL13Rα2-CAR T cells re le to recognize nd kill only IL13Rα2- positive nd not IL13Rα1-positive trget cells in vitro. In ddition, IL13Rα2-CAR T cells induce tumor regression in n orthotopic xenogrft mouse model of GBM, which ws ssocited with significnt survivl dvntge. Correspondence: Stephen Gottschlk, Center for Cell nd Gene Therpy, Bylor College of Medicine, 112 Btes Street, Suite 177, Houston, Texs 773, USA. E-mil: smgottsc@txch.org vol. 24 no. 2, fe. 216

2 The Americn Society of Gene & Cell Therpy IL13Rα2-specific T Cells for Gliolstom RESULTS Genertion of IL13Rα2-CAR T cells We initilly generted two retrovirl vectors encoding CARs sed on the IL13Rα2-specific MA clone 47 (Figure 1). 23,24 Both CARs contined N-terminl leder sequence, codonoptimized synthetic gene encoding, spcer region, CD28 trnsmemrne domin, nd signling domins derived from CD28 nd CD3.ζ (Figure 1). As the spcer region, we either used the IgG1 hinge (16 mino cids; short spcer region; IL13Rα2-CAR.) or the IgG1-CH2CH3 domin (239 mino cids; long spcer region; IL13Rα2-CAR.). As controls, LSR nd SSR IL13Rα2-CARs without signling domins were constructed (IL13Rα2-CAR., IL13Rα2-CAR.; Figure 1). CD3/CD28-ctivted T cells from helthy donors were trnsduced with RD114-pseudotyped retrovirl prticles, nd 4 to 5 dys post-trnsduction T-cell phenotype nd CAR expression ws determined y fluorescence ctivted cell sorting (FACS) nlysis. CARs were expressed on the cell surfce with the trnsduction efficiency rnged from 74.1 to 93.3% nd no significnt differences etween constructs (Figure 1,c). Expression of full-length IL13Rα2-CAR. nd IL13Rα2-CAR.LSR. CD28.ζ ws confirmed y western lot using CD3.ζ ntiody for detection (Figure 1d). Western lot nlysis under nonreducing conditions reveled no significnt differences in dimer/multimer formtion etween LSR AND SSR CARs (Supplementry Figure S2). Phenotypic nlysis reveled mixture CD4- nd CD8-positive T cells. While the rtio of CD8- to CD4-positive T cells ws ~3:1 for IL13Rα2-CAR., IL13Rα2-CAR., nd IL13Rα2-CAR. T-cell lines, it ws ~1.5:1 for IL13Rα2-CAR. (Supplementry Figure S1). IL13Rα2-CAR T cells recognize IL13Rα2 ut not IL13Rα1 To initilly determine the specificity of IL13Rα2-CARs, we cultured T cells expressing IL13Rα2-CAR., IL13Rα2-CAR., IL13Rα2-CAR., or IL13Rα2-CAR. on tissue culture pltes tht were uncoted or coted with recominnt proteins IL13Rα1, IL13Rα2, or IL4Rα. Nontrnsduced () T cells nd T cells expressing n IL13mutein-CAR. 1 tht recognizes IL13Rα1 nd IL13Rα2 served s controls. T cells expressing IL13Rα2-CAR. or IL13Rα2-CAR.LSR. CD28.ζ produced significnt levels of IFNγ (n = 4, P <.1) when stimulted with recominnt IL13Rα2 proteins in comprison to IL13Rα1- or IL4Rα-stimulted T cells (Figure 2). In contrst, T cells expressing IL13Rα2-CAR. or IL13Rα2-CAR. produced no IFNγ in response to ll three proteins, indicting tht IFNγ production depends on n intct IL13Rα2-CAR signling domin. IL13Rα2-CAR. T cells lso produced low levels of IFNγ without ctivtion, indicting seline T-cell ctivtion, which ws confirmed y intrcellulr stining for SSR CD28 TM c 1 ns 8 LSR CD28 TM SSR CD28 TM CD28 ζ Positive (%) LSR CD28 TM CD28 ζ Δ CD28.ζ d SSR 76 LSR Cell numer αcd3.ζ 52 αgapdh CAR Figure 1 Genertion of IL13Rα2 CAR T cells. () Scheme of IL13Rα2 CARs. All CARs contined n N-terminl leder sequence, codon-optimized synthetic gene encoding for, spcer region, CD28 trnsmemrne domin, nd signling domins derived from CD28 nd CD3.ζ. Spcer region ws either the IgG1 hinge (16 mino cids; short spcer region; IL13Rα2-CAR.) or the IgG1-CH2CH3 domin (239 mino cids; long spcer region; IL13Rα2-CAR.). nd SSR. IL13Rα2-CARs without signling domins were constructed nd served s controls. (,c) CAR expression ws confirmed using FACS nlysis. Representtive plots () nd summry dt (c) is shown (men %, n = 5 6 per CAR construct). (d) Expression of full-length IL13Rα2-CAR. nd IL13Rα2-CAR. y western lot nlysis using CD3-ζ ntiody. CAR, chimeric ntigen receptor. Moleculr Therpy vol. 24 no. 2 fe

3 IL13Rα2-specific T Cells for Gliolstom The Americn Society of Gene & Cell Therpy phosphorylted CD3.ζ (Supplementry Figure S3). IL13mutein- CAR. T cells produced significnt levels of IFNγ in the presence of IL13Rα1 (n = 4, P <.1) nd IL13Rα2 (n = 4, P <.5) in comprison to T cells. We next confirmed the specificity of IL13Rα2-CAR T cells using cell lines tht were negtive for IL13Rα1 nd IL13Rα2 (Rji), positive for IL13Rα1 (293T-GFP cells), or positive for IL13Rα1 nd IL13Rα2 (, 293T-GFP/IL13Rα2; Supplementry Figure S4). T cells expressing IL13Rα2-CAR., IL13Rα2-CAR., IL13Rα2-CAR., or IL13Rα2-CAR. were cocultured with Rji, 293T-GFP, or 293T-GFP/IL13Rα2 cells. T cells served s controls. After 24 hours, medi ws collected nd the concentrtion of IFNγ nd IL2 ws determined y ELISA. IL13Rα2- CAR. nd IL13Rα2-CAR. T cells produced significnt mounts of IFNγ only in the presence of or 293T-GFP/IL13Rα2 cells (Figure 2) with SSR.CAR T cells producing significnt more IFNγ thn LSR.CAR T cells (n = 6, P <.1). IL13Rα2-CAR. T cells produced lso significnt mounts of IL2 in the presence of 293T-GFP/ IL13Rα2 nd cells, while IL13Rα2-CAR. T cells did not (Figure 2c). T cells nd T cells expressing IL13Rα2-CAR. or IL13Rα2-CAR. produced no IFNγ or IL2 in response to ny trget cells. Finlly, we confirmed the specificity of IL13Rα2-CAR T cells in stndrd cytotoxicity ssys using Rji, 293T-GFP, 293T-GFP/IL13Rα2, (Figure 2d). In ddition, IL13Rα2-CAR T cells killed the IL13Rα2-positive gliom cell line U87 nd primry gliom cells, while IL13Rα2-negtive primry gliom cells were not killed (Supplementry Figure S5). 8, 6, Mutein.CD28.ζ c 6, P <.1 IFNγ pg/ml 4, 2, 1, IL2 pg/ml 4, 2, P <.5 5, Medi IL4R IL 13Rα1 IL 13Rα2 Medi Rji 293T-GFP 293T-GFP/IL 13Rα2 4, d 8 3, P <.1 P <.1 6 P <.1 P <.1 IFNγ pg/ml 2, 1, % Lysis 4 5, 2 ns ns Medi Rji 293T-GFP 293T-GFP/IL 13Rα2 Rji 293T-GFP 293T-GFP/IL 13Rα2 Figure 2 IL13Rα2-CAR T cells relese cytokines fter stimultion with recominnt IL13Rα2 protein or IL13Rα2-positive cells. IL13Rα2-CAR or nontrnsduced () T cells were stimulted with recominnt IL13Rα1, IL13Rα2, or IL4Rα proteins. After 24 hours, IFNγ () ws mesured y ELISA (n = 4). T cells expressing IL13Rα2-CAR constructs, ut not controls, expressed significnt levels of IFNγ (P <.1) when stimulted with recominnt IL13Rα2 protein in comprison to IL13Rα1 nd IL4Rα stimulted T cells. IL13Rα2-CAR T cells were cocultured with Rji, cells, 293T-GFP, nd 293T-GFP/IL13Rα2 t 2:1 E:T rtio. nd CAR.Δ T cells served s controls. (,c) After 24h cytokines (IFNγ, IL2) were mesured y ELISA. () nd 293T-GFP-IL13Rα2 (IFNγ); versus : n = 6, P <.1; versus : n = 6, P <.5. (c) nd 293T-GFP-IL13Rα2 (IL2); versus : n = 4, P <.1; versus : n = 4, NS. (d) Four hours cytotoxicity ssy t n E:T rtio of 1:1 (n = 4). CAR, chimeric ntigen receptor vol. 24 no. 2 fe. 216

4 The Americn Society of Gene & Cell Therpy IL13Rα2-specific T Cells for Gliolstom Genertion of SSR IL13Rα2-CARs with CD28.OX4.ζ, CD28.41BB.ζ, or 41BB.ζ endodomins While the results descried ove demonstrted tht IL13Rα2- CAR T cells only recognize IL13Rα2 s judged y cytokine production nd cytolytic ctivity, they lso highlighted differences etween LSR nd SSR IL13Rα2-CARs. Since only IL13Rα2-CAR. SSRs produced IL2 in the presence of IL13Rα2-positive trget cells, we focused in the next set of experiments on IL13Rα2-CARs with SSRs, nd generted dditionl CARs with CD28.OX4.ζ, CD28.41BB.ζ, or 41BB.ζ endodomins (Figure 3). CAR T cells were generted y retrovirl trnsduction nd CAR expression on the cell surfce ws determined y FACS nlysis (Figure 3,c) nd western lot (Figure 3d). While ll CARs were expressed y T cells s judged y western lot nlysis, IL13Rα2-CAR.SSR. CD28.41BB.ζ ws not expressed on the cell surfce, nd ws excluded from further nlysis. Functionl comprison of IL13Rα2-CAR., IL13Rα2-CAR.SSR.41BB.ζ, nd IL13Rα2-CAR.SSR. CD28.OX4.ζ T cells To compre the ility of IL13Rα2-CAR.SSR T cells to produce IFNγ nd IL2 in response to ntigen exposure, we performed coculture ssy with cells. nd T cells expressing IL13Rα2-CAR. served s controls. All IL13Rα2-CAR.SSRs with functionl endodomins induced IFNγ nd IL2 production in the presence of cells, however IL13Rα2-CAR.SSR.41BB.ζ T cells produced significntly less (n = 5, P <.5) IFNγ in comprison to IL13Rα2-CAR. nd IL13Rα2-CAR.SSR. CD28.OX4.ζ T cells (Figure 4). IL13Rα2-CAR. T cells produced the highest mount of IL2, followed y IL13Rα2- CAR.SSR.41BB.ζ nd IL13Rα2-CAR.SSR.CD28.OX4.ζ T cells. In cytotoxicity ssys, no significnt difference ws oserved etween ll three constructs using Rji, 293T-GFP, 293T-GFP/ IL13Rα2, nd s trgets (Figure 4). Since ll three IL13Rα2-CAR.SSRs T cells with functionl endodomins produced IL2, we tested ll three constructs in our orthotopic gliom xenogrft mouse model in which T cells re directly injected into tumors. 6 The model llows for seril ioluminescence imging since re geneticlly modified to express n egfp.ffluc fusion protein (.egfp.ffluc). On dy,.egfp.ffluc cells were injected stereotcticlly into rins of SCID mice, nd on dy 7 T cells expressing IL13Rα2-CAR.SSR. CD28.ζ, IL13Rα2-CAR.SSR.41BB.ζ, IL13Rα2-CAR.SSR.CD28. OX4.ζ, or IL13Rα2-CAR. were injected intrtumorlly. While mice treted with IL13Rα2-CAR. T cells showed continuous tumor growth within 4 dys of T-cell injection, mice treted with IL13Rα2-CAR.SSR T cells tht hd functionl endodomins did not (Figure 5,). Comprison of ioluminescence imging results reveled no significnt difference etween IL13Rα2-CAR. T cells nd the IL13Rα2-CAR.SSR T cells groups on the dy of T-cell injection. However, mice treted with IL13Rα2-CAR. or IL13Rα2-CAR.SSR.CD28.OX4.ζ T cells hd significntly lower tumor signls s erly s 1 dy post-tretment in comprison to mice treted with IL13Rα2- CAR. T cells (dy 8, P =.12 nd P =.23; Tle 1). This resulted in significnt survivl dvntge of IL13Rα2-CAR.SSR. CD28.ζ or IL13Rα2-CAR.SSR.CD28.OX4.ζ T-cell-treted mice (P =.2 nd P =.92; Figure 5c). While IL13Rα2-CAR. SSR.41BB.ζ SSR CD8α TM 41BB ζ c 1 ns 8 SSR.CD28.OX4.ζ SSR CD28 TM CD28 OX4 ζ Positive (%) 6 4 SSR.CD28.41BB.ζ SSR CD28 TM CD28 41BB ζ 2 41BB.ζ CD28.OX4.ζ CD28.41BB.ζ 41BB.ζ CD28.OX4.ζ CD28.41BB.ζ d 41BB.ζ CD28.OX4.ζ CD28.41BB.ζ Cell numer CAR αcd3.ζ αgapdh Figure 3 Genertion of SSR IL13Rα2-CARs with CD28.OX4.ζ, CD28.41BB.ζ or 41BB.ζ endodomins. () Scheme of SSR IL13Rα2-CARs. (,c) CAR expression ws confirmed using FACS nlysis. Representtive plots () nd summry dt (c) is shown. IL13Rα2-CAR.SSR.CD28.OX4.ζ nd IL13Rα2-CAR.SSR.CD28.41BB.ζ: men: % (n = 4); IL13Rα2-CAR.SSR.CD28.41BB.ζ: men: 4.9% (n = 3). (d) Expression of IL13Rα2-CAR. SSR.41BB.ζ, IL13Rα2-CAR.SSR.OX4.CD28.ζ, nd IL13Rα2-CAR.SSR.41BB.CD28.ζ y western lot nlysis. CAR, chimeric ntigen receptor. Moleculr Therpy vol. 24 no. 2 fe

5 IL13Rα2-specific T Cells for Gliolstom The Americn Society of Gene & Cell Therpy SSR.41BB.ζ SSR.CD28.OX4.ζ SSR.41BB.ζ SSR.CD28.OX4.ζ 6 P <.1 P <.1 IFNγ pg/ml 4, P <.1 4, P <.5 3, 3, 2, 1, IL2 pg/ml 2, 1, P <.1 P <.1 P <.5 % Lysis 4 2 ns ns Medi Medi Medi 293T-GFP 293T-GFP/ IL13Rα2 Figure 4 Comprison of IL13Rα2-CAR., IL13Rα2-CAR.SSR.41BB.ζ, nd IL13Rα2-CAR.SSR.CD28.OX4.ζ T cells. () IL13Rα2-CAR T cells were cocultured with cells t 2:1 E:T rtio. nd CAR.Δ T cells served s controls. After 24 hours, IFNγ nd IL2 ws mesured y ELISA (n = 5); versus (; IFNγ): P <.1; versus SSR.41BB.ζ (; IFNγ): P <.5; versus SSR.CD28.OX4.ζ for (; IFNγ): P <.1; versus (; IL2): P <.1; versus SSR.41BB.ζ (; IL2): P <.1; versus SSR.CD28. OX4.ζ (; IL2): P <.1. () Four hours cytotoxicity ssy t n E:T rtio of 1:1 (n = 4). CAR, chimeric ntigen receptor; ELISA, enzyme-linked immunosorent ssy. SSR.41BB.ζ T-cell-treted mice responded slower resulting in significnt difference etween IL13Rα2-CAR. T-celltreted on dy 14 (P =.5; Tle 1), tretment lso resulted in significnt survivl dvntge (P =.39; Figure 5c). IL13Rα2- CAR. T-cell-treted mice hd the longest medin survivl (84 dys). However, there ws no sttisticl difference in comprison to the medin survivl of IL13Rα2-CAR.SSR.41BB.ζ (63 dys) or IL13Rα2-CAR.SSR.CD28.OX4.ζ (56 dys) T- cell-treted mice. While IL13Rα2-CAR T cells hd potent nti-gliom ctivity, mice eventully developed recurrent glioms. To investigte the etiology of tumor recurrence, cells were isolted from two tumor-ering mice tht hd een treted either with IL13Rα2- CAR. or IL13Rα2-CAR.SSR.CD28.OX4.ζ T cells. FACS nlysis fter short-term culture reveled cell surfce expression of IL13Rα2, nd these cells were redily killed y IL13Rα2- CAR T cells in cytotoxicity ssy (Figure 6). We next determined T-cell persistence y ioluminescent imging of geneticlly modified T cells expressing IL13Rα2-CAR. nd egfp.ffluc (Luc/IL13Rα2-CAR T cells) injected into tumors. T cells were detected for out 6 dys mking limited persistence the most likely explntion for tumor recurrence (Figure 7). DISCUSSION Here, we descrie the development nd chrcteriztion of new scfv-sed CAR, IL13Rα2-CAR tht is specific for IL13Rα2. We show tht T cells expressing this CAR cn effectively trget nd kill IL13Rα2, ut not IL13Rα1-positive trget cells nd tht only IL13Rα2-CARs with SSR induce IL2 production in strictly IL13Rα2-dependent mnner. Finlly, we demonstrte tht IL13Rα2-CAR.SSR T cells hve potent ntitumor ctivity in vivo. IL13Rα2, cncer testis ntigen, is errntly expressed in GBM nd other mlignncies such s melnom, drenocorticl crcinom, colorectl, pncretic, ovrin cncers. 15,25 27 It is promising immunotherpy trget since it is not expressed in most norml tissues nd in one study, using nnostring digitl RNA counting, elonged to the top 1% of 73 evluted tumor ssocited ntigens tht were differentilly expressed etween tumors nd norml tissues. 25 While IL13Rα2 hs een trgeted with immunotoxins, vccines, nd ntigen-specific T cells with encourging results, 1,28,29 few pproches so fr hve used IL13Rα2-specific mas or scfvs s trgeting moieties for therpeutics. 24,3 We constructed IL13Rα2-specific, scfv-sed CARs nd determined the influence of long nd short spcer regions, s well s endodomins on their function. While IL13Rα2-CAR. nd IL13Rα2-CAR. recognized trget cells s judged y IFNγ production, only IL13Rα2-CAR.SSR. CD28.ζ induced IL2 production, indicting etter T-cell ctivtion. We confirmed the inility to induce IL2 expression for one dditionl LSR IL13Rα2-CAR contining CD28.41BB.ζ endodomin (Supplementry Figure S6). Other groups hve lso reported tht the length of the spcer region contriutes to CAR function. 31,32 For exmple, scfvs tht ind to n epitope in close proximity to the cncer cell memrne, require long spcer regions for optiml CAR function in contrst to scfvs tht ind to epitopes distl to the cell memrne. While the precise epitope within the IL13Rα2 molecule for is not defined, 23,24 it inds to the sme epitope s the prentl MA 47 tht competes with IL13 for its inding site. 24 Our finding tht SSR confers optiml CAR ctivity suggests tht the epitope is locted distl to the cell memrne. We constructed four SSR IL13Rα2-CARs with different endodomins, CD28.ζ, 41BB.ζ CD28.OX4.ζ, nd CD28.41BB.ζ. While ll four CARs were expressed s judged y western lot nlysis, however no significnt cell surfce expression ws oserved for IL13Rα2-CAR.SSR.CD28.41BB.ζ. We explored if chnging the trnsmemrne domin from CD28 to CD8α in IL13Rα2-CAR.SSR.CD28.41BB.ζ would result in etter cell surfce expression, however no incresed expression ws oserved (Supplementry Figure S7). Since IL13Rα2-CARs.LSR vol. 24 no. 2 fe. 216

6 The Americn Society of Gene & Cell Therpy IL13Rα2-specific T Cells for Gliolstom Dy 7 Dy 7 Dy 8 Dy 8 Dy 14 Dy 14 Dy 21 Dy 21 Dy 28 Dy 28 Dy 35 Dy 35 SSR.41BB.ζ Dy 7 Dy 7 Dy 8 Dy 8 Dy 14 Dy 14 Dy 21 Dy 21 Dy 28 Dy 28 Dy 35 Dy Rdince Rdince SSR.41BB.ζ SSR.CD28.OX4.ζ Dys posttumor injection Dys posttumor injection SSR.41BB.ζ SSR.CD28.OX4.ζ Rdince Rdince c Survivl (%) SSR.CD28.OX4.ζ Dys posttumor chllenge Dys posttumor injection Dys posttumor injection Figure 5 Tretment of gliom xenogrft with T cells expressing IL13Rα2-CARs results in tumor regression nd improved overll survivl. gliom-ering mice were treted on dy 7 with (n = 9), SSR.41BB.ζ (n = 9), or SSR.OX4.CD28.ζ (n = 9) T cells. CAR T cells (n = 7) served s controls. () Representtive imges for ech group nd () quntittive ioluminescence (rdince = photons/sec/cm2/sr) imging dt for ll mice re shown (dotted lines: individul mice; solid lines: medin). (c) Kpln-Meier survivl nlysis ( versus : P =.2; versus SSR.41BB.ζ: P =.39; versus SSR.OX4.CD28.ζ: P =.92; versus SSR.41BB.ζ: P =.4723; versus SSR.OX4. CD28.ζ: P =.3582; SSR.41BB.ζ versus SSR.OX4.CD28.ζ: P =.8374). CAR, chimeric ntigen receptor. Moleculr Therpy vol. 24 no. 2 fe

7 IL13Rα2-specific T Cells for Gliolstom The Americn Society of Gene & Cell Therpy CD28.41BB.ζ were expressed on the cell surfce (Supplementry Figure S6), our results suggests tht the SSR interferes with efficient CAR trfficking to the cell surfce. Locl or systemic injections of conventionl s well s CAR T cells re ctively eing explored for high-grde gliom. 1,33,34 We focused on locl injections since in our previous study with humn EphA2-CAR T cells in SCID mice we only oserved ntitumor ctivity fter locl nd not fter til vein injection, 5 most likely reflecting the inility of humn T cells to trnsverse the murine lood rin rrier. IL13Rα2-CAR., IL13Rα2-CAR. SSR.41BB.ζ, nd IL13Rα2-CAR.SSR.CD28.OX4.ζ T cells hd potent ntitumor in vivo resulting in significnt survivl dvntge. While mice treted with IL13Rα2-CAR. T cells Tle 1 Tumor signl comprison Tumor signl comprison P * Dy 7 versus SSR.41BB.ζ.917 versus SSR.CD28 ζ.111 versus SSR.CD28.OX4 ζ.917 Dy 8 versus SSR.41BB ζ.835 versus SSR.CD28 ζ.12 versus SSR.CD28.OX4 ζ.23 Dy 14 versus SSR.41BB ζ.5 versus SSR.CD28 ζ.15 versus SSR.CD28.OX4 ζ.15 Dy 21 versus SSR.41BB ζ.1 versus SSR.CD28 ζ.1 versus SSR.CD28.OX4 ζ.12 Dy 28 versus SSR.41BB ζ.51 versus SSR.CD28 ζ.8 versus SSR.CD28.OX4 ζ.34 * Wilcoxon rnk-sum test. hd the longest medin survivl in comprison to IL13Rα2-CAR. SSR.41BB.ζ or IL13Rα2-CAR.SSR.CD28.OX4.ζ T-cell-treted mice, this difference did not rech significnce. Our finding tht ddition of second costimultory endodomin does not improve ntitumor ctivity in vivo is in greement with recent findings y others. 35 While we fvor one of our second-genertion CARs to move forwrd for further preclinicl testing, we re conducting dditionl experiments to estlish the miniml effective dose of ech CAR T-cell popultion, test their efficcy in dditionl gliom models, nd evlute their ility to kill gliom cells fter repeted stimultions. Our dt suggest tht tumor recurrence is most likely due to limited T-cell persistence in the tumor milleu in vivo. This limittion my e overcome y modifying the gliom cells to secrete IL While effective, this strtegy is difficult to trnslte into the clinicl setting. We therefore re exploring dditionl genetic modifictions of IL13Rα2-CAR T cells to enhnce their expnsions nd persistence like trnsgenic expression of cytokines 36,37 nd/or silencing negtive regultors. For exmple, glioms express PD-L1, 38,39 nd expresses PD-L1, which is upregulted in the presence of IFNγ (Supplementry Figure S8), nd could e trgeted in future studies. While we did not oserve immune escpe, trgeting single ntigen is ssocited with the risk of selecting tumor cells tht hve down regulted the expression or hve deleted the trgeted ntigen. 4,41 In the context of CAR T cells severl groups including ours hve developed strtegies to overcome this limittion y infusing two T-cell products with different specificity, expressing multiple CARs in T cells or expressing single CAR with dul specificity We evluted IL13Rα2-CAR T cells in immune-deficient mice, which re widely used to study the efficcy of CAR T cells. 19 While idel to study the interction etween humn T cells nd humn tumor cells, immune deficient mice to not recpitulte relisticlly the complex interctions etween doptively trnsferred T cells, tumor cells, nd the resident immune system, tht often contriutes to the immunosuppressive tumor microenvironment. To overcome this limittion, severl groups hve developed immune-competent mouse models to study CAR T cells. 11,45 For our IL13Rα2-CAR T cells, such studies would require the genetic modifiction of gliom cells to express humn IL13Rα2, since -sed CAR T cells do not recognize murine IL13Rα2 Cell numer IL13Rα2 No T-cell therpy SSR.CD28.OX4.ζ 96.8% 91.9% 92.5% Lysis % :1 2:1 1:1 E:T rtio 5:1 Rji No T-cell Therpy SSR.CD28.OX4.ζ Figure 6 Anlysis of cells isolted from recurrent tumors. cells were isolted from recurrent tumor of mice tht were treted with IL13Rα2-CAR T cells. After short-term culture (2 7 dys), FACS nlysis nd cytotoxicity ssys were performed. () FACS nlysis for IL13Rα2. () IL13Rα2-CAR T cells killed tumor cells isolted from recurrent tumors in contrst to Rji cells in stndrd 4-hour cytotoxicity ssy. CAR, chimeric ntigen receptor vol. 24 no. 2 fe. 216

8 The Americn Society of Gene & Cell Therpy IL13Rα2-specific T Cells for Gliolstom 1 4 Q1 2.4% Luc T cells Q2 7.74% 1 4 Q1 28.3% Luc/IL13Rα2-CAR T cells Q2 48.2% IL13Rα2-CAR Q4 24.3% Q3 65.9% Q4 8.96% Q3 14.6% GFP d1 Luc T cells Luc/IL13Rα2-CAR T cells c Luc T cells +Luc/IL13Rα2-CAR T cells d2 d4 Rdince d Dys post T-cell injection Figure 7 Limited persistence of IL13Rα2-CAR T cells in vivo. -CAR T cells were trnsduced to express egfp.ffluc. () FACS nlysis confirmed the expression of CAR nd egfp.ffluc trnsgenes. (,c) unmodified cells were injected intrcrnilly into mice. On dy 7, mice received eGFP.ffLuc CAR T cells intrcrnilly using the sme tumor coordintes. Bioluminescence imging ws used to monitor T-cell persistence. CAR, chimeric ntigen receptor. (Krenciute et l., unpulished dt), or the genertion of murine IL13Rα2-specific scfv. In conclusion, T cells redirected to IL13Rα2 with - sed CARs hve potent ntitumor ctivity ginst gliom cells in vitro, nd induce the regression of estlished GBM xenogrfts in vivo. Our study dds to the growing literture 31,32 tht there is n intricte interply etween scfvs, spcer region, trnsmemrne domin, nd endodomin tht determines CAR function, nd tht there is no single optiml configurtion tht is one size fits ll. IL13Rα2-CAR T cells my e of vlue in the tretment of not only IL13Rα2-positive GBMs ut lso other mlignncies in which IL13Rα2 is expressed. MATERIALS AND METHODS Cell lines. (GBM), U87 (GBM), 293T (humn emryonic kidney), nd Rji (Burkitt s lymphom) cell line were purchsed from the Americn Type Culture Collection (ATCC; Mnsss, VA). GBM4687 nd GBMR31 re primry peditric GBM cell lines, 46 nd GBM6 nd GBM39 re primry dult GBM cell lines. 47 The genertion of cells expressing enhnced green fluorescent protein nd firefly luciferse (.egfp.ffluc), 293T cells expressing green fluorescent protein (293T.GFP) or IL13Rα2 nd GFP (293T.IL13Rα2.GFP) ws previously reported. 5,7 Cell lines were grown in Roswell Prk Memoril Institute or Dulecco's Modified Egle Medium (GE Helthcre Life Sciences HyClone Lortories, Logn, UT) with 1% fetl ovine serum (FBS; GE Helthcre Life Sciences HyClone) nd 2 mmol/l GlutMAX-I (Invitrogen, Crlsd, CA). The Chrcterized Cell Line Core Fcility t MD Anderson Cncer Center, Houston, Texs, performed cell line vlidtion. Genertion of retrovirl vectors encoding IL13Rα2-scFv-specific CARs. A codon-optimized gene ws synthesized y GeneArt (Invitrogen) contining the immunogloulin hevy-chin leder peptide, nd flnked y 5 NcoI nd 3 BmHI sites. This mini gene ws sucloned into SFG retrovirl vector contining IL13Rα2-specific CARs (IL13Rα2- CARs) with short or long spcer regions (SSRs, LSRs) nd CD28.ζ, CD28. OX4.ζ, CD28.41BB.ζ, or 41BB.ζ endodomins. 4,48,49 All CARs contined CD28 trnsmenrne domin except for IL13Rα2-CAR.SSR.41BB.ζ, which hd CD8α trnsmemrne domin. IL13Rα2-CAR. nd IL13Rα2-CAR. without n endodomin were generted y PCR cloning. All cloning of the CARs were verified y sequencing (Seqwright, Houston, TX). RD114-pseudotyped retrovirl prticles were generted y trnsient trnsfection of 293T cells s previously descried. 5 Genertion of CAR T cells. Humn peripherl lood mononucler cells from helthy donors were otined under Bylor College of Medicine Institutionl Review Bord-pproved protocol, fter informed consent ws otined in ccordnce to the Declrtion of Helsinki. To generte IL13Rα2-CAR T cells, peripherl lood mononucler cells were isolted y Lymphoprep (Greiner Bio-One, Monroe, NC) grdient centrifugtion nd then stimulted on treted nontissue culture 24-well pltes, which were precoted with OKT3 (CRL-81, ATCC) nd CD28 (BD Bioscience, Mountin View, CA) ntiodies. Recominnt humn interleukin-7 nd -15 (IL7, 1 ng/ml; IL15, 5 ng/ml; Proleukin; Chiron, Emeryville, CA) ws dded to cultures on dy 2. 5 On dy 3, OKT3/CD28-stimulted T cells ( cells/well) were trnsduced on RetroNectin (Clontech, Mountinview, CA) coted pltes in the presence of IL7 nd IL15. On dy 5 or 6, T cells were trnsferred into new wells nd susequently expnded with IL-7 nd IL15. Nontrnsduced () T cells were ctivted with OKT3/CD28 nd expnded in prllel with IL-7 nd IL-15. IL13Rα2-CAR expression ws determined 4 to 5 dys post-trnsduction. Flow cytometry. A FACSCliur (BD Bioscience) or BC Gllios (Beckmn Coulter, Bre, CA) instruments were used to cquire immunofluorescence dt which were nlyzed with CellQuest (BD Bioscience) Moleculr Therpy vol. 24 no. 2 fe

9 IL13Rα2-specific T Cells for Gliolstom The Americn Society of Gene & Cell Therpy or BC Gllios (Beckmn Coulter) respectively. FlowJo v.7 (FlowJo, LLC Ashlnd, OR) or Kluz v1.2 (Beckmn Coulter) were used for finl dt nlysis nd grphic representtion. Isotype controls were immunogloulin G1 fluorescein isothiocynte (IgG1-FITC; BD Bioscience), IgG1 phycoerythrin (IgG1-PE; BD Bioscience). SSR IL13Rα2-CAR expression ws detected y stining T cells with humn IL13Rα2 chimer (R&D Systems, Minnepolis, MN) followed y Fc-FITC (Milipore, Billeric, MA) or Fc-PE (SouthernBiotech, Birminghm, AL). LSR IL13Rα2-CARs were detected using Fc-FITC or Fc-PE. cells were nlyzed for PD-L1 expression using CD271 PE ntiody (BD Bioscience). Forwrd nd side sctter gting were used to discriminte live cells from ded cells. Cells were collected nd wshed once with PBS (Sigm, St. Louis, MO) contining 1% FBS (GE Helthcre Life Sciences HyClone Lortories; FACS uffer) prior to the ddition of ntiodies. Cell were incuted for 3 minutes on ice in the drk, wshed once, nd fixed in FACS uffer with.5% prformldehyde (BD Bioscience) prior to nlysis. Western lot. Cells were dissocited with PBS + 3 mmol/l ethylenediminetetrcetic cid nd lysed in uffer contining 5 mmol/l Tris, 15 mmol/l NCl, 5 mmol/l Ethylenediminetetrcetic cid, 1% Triton X-1 (ll from Sigm), nd protese inhiitors (Thermo Scientific, Wlthm, MA). Protein concentrtions were determined using Bio- Rd protein ssy (Bio-Rd, Hercules, CA) with ovine serum lumin s the stndrd. Smples were dentured in Lemmli uffer (Bio-Rd) with βme (2-mercptoethnol, Bio-Rd; reducing conditions) or without βme (nonreducing condition) t 95 C for 5 minutes. Cell lyste (5 μg per lne) were run on 1% SDS polycrylmide gel nd trnsferred to nitrocellulose memrnes (BioRd). Memrnes were locked with 5% milk powder in Tris-uffered sline +.1% Tween-2 (ll from Sigm) nd then proed with nti-cd3.ζ (sc-1239, Snt Cruz Biotechnology, Snt Cruz, CA) or glycerldehyde-3-phosphte dehydrogense (sc-47724, Snt Cruz Biotechnology) mouse monoclonl ntiodies followed y horserdish peroxidse-conjugted got mouse IgG ntiody (sc-25, Snt Cruz Biotechnology). Blots were developed using SuperSignl West Dur Extended Durtion Sustrte (Thermo Scientific) nd exposed to GeneMte Blue Bsic Autordiogrphy Film (BioExpress, Kysville, UT). Coculture ssys for coculture ssys, ech CAR ws expressed in T cells from the sme donor. Biologicl repets were done using different donors nd dt presented in the figures is the verge of three to five donors. Recominnt protein coculture ssy. Nontissue culture 24-well pltes were precoted with recominnt humn IL13Rα1, IL13Rα2, or IL4Rα proteins, (R&D Systems) t finl concentrtion of 5 ng/well. Pltes were wshed once using Roswell Prk Memoril Institute, nd CAR or T cells were plted. After 24 hours, superntnts were hrvested nd IFNγ nd IL2 relese ws mesured y ELISA per the mnufcturer s instructions (R&D Systems). Cell culture coculture ssy. CAR T cells were cocultured with trget cells t 2:1 effector to trget (E:T) rtio in 24-well plte. T cells served s controls. After 24 hours, culture superntnts were hrvested, nd the presence of IFNγ nd IL2 ws determined y ELISA s per the mnufcturer s instructions (R&D Systems). Cytotoxicity ssy. Stndrd chromium ( 51 Cr) relese ssys were performed s previously descried. 5 Briefly, trget cells were leled with.1 mci (3.7MBq) 51 Cr nd mixed with decresing numers of effector cells to give effector to trget rtios of 4:1, 2:1, 1:1, nd 5:1. Trget cells incuted in complete medium lone or in 1% Triton X-1 were used to determine spontneous nd mximum 51 Cr relese, respectively. After 4 hours, superntnts were collected nd rdioctivity ws mesured in gmm counter (Cor Quntum; PerkinElmer; Wellesley; MA). The men percentge of specific lysis of triplicte wells ws clculted ccording to the following formul: (test relese spontneous relese)/(mximl relese spontneous relese) 1. Orthotopic xenogrft SCID mouse model. All niml experiments followed protocol pproved y the Bylor College of Medicine Institutionl Animl Cre nd Use Committee. Experiments were performed s descried previously 5 with few modifictions. ICR-SCID mice were purchsed from Tconic (IcrTc:ICR-Prkdc scid ; Fox Chse C.B-17 SCID ICR; Tconic, Hudson, NY). Mle 7- to 9-week-old mice were nesthetized, the hed ws shved nd the mice were immoilized in Cunninghm Mouse/ Neontl Rt Adptor (Stoelting, Wood Dle, IL) stereotctic pprtus fitted into n E156 L Stndrd Stereotxic Instrument (Stoelting), nd then scrued with 1% povidone-iodine. A 1-mm skin incision ws mde long the midline. The tip of 3G ½ inch needle mounted on Hmilton syringe (Hmilton, Reno, NV) served s the reference point. A 1 mm urrhole ws drilled into the skull 1 mm nterior nd 2 mm to the right of the regm eGFP.ffLuc cells in 2. μl were injected 3-mm deep to the regm, corresponding to the center of the right cudte nucleus over 5 minutes. The needle ws left in plce for 3 minutes, to void tumor cell extrusion, nd then withdrwn over 5 minutes. Seven dys fter tumor cell injection, nimls were treted with effector cells in 2 μl to the sme tumor coordintes. The incision ws closed with 2 3 interrupted 7. Ethilon sutures (Ethicon, Somerville, NJ). A sucutneous injection of.3.1 mg/kg uprenorphine (Buprenex RBH, Hull, Englnd) ws given for pin control. Bioluminescence imging. Isofluorne nesthetized nimls were imged using the IVIS system (IVIS, Xenogen, Almed, CA) 1 15 minutes fter 15 mg/kg D-luciferin (Xenogen) ws injected per mouse intrperitonelly. The photons emitted from the luciferse-expressing tumor cells were quntified using Living Imge softwre (Cliper Life Sciences, Hopkinton, MA). A pseudo-color imge representing light intensity (lue lest intense nd red most intense) ws generted nd superimposed over the gryscle reference imge. Mice were euthnized when the tumor rdince ws greter thn on two occsions or when they met euthnsi criteri (neurologicl deficits, weight loss, signs of distress) in ccordnce with the Center for Comprtive Medicine t Bylor College of Medicine. Sttisticl nlysis. All in vitro experiments were performed t lest in triplicte, GrphPd Prism 5 softwre (GrphPd softwre, L Joll, CA) ws used for sttisticl nlysis. Mesurement dt were presented s men ± stndrd devition. The differences etween mens were tested y pproprite tests. The significnce level used ws P <.5. For the mouse experiments, chnges in tumor rdince from seline t ech time point were clculted nd compred etween groups using t-test or Wilcoxon rnk-sum test, whichever pproprite. Survivl determined from the time of tumor cell injection ws nlyzed y the Kpln-Meier method nd differences in survivl etween groups were compred y the Wilcoxon test. SUPPLEMEARY MATERIAL Figure S1. Phenotypic nlysis of IL13Rα2-CAR T-cell lines. Figure S2. Western lot of IL13Rα2-CAR T cells. Figure S3. IL13Rα2-CAR. induces constitutive CD3.ζ phosphoryltion. Figure S4. Cell surfce expression of IL13Rα1 nd IL13Rα2. Figure S5. Cell surfce expression of IL13Rα2 in rin tumor cell lines. Figure S6. Genertion nd chrcteriztion of LSR.CD28.41BB.ζ CAR T cells. Figure S7. Genertion of SSR.CD28.41BB.ζ CAR T cells. Figure S8. FACS nlysis of PD-L1 expression on cell surfce with nd without IFNγ stimultion. ACKNOWLEDGMES This work ws supported y NIH Grnts 1R1CA nd 1R21NS8982-1, Cookies for Kids Cncer, nd the Jmes S McDonnell Foundtion. The Center for Cell nd Gene Therpy hs reserch collortion with Celgene nd Blueird Bio. GD, MSL, IVB, nd SG hve ptent pplictions in the field of T-cell nd gene-modified T-cell therpy for cncer nd/or IL13Rα2-trgeted therpies vol. 24 no. 2 fe. 216

10 The Americn Society of Gene & Cell Therpy IL13Rα2-specific T Cells for Gliolstom REFERENCES 1. Surydevr, CM, Verl, T, Snchez-Perez, L, Rep, EA, Choi, BD, Fecci, PE et l. (215). Immunotherpy for mlignnt gliom. Surg Neurol Int 6(Suppl 1): S68 S Vn Gool, SW (215). Brin Tumor Immunotherpy: Wht hve We Lerned so Fr? Front Oncol 5: Rerdon, DA, Freemn, G, Wu, C, Chiocc, EA, Wucherpfennig, KW, Wen, PY et l. (214). Immunotherpy dvnces for gliolstom. Neuro Oncol 16: Ahmed, N, Slsmn, VS, Kew, Y, Shffer, D, Powell, S, Zhng, YJ et l. (21). HER2-specific T cells trget primry gliolstom stem cells nd induce regression of utologous experimentl tumors. Clin Cncer Res 16: Chow, KK, Nik, S, Kkrl, S, Brwley, VS, Shffer, DR, Yi, Z et l. (213). T cells redirected to EphA2 for the immunotherpy of gliolstom. Mol Ther 21: Johnson, LA, Scholler, J, Ohkuri, T, Kosk, A, Ptel, PR, McGettign, SE et l. (215). 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