Adenovirus Expressing Both Thymidine Kinase and Soluble PD1 Enhances Antitumor Immunity by Strengthening CD8 T-cell Response

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1 originl rticle The Americn Society of Gene & Cell Therpy Adenovirus Expressing Both Thymidine Kinse nd Solule PD1 Enhnces Antitumor Immunity y Strengthening CD8 T-cell Response Seung-Pil Shin 1,2, Hye-Hyun Seo 1, Je-Hun Shin 3, Hyung-Be Prk 4, Dong-Pyo Lim 3, Hyeon-Seok Eom 4, Yong-Soo Be 2, In-Hoo Kim 5, Kyungho Choi 3,6,7 nd Sng-Jin Lee 1 1 Genitourinry Cncer Brnch, Reserch Institute Ntionl Cncer Center, Gyeonggi-do, Kore; 2 Deprtment of Life Science, Sungkyunkwn University, Suwon, Kore; 3 Specific Orgns Cncer rnch, Reserch Institute Ntionl Cncer Center, Gyeonggi-do, Kore; 4 Hemtologic Mlignncy rnch, Reserch Institute Ntionl Cncer Center, Gyeonggi-do, Kore; 5 Moleculr Imging nd Therpy rnch, Reserch Institute Ntionl Cncer Center, Gyeonggi-do, Kore; 6 Present ddress: Deprtment of Biochemistry nd Moleculr Biology, Seoul Ntionl University College of Medicine, Seoul, Kore; 7 Deprtment of Biomedicl Sciences, Seoul Ntionl University College of Medicine, Seoul, Kore Adenoviruses hroring the herpes simplex virus thymidine kinse (HSVtk) gene under the regultion of trnssplicing riozyme trgeting humn telomerse reverse trnscriptse (htert-tr) show mrked nd specific ntitumor ctivity. In ddition to inducing tumor cell deth y direct cytotoxicity, it is ecoming cler tht HSVtk lso induces ntitumor immunity. Progrmmed deth lignd 1 (PD-L1) expressed on tumor cell surfces medites tumorinduced immunoresistnce y inhiiting PD1-expressing tumor-infiltrting T cells. Here, we explored whether solule form of PD1 (spd1-ig), which locks PD-L1, could synergize with TERT-TR-regulted HSVtk to enhnce the denovirl therpeutic efficcy y oosting ntitumor immunity. Tumor ntigen relesed y HSVtk-trnsduced tumors successfully primed tumor ntigen specific CD8 T cells vi dendritic cells (DC). Regression of murine tumors ws mrkedly enhnced when spd1-ig ws incorported into the denovirus s compred with single-module denovirus expressing only HSVtk. This effect ws olished y CD8 T-cell depletion. Consistent with this, following doptive trnsfer of tumor ntigen specific CD8 T cells into tumor-ering Rg1 / mice, dul-module denovirus significntly enhnced CD8 T cell medited tumor rejection. In ddition, secondry tumor chllenge t distl site ws completely suppressed in mice treted with dul-module denovirus. These results suggest tht dul-trgeting strtegy to elicit oth tumor ntigen priming nd tumor-induced immunoresistnce enhnces CD8 T cell medited ntitumor immunity. Received 25 June 212; ccepted 21 Octoer 212; dvnce online puliction 22 Jnury 213. doi:1.138/mt Introduction Adenoviruses hroring herpes simplex virus thymidine kinse (HSVtk) hve een proposed s therpeutic pproch to the tretment of vrious cncers. Using humn telomerse reverse trnscriptse (htert)-trgeting riozyme (htert-tr) to control tumor-specific expression of HSVtk, we successfully chieved HSVtk expression in primry liver nd colorectl cncer cells. 1,2 When HSVtk-expressing cells re exposed to gnciclovir (GCV), prodrug, HSVtk phosphoryltes GCV t single site. This monophosphorylted form of GCV is trpped inside the cell nd converted into tri-phosphorylted GCV y cellulr kinses. The tri-phosphorylted GCV in turn inhiits DNA polymerse, cusing singlestrnd DNA reks, eventully leding to poptosis. 3 6 Although this direct cytotoxic effect is thought to e the min mechnism of HSVtk ntitumor ctivity, it hs ecome cler from recent reports tht HSVtk-medited tumor cell lysis elicits ntitumor immunity. 7 9 New strtegies to potentite this ntitumor immunity y comining vrious immune modultors such s FMS-like tyrosine kinse 3 lignd nd grnulocyte-mcrophge-colony stimulting fctor with denoviruses hroring HSVtk hve shown gret promise s immunotherpeutic vccines Most of these studies focused on tumor ntigen priming nd enhncing the recruitment or ctivtion of ntigen presenting cells such s dendritic cells (DCs). However, to mximize HSVtk-sed ntitumor immunity, tumor immunoresistnce mechnisms such s immunosuppressive cytokines, mjor histocomptiility complex downregultion, nd immunosuppressive cell-surfce molecules expressed on the cncer cell surfce must e overcome To dte, these issues hve not een ddressed in the context of HSVtk-sed gene therpy. Progrmmed deth lignd 1 (PD-L1) is cell-surfce glycoprotein nd memer of the B7 fmily of T-cell costimultory molecules. 17 Although PD-L1 mrna is uiquitously expressed in humns, cellsurfce expression of PD-L1 is restricted to cells of the mcrophge linege. 17 Of note, PD-L1 is lso expressed on the surfce of mny humn tumor cells. 18,19 The PD-L1 receptor (progrmmed deth-1, PD-1) is expressed on T cells. 2 Binding of PD-L1 trnsduces negtive regultory signls vi the PD-1 inhiitory cytoplsmic domin. 21,22 PD-L1 expressed on the surfce of tumor cells rogtes cell-medited ntitumor immunity y inducing T-cell poptosis nd inhiiting cytokine production nd tumor-killing effect of ctivted T cells. 23,24 Therefore, PD-L1 expression on the tumor cell surfce ppers to Correspondence: Sng-Jin Lee, Reserch Institute Ntionl Cncer Center, Gyeonggi-do, , Repulic of Kore. E-mil: leesj@ncc.re.kr or Kyungho Choi, Seoul Ntionl University College of Medicine, Seoul, , Repulic of Kore. E-mil: khchoi@snu.c.kr vol. 21 no. 3, mr. 213

2 The Americn Society of Gene & Cell Therpy spd1-ig synergizes ntitumor immunity of HSVtk medite tumor-induced immunoresistnce. Consistent with this, neutrlizing ntiodies ginst either PD-1 or PD-L1 overcome this resistnce nd enhnce ntitumor immunity in preclinicl mouse models Therpeutic ntiodies trgeting oth molecules re currently eing tested in phse I clinicl trils. 23,28 However, systemic downregultion of the PD-L1/PD-1 xis cn lso trigger systemic utoimmunity vi uncontrolled ctivtion of utorective T cells, s ws shown for ntiodies to cytotoxic T lymphocyte ssocited ntigen 4, nother negtive regultor of T cells. 29,3 Thus, loclized inhiition of the PD-L1/PD-1 xis in the tumor microenvironment my e more preferle nd sfer strtegy for overcoming tumorinduced T-cell tolernce. In the current study, s strtegy for locking PD-L1 in the tumor microenvironment, we generted recominnt solule PD-1 in which the extrcellulr domin of PD-1 ws fused to the Fc portion of mouse IgG2 (spd1-ig). The spd1-ig cdna ws integrted into repliction-deficient denovirus hroring HSVtk under the regultion of mouse TERT-TR to chieve the dul gol of enhncing tumor ntigen priming nd locking locl immune resistnce. In ddition to the direct cytotoxic effects of HSVtk, the dul-module denovirus ws designed to enhnce ntitumor vccine efficiency y mximizing ntitumor immunity. Our experimentl evidence suggests tht the dul-module denovirus enhnces ntitumor immunity y mplifying the CD8 T-cell response. Results Construction of n denovirus hroring mouse TERT-TR- HSVtk We previously developed novel strtegy for tumor-specific denovirl expression of HSVtk using well-known tumor mrker, TERT. In this strtegy, riozyme tht recognizes nd digests mrna sequence of htert is delivered to tumor cells using recominnt denovirus. In ddition, this riozyme is designed to ligte HSVtk mrna to the digested 3 end of htert mrna, which leds to new trnsltion of HSVtk mrna in the tumor cells. This kind of riozyme is clled trns-splicing riozyme. 1,2 (see lso Supplementry Figure S1). In theory, trns-splicing riozyme controlled HSVtk expression is restricted to cells tht express high levels of htert mrna, which include tumor cells. Thus, this system represents n efficient wy to deliver HSVtk into tumor cells without ffecting cells in the surrounding norml tissues, which express low levels of htert. In fct, this strtegy ws highly effective in humn colorectl cncer cells in xenogrft mouse models. One drwck of this system in immunologicl nlysis is tht experiments with humn cncer cells hve to e performed in immune-deficient mice to prevent xenogrft rejection. Hence, this system is not pproprite for evluting the effect of HSVtk on ntitumor immunity. To overcome this limittion, we designed similr system using trns-splicing riozyme tht trgets mouse TERT mrna (mtert-tr) to evlute the immunologicl effects of TERT-TRregulted HSVtk using syngeneic mouse tumor models. The TERT recognition sequence ws plced proximl to the HSVtk coding sequence such tht HSVtk expression ws dependent on the presence of mtert trnscripts (Supplementry Figure S1). 31 This entire expression cssette, under the control of the cytomeglovirus (CMV) promoter, ws integrted into n E1/E3-deficient denovirus genome to generte n denovirus () hroring mtert-tr-regulted HSVtk (mtert-tr-hsvtk) (Figure 1). An denovirus () lcking the expression cssette ws lso generted s control. To determine whether HSVtk expression medited y ws cytotoxic to murine tumor cells, we used CT26 cells, colon cncer cell line derived from BALB/c mouse, ecuse this cell line expresses high levels of mtert mrna (dt not shown). CT26 cells were exposed to t vrying multiplicity of infection (MOIs) in the presence or sence of GCV. An MOI of 2.5 in the presence of GCV ws sufficient to kill most of the cells, wheres exhiited no cytotoxicity up to n MOI of 5 (Figure 1). Consistent with this in vitro cytotoxicity, tumor growth ws significntly inhiited when ws injected intrtumorlly into sucutneous CT26 tumors in BALB/c mice (Figure 1c). Thus, showed similr ntitumor effects to those of denoviruses hroring humn TERT-TR-HSVtk, which indicted tht it would e pproprite for studies of ntitumor immunity in mice. Enhnced DC-medited tumor ntigen presenttion y HSVtk Some reports suggest tht HSVtk expression in tumor cells, either y DNA trnsfection or denovirus infection, enhnces ntigen priming of cytotoxic CD8 T cells. 9,32 Becuse HSVtk expression in c ITR ψ E1 CMV Rz HSVtk pa CMV pa E3 ITR ITR ψ E1 E3 ITR OD /GCV(+) /GCV( ) Virus (MOI) , Figure 1 Regultion of denovirl HSVtk gene expression y mouse telomerse reverse trnscriptse trgeting riozyme (TERT-TR). (), n E1/E3-deficient denovirus, expressing HSVtk under the regultion of mouse TERT-TR. is control E1/E3-deficient denovirus. () CT26 cells (3 1 3 ) were seeded in 96-well pltes nd then exposed to (closed circles) or denovirus t the indicted multiplicity of infections in the presence (closed squres) or sence of 1 µmol/l gnciclovir (GCV) (closed tringles). On dy 3 postinfection, cytotoxicity ws mesured using cell prolifertion ssy kit. Dt represent the men ± SD of triplicte ssys. (c) BALB/c mice were inoculted sucutneously in oth flnks with CT26 cells (1 1 6 ) followed y intrtumorl injection of plque forming units of either (closed squres) or (closed circles), nd GCV (75 mg/kg) ws dministered intrperitonelly twice per dy. Dt represent the men ±SEM (n = 5). ITR, inverted terminl repets; CMV, cytomeglovirus promoter; HSVtk, herpes simplex virus thymidine kinse; pa, poly A; Rz, mouse TERT trgeting trns-splicing riozyme; ΔE1 nd ΔE3, deletion of E1 nd E3; Ψ, pckging signl. Moleculr Therpy vol. 21 no. 3 mr

3 spd1-ig synergizes ntitumor immunity of HSVtk The Americn Society of Gene & Cell Therpy the presence of GCV elicits cell deth in significnt proportion, if not ll, of the tumor mss in vivo, tumor ntigens derived from ded cells my e cptured y APCs such s DCs, nd these cells would then migrte to drining lymph nodes to prime tumor ntigen specific T cells. Although this model ws suggested in the literture, 33 we decided to test this possiility using defined ntigen-specific T cells. For this experiment, we used the murine tumor cell line E.G7 ( derivtive of EL4 cells tht stly expresses ovlumin; OVA) s model tumor ntigen, nd T cells purified from OVA-specific T-cell receptor trnsgenic mice (OT-I mice) s the model tumor-ntigen (OVA)-specific CD8 T-cell popultion. Most of CD8 T cells purified from OT-1 mice re OVA-specific nive cytotoxic T cells. We first tested the ility of to induce cell deth in sucutneous E.G7 tumors in syngeneic B6 mice. When tumors were isolted nd exmined histologiclly following intrtumorl injection of, there ws significnt degree of cell deth, which suggested tht tumor ntigen relese ws likely following virl infection (Figure 2). Next, we purified DCs from the tumor-drining lymph nodes nd exmined whether these cells were loded with OVA using DC/CD8 T-cell coculture ssy for interferon-γ (IFN-γ) production. Purified OT-I T cells cultured with DCs from -treted tumor-ering mice produced much more IFN-γ thn OT-I T cells cultured with DCs from control virus treted tumor-ering mice. These results indicted tht DCs from -treted mice presented sufficient level of OVA ntigen to stimulte OVA-specific T cells (Figure 2). Thus, expression of HSVtk in tumors nd susequent GCV-induced cell deth resulted in the relese of tumor ntigens, nd these ntigens were efficiently cptured y DCs cple of stimulting tumor ntigen specific cytotoxic T cells. Thus, tumor-specific expression of HSVtk cn effectively stimulte ntitumor T-cell ctivity vi DCs, in ddition to inducing direct tumor cell cytotoxicity. Construction of n denovirus hroring oth mtert-tr-hsvtk nd spd1-ig stimulted ntitumor CD8 T-cell rectivity, rising the intriguing possiility tht comining this pproch with nother strtegy for inctivting tumor-induced immune resistnce could further enhnce ntitumor T-cell rectivity. PD-L1 is well-known immune suppressor expressed on tumor cell surfces. We generted solule form of the PD-L1 receptor, spd1, which neutrlizes PD-L1 nd rogtes PD-L1-medited T-cell inhiition. To increse the stility of spd1 in vivo, spd1 ws fused to the Fc portion of IgG2 to generte spd1-ig. We constructed dul-module denovirus (.spd1) contining HSVtk under the regultion of mtert-tr in the E1 region nd spd1-ig in the E3 region of the denovirl genome (Figure 3)..sPD1 c ITR ψ E1 CMV Rz HSVtk pa Ad5EF1α.sPD1 [H 3 ] PCV uptke 1 6 ITR ψ E sPD Cell Virus (MOI) E3 EF1α spd1-ig E3 EF1α spd1-ig ITR pa ITR pa (2MOI) spd1-ig in superntnt spd1-ig in cell lyste d INF-γ (pg/ml) β-ctin in cell lyste 1,5 1, P =.2244 Ad5EFα spd1 spd1 spd1 IFN-γ (pg/ml) 2 P = OT-1 + DC- OT-1 OT-1 only + DC- Figure 2 Enhnced dendritic cells (DC)-medited ntigen presenttion elicited y HSVtk. () E.G7 tumors in C57/BL6 mice were injected intrtumorlly with either or. Tumors were hrvested 4 dys lter for histologicl nlysis y hemtoxylin nd eosin stining. () Two dys fter the injection of denoviruses, DCs (5 1 4 ) were hrvested from drining lymph nodes round E.G7 tumors y MACS chromtogrphy using nti-cd11c microeds nd then cocultured with ovlumin-specific CD8 T cells (1 1 6 ) from OT-1 trnsgenic mice. After 72 hours, culture superntnts were collected nd the mount of interferon-γ ws mesured. DC-, DCs from injected mice; CD-, DCs from -injected mice were lso mesured. Dt represent the men ±SD. P, unpired t-test. Figure 3 Construction of dul-module denovirus expressing mouse telomerse reverse trnscriptse-trgeting riozyme (mtert- TR)-regulted HSVtk nd spd1-ig. ().spd1 encodes mtert- TR-HSVtk under the control of the CMV promoter in the E1 region nd spd1-ig under control of the EF1α promoter in the E3 region. Detils of the construction re descried in Mteril nd Methods section. () HEK293 cells (4 1 5 ) were infected with the indicted denovirus s n multiplicity of infection of 1 for 24 hours, followed y lysis in RIPA uffer nd immunolot nlysis. spd1-ig expression y.spd1 (totl of 8 µg protein per lne) ws confirmed using n nti-pd1 ntiody. (c) The enzymtic ctivity of HSVtk in phosphorylting gnciclovir ws evluted in CT26 cells y mesuring the ccumultion of rdio-leled [H 3 ]PCV (1 µci/ml) s descried in Mterils nd Methods section. Dt represent the men ± SD of triplicte ssys. (d) Culture superntnt hrvested from cells infected with denovirus (5 or 1 MOI) ws mixed with cocultures of ovlumin (OVA)-specific CD8 T cells (1 1 6 ) purified from OT-1 mice nd MC38/OVA cells (1 1 4 ) in 6-well pltes. After 72 hours, culture superntnts were collected nd interferon-γ levels were mesured. Dt represent the men ± SD. P, unpired t-test. Ψ, pckging signl; ΔE1 nd ΔE3, deletion of E1 nd E3; CMV, cytomeglovirus promoter; EF1α, elongtion fctor 1α promoter; HSVtk, herpes simplex virus thymidine kinse; ITR, inverted terminl repets; MOI, multiplicity of infection; Rz, mouse TERT trgeting trns-splicing riozyme; pa, poly A; PCV, penciclovir vol. 21 no. 3 mr. 213

4 The Americn Society of Gene & Cell Therpy spd1-ig synergizes ntitumor immunity of HSVtk HEK293 cells infected with.spd1 expressed nd secreted spd1-ig into the extrcellulr milieu, s confirmed y immunolot nlysis (Figure 3). The expression level of HSVtk in.spd1-infected cells ws comprle with tht of infected cells s mesured y enzymtic ctivity (Figure 3c). 34 We lso tested whether secreted spd1-ig could enhnce ntitumor T-cell rectivity y neutrlizing PD-L1 on the tumor cell surfce in vitro. Culture superntnt from Ad5EF1α.sPD1-infected 293HEK cells ws dded to cocultures of OVA-expressing tumor cells nd OVAspecific OT-I T cells, nd T-cell rectivity ws mesured ccording to the mount of IFN-γ secretion. As expected, IFN-γ secretion y ntigen-specific T cells in response to cncer cell chllenge ws enhnced y spd1-ig-contining superntnt (Figure 3d). The expression of PD-L1 on the cell surfce of cncer cells nd the inding of spd1-ig in the culture superntnt to the cncer cell surfce were confirmed y flow cytometry (Supplementry Figure S2). These results suggest tht the dul-module denovirus my enhnce ntitumor T-cell rectivity within the tumor microenvironment in vivo. Effective suppression of tumor growth y.spd1 To exmine whether spd1-ig enhnced the ntitumor ctivity of HSVtk in vivo, we used CT26 colon cncer model (see Figure 1c). OD ,5 2, 1,5 1,.sPD1/GCV(+) Ad5EF1α.sPD1 /GCV(+) 5.sPD1/GCV( ) Virus (MOI).sPD1 Ad5EF1α.sPD P =.142 Figure 4 In vitro nd in vivo nticncer ctivity of.spd1. () CT26 cells were infected with (inverted closed tringle), (closed tringle), Ad5EF1.sPD1 (closed squre), or. spd1 (closed circle, closed dimond) t vrious MOIs together with 1 µmol/l gnciclovir (GCV) (closed tringle, closed circle), or without GCV (closed tringle, closed squre, closed dimond). After 3 dys, cell cytotoxicity ws mesured s descried for Figure 1. Dt represent the men ± SD of triplicte ssys. () BALB/c mice were inoculted sucutneously in oth flnks with CT26 cells (1 1 6 ). Tumors were treted with plque forming units of (closed inverted tringle), Ad5EF1.sPD1 (closed squre), (closed tringle), or.spd1 (closed circle). GCV (75 mg/kg) ws dministered intrperitonelly twice dy. Tumor volumes were clculted nd plotted t the indicted time points. Dt represent the men ±SEM (n = 1). P, unpired t-test. GCV, gnciclovir; MOI, multiplicity of infection. This model ws chosen in prt ecuse CT26 cells express high levels of PD-L1 on their cell surfce (Supplementry Figure S2). CT26 cells infected with.spd1 showed similr degree of in vitro cytotoxicity to those infected with in the presence of GCV, which suggested tht spd1-ig does not contriute to direct cytotoxicity y HSVtk (Figure 4). By contrst,. spd1 significntly enhnced tumor regression s compred with following intrtumorl injection into CT26 tumors in BALB/c mice; indeed, ner-complete tumor regression ws oserved (Figure 4). A control denovirus hroring spd1-ig lone (Ad5EF1.sPD1) did not show ny ntitumor effects in this model, which suggests tht spd1-ig expression in the tumor tissue is not sufficient to induce tumor regression, nd tht ntigen priming y HSVtk in vivo is required for the effects of spd1-ig. Enhnced suppression of tumor growth y spd1-ig is medited y CD8 T cells HSVtk enhnced ntigen-specific CD8 T-cell priming y DCs (Figure 2) nd spd1-ig enhnced ntitumor CD8 T cell rectivity in vitro (Figure 3d). These oservtions suggest tht enhnced CD8 T-cell rectivity is responsile for the enhnced ntitumor effects of.spd1 in vivo. To test this possiility, CT26 tumor ering mice were treted with nti-cd8 ntiody to deplete the CD8 T-cell popultion. Enhnced tumor growth suppression y dul-module.spd1 ws nerly olished in CD8 T cell depleted mice (Figure 5). Of note, the ntitumor ctivity of ws lso decresed, suggesting tht the 1,2.sPD1 w/o A w/o A 9 w/o A Tumor growth reduction (%) w/ CD8-depleting As 1 w/o fold 1.9-fold.sPD1 P = sPD1 w/ A w/ A w/ A Figure 5 Tumor growth suppression y spd1-ig is medited y CD8 T cells. () Sucutneous CT26 tumors in BALB/c mice were injected intrtumorlly with (closed tringle), (closed squre), or.spd1 (closed circle) (left pnel). To determine the involvement of CD8 T cells in the ntitumor ctivity of spd1-ig, sucutneous CT26 tumors in C57/BL6 mice were injected intrtumorlly with (closed tringle), (closed squre), or.spd1 (closed squre) (right pnel). Two dys efore virus tretment nd every 5 dys fter tretment, mice were injected intrvenously with the 2.43 nti-cd8 ntiody (5 μg) to deplete CD8 T cells (right pnel). Arrow indictes the time of ntiody injection. Tumor growth ws clculted nd plotted t the indicted time points. The rrows indicte the time when the ntiody ws injected. () Reduction of tumor growth in the presence (closed squre) or sence (open squre) of 2.43 nti-cd8 ntiody ws clculted reltive to on dy 12. Dt represent the men ± SEM. P, unpired t-test. As, ntiodies. Moleculr Therpy vol. 21 no. 3 mr

5 spd1-ig synergizes ntitumor immunity of HSVtk The Americn Society of Gene & Cell Therpy effect of is prtilly dependent on n ntitumor immune responses medited y CD8 T cells in the CT26 mouse tumor model. However, the extent to which CD8 depletion ffected ntitumor ctivity ws fr greter for.spd1 thn for (4.76-fold versus 1.9-fold), underscoring the importnce of CD8 T cells in ntitumor ctivity induced y the former versus the ltter (Figure 5). To exmine the role of tumor-specific CD8 T cells more directly, we used the E.G7 tumor model nd OVA-specific OT-I T cells. Intrtumorl injection of sucutneous E.G7 tumors in syngeneic B6 mice with.spd1 resulted in enhnced tumor regression, similr to tht oserved in the CT26 model. By contrst, when the sme experiment ws performed in syngeneic lymphocyte-deficient Rg1 / mice, the ntitumor effect of oth viruses ws mrkedly reduced (Figure 6). The degree of reduction ws gin fr stronger for.spd1 thn for (6.58-fold versus 1.61-fold), consistent with the effects of CD8 T-cell depletion in the CT26 model (Figure 6). When ntigenspecific T-cell response ws reconstituted y introducing OT-I T cells intrvenously into E.G7-ering Rg1 / mice, the ntitumor effect of intrtumorl injection of denovirus ws restored, nd the effect ws more prominent following injection of. spd1 thn following injection of (Figure 6c). Notly, the numer of OT-I T cells in the lood ws gretly incresed in mice treted with.spd1 s compred with (Figure 6d). These results suggest tht.spd1 enhnces tumor regression y directly enhncing tumor-specific CD8 T-cell function. Systemic ntitumor effects of loclized.spd1 tretment There ws systemic increse in tumor-specific T cells in the lood of.spd1-treted mice, which rised the possiility tht loclized injection of virus t the primry tumor site my inhiit secondry tumor growth t distl site in the sme mouse. CT26 tumors in BALB/c mice were treted with.spd1 three times t 3-dy intervls. Fourteen dys fter the initil tretment, secondry CT26 tumor chllenge ws performed in the opposite flnk of the sme mouse. At this time, there ws miniml growth of the primry tumor in.spd1-treted mice, wheres -treted mice hd developed lrge primry tumors (>6 mm 3 ), which hmpered secondry tumor injection in these mice. Therefore, new cohort of norml BALB/c mice ws used s control group for secondry tumor chllenge. Secondry tumors grew well in the control group, ut did not grow t ll in. spd1-treted mice (Figure 7,c). The sme result ws oserved in the E.G7 tumor model in B6 mice (Figure 7,c). For the more rigorous comprison, when the secondry tumor ws chllenged to the mice ering smll primry tumor for the control group, similr results were otined (Supplementry Figure S3). Thus, loclized injection of dul-module denovirus elicits systemic ntitumor effects y enhncing ntitumor immunity. Tumor growth reduction (%) c d Wild type Rg1 / fold 1,5 1, fold.sPD1 Wild type.spd P =.156.sPD1 6,.sPD1 4,5 3, 1, Virus injection T cell nd virus injection Rg1 / P <.1 OT-1 CD T cells (µl) sPD1 P =.39 Figure 6 Tumor growth suppression y spd1-ig in T nd B cell deficient Rg1 / mice. () Sucutneous E.G7 tumors in C57/BL6 mice were injected intrtumorlly with (closed tringle), (closed squre), or.spd1 (closed circle) (left pnel). To mesure lymphocyte involvement in the ntitumor ctivity of spd1-ig, sucutneous E.G7 tumors in Rg1 / mice were injected intrtumorlly with (closed tringle), (closed squre), or.spd1 (closed circle) (right pnel). Tumor growth ws clculted nd plotted t the indicted time points. P, unpired t-test. () Reduction of tumor growth in Rg1 / (closed squre) or wild type mice (open squre) ws clculted reltive to on dy 12. (c) E.G7 tumors (1 1 6 cells) in Rg1 / mice were injected intrtumorlly twice t 7 dy intervls with plque forming units of (closed tringle), (closed squre), or.spd1 (closed circle). CD8 T cells (5 1 4 ) from OT-1 mice were injected intrvenously t the sme time s the first denovirus injection. Tumor growth ws clculted nd plotted t the indicted time points. Dt represent the men ± SEM (n = 6). P, unpired t-test. (d) For ex vivo nlysis of immune cells, E.G7 tumors in Rg1 / mice were cotreted with CD8 T cells from OT-1 mice nd the indicted denovirus. Eight dys fter the first denovirus injection, peripherl lood mononucler cells were hrvested from the mouse eye vein. Asolute numers of OT-1 CD8 T cells were clculted from totl numers of live cells nd percentges of TCR Vα2 +, Vβ5 +, T cells (OT-1 cells) in flow cytometry profile. P, Wilcoxon mtched-pirs signed rnk test vol. 21 no. 3 mr. 213

6 The Americn Society of Gene & Cell Therpy spd1-ig synergizes ntitumor immunity of HSVtk (1st tumor chllenge).spd1 (1st tumor chllenge).spd1 (2nd tumor chllenge) No tretment (2nd tumor chllenge) ,5 1,25 1, (1st tumor chllenge).spd1 (1st tumor chllenge).spd1 (2nd tumor chllenge) No tretemnt (2nd tumor chllenge) c Avg = ± Avg = ± Avg = Avg = No tretment.spd1 No tretment.spd1 CT-26 E.G7 Figure 7 Inhiition of secondry tumors y.spd1 tretment. () CT26 tumors were implnted sucutneously in BALB/c mice nd then injected intrtumorlly with plque forming units of.spd1 three times t 3-dy intervls. Two weeks fter the initil tretment, the opposite flnk ws chllenged with tumor cells. Gnciclovir (75 mg/kg) ws dministered intrperitonelly during virus tretment strting 1 dy fter the first virus injection nd continuing for 12 dys. Tumor size ws clculted every 3 dys. () E.G7 tumors were implnted sucutneously in C57/BL6 mice nd then treted s descried for (). (c) On dy 7 fter the second secondry chllenge, tumor size t the chllenge site in mice previously treted with.spd1 or in untreted mice ws mesured. Dt represent the men ± SD. Avg, verge. Discussion As fvored strtegy for delivering trnsgenes into cells in situ, denoviruses re generlly considered oth efficient nd sfe. However, there hs een limited success in trnsferring suicide genes, such s HSVtk, into tumor cells vi repliction-incompetent denoviruses due to low rtes of infection nd the consequent low potency in terms of clinicl ppliction. 35 Nonetheless, it is now cler tht, in ddition to direct cytotoxicity, prtil tumor cell deth induced y denovirl HSVtk in the infected re cn efficiently potentite ntitumor immunity nd chieve indirect killing of tumors. Therefore, HSVtk-sed denoviruses could e considered for use s tumor vccines s well s cytotoxic therpeutics. In ddition, denovirl vectors hve the dvntge of eing le to ccommodte multiple genes within single vehicle, which enles delivery of comintion therpeutics s single gent. The efficcy of HSVtk-sed denoviruses s tumor vccines could e gretly improved y incorporting other immune modultors within the sme vector. Tumor vccines imed t oosting ntitumor immunity generlly fce one mjor rrier to mximum efficcy; nmely, immunoresistnce, imed t voiding recognition nd ttck y the immune system. This is most likely one of the min resons why numerous ttempts to develop efficient therpeutic vccines hve filed. The role of the PD-L1/PD1 signling xis in tumor protection hs een well demonstrted in severl studies, mking it good trget for disrupting tumor immunoresistnce. PD1 knockout mice exhiit spontneous utoimmune responses due to enhnced T-cell rectivity. 36,37 Consistent with this, implnted tumors re efficiently rejected y PD1 knockout mice. 25 Likewise, nti-pd-l1 ntiody tretment of tumor-ering mice efficiently rogtes tumor grfts. 25,27 However, dt from PD1 knockout mice lso indicte tht systemic lockde of the PD1/PD-L1 xis my induce dverse utoimmune side effects. In this sense, loclized disruption of PD-L1 my e more dvntgeous in terms of sfety. The concept of solule PD1 s neutrlizing gent for PD-L1 hs een demonstrted y severl groups. 38,39 However, these studies used primrily plsmid DNA to express spd1 systemiclly in n effort to ugment other therpeutic DNA or denovirl ntigenic vccines. Here, we demonstrted n lterntive method which delivered spd1 directly into the tumor microenvironment using n denovirl vector contining HSVtk. In this system, tumor cells infected with the virus relese tumor ntigens on HSVtk-medited cell deth. These ntigens re then processed y locl DCs, which then efficiently prime tumor ntigen specific cytotoxic T cells. Once primed, ntitumor cytotoxic T cells infiltrte the tumor tissue nd encounter virus-infected tumor cells expressing spd1. Within the tumor microenvironment, spd1 cn enhnce the T-cell ntitumor rectivity with miniml side effects. In the current study, this type of dul-module denovirus hroring HSVtk nd spd1-ig efficiently suppressed estlished tumor growth in two different murine tumor models. This effect ws medited y tumor-specific CD8 T cells, s demonstrted y oth CD8 T-cell depletion experiments (loss-of-function experiments) nd tumor-specific T-cell reconstitution experiments (ginof-function experiments). Although previous studies reported the systemic T-cell ctivtion during HSVtk/GCV tretment, 4,41 to our knowledge, this is the first demonstrtion tht CD8 T cells specific to defined tumor ntigen cn directly medite the ntitumor effects of HSVtk-sed denovirl therpy. Upon CD8 T-cell depletion, or in Rg1 / mice, the oservtion tht tretment with.spd1 still resulted in mildly enhnced ntitumor ctivity ws intriguing. This suggests tht lthough CD8 T cells ply mjor role in the ntitumor ctivity of spd1, other immune mechnisms my lso e involved. spd1 my hve direct effect on other immune cells, such s nturl killer cells, 42 or, lterntively, ntiody-dependent cell cytotoxicity medited y the Fc portion of immunogloulin fused to spd1 my ply role in this phenomenon. 43 A numer of previous studies demonstrte the synergistic effect of denovirus-medited expression of HSVtk nd other cncer therpies such s rdition, surgery, nd some chemotherpeutic gents. 44 Such synergistic effects resulted from n incresed level of phosphorylted GCV incorported into the DNA during DNArepir processes, leding to enhnced cell deth. This rises the possiility tht.spd1 could lso e comined with stndrd therpy, ecuse it is likely to potentite tumor-infiltrting immune cells, which re primed y surgery or rdition-induced ntigen relese nd inflmmtion. Alterntively, AdmTR.sPD1 could e Moleculr Therpy vol. 21 no. 3 mr

7 spd1-ig synergizes ntitumor immunity of HSVtk The Americn Society of Gene & Cell Therpy modified to ttrct or ctivte ntigen presenting cells vi incorportion of third immune modultor, such s FMS-like tyrosine kinse 3 lignd or grnulocyte-mcrophge-colony stimulting fctor. This pproch my provide n dditionl DC oost in our immune ctivtion xis of.spd1 including tumor cell lysis, dendritic cell priming, nd T-cell ctivtion. In fct, tretment sed on n HSVtk denovirl vector in conjugtion with FMS-like tyrosine kinse 3 lignd-expressing denovirus is very close to entering phse I clinicl trils. 1 Thus, denovirl vectors hroring HSVtk, FMSlike tyrosine kinse 3 lignd, nd spd1 within single vector my soon e relized s the next-genertion of denovirl tumor vccines. This venue is currently under ctive investigtion. For clinicl pplictions, lthough denovirl gene therpy hs generlly een considered loclized therpeutic, our results suggest tht.spd1 could e used to tret tumors with multiple metsttic foci. As suggested y Figure 7, tretment of primry tumor, even sizele one, with.spd1 my elicit systemic ntitumor response to other smll metsttic foci. We propose tht the dul-module denovirus descried herein cn oth enhnce ntigen priming nd overcome tumor immune resistnce. It, therefore, represents promising strtegy for strengthening the clinicl pplictions of HSVtk-sed gene therpy. Mterils nd Methods Cells nd mice. All cells were cultured in RPMI supplemented with 1% fetl ovine serum nd 1% penicillin/streptomycin. Six-week-old femle BALB/c nd C57/BL6 mice were purchsed from SLC (Jpn). OT-1 (B6 ckground) nd Rg1 / mice (B6 ckground) were from the Jckson lortory (Br Hror, ME). All niml experiments were performed in ccordnce with the Guidelines for the Cre nd Use of Lortory Animls of the Ntionl Cncer Center (Kore). Adenovirus construction. The AdenoZAPTM nd Adeno QuickTM systems (OD26; Boise, ID) were used to generte hroring mouse TERT-TR-HSVtk under the control of the CMV promoter (CMV.mTR. HSVtk). A DNA frgment contining CMV.mTR.HSVtk ws otined y digesting pavq-cmv-mtert AS1 Ri (+67) TK 31 with SpeI/ScII nd then inserted the resulting frgment into the SpeI/EcoRV sites of pzap1.1 (OD26) to generte pzap1.1.cmv.mtr.hsvtk. pzap1.1.cmv.mtr. HSVtk ws digested with PcI/DrIII, ligted with RightZAP1.2, nd then trnsfected into HEK293 cells. To generte spd1-ig, the extrcellulr domin of PD-1 ws mplified y PCR using the primers: 5 -CCG CTC GAG CTC ACC ATG TGG GTC CGG CAG GTA CCC TGG-3 nd 5 -AGA TCT TCC TCC TCC TCC TTG AAA CCG GCC TTC TGG TTT GGG-3. The mplified product ws inserted into the XhoI/BglII sites of pfuse-migg2a.fc1 (Invitrogen, Sn Diego, CA) to generte pfusemigg2a.fc1.ef1α.spd-1. EF1.sPD1-Ig from pfuse-migg2a.fc1. EF1.sPD1-Ig ws sucloned into the EcoRI/SwI sites of pe3.1 (OD26) to generte pe3.1.ef1.spd1-ig. The CMV.mTR.HSVtk frgment of pzap1.1.cmv.mtr.hsvtk ws ligted into the BmHI/SpeI sites of pe1.2 (OD26) to generte pe1.2.cmv.mtr.hsvtk. To generte.spd1, pe1.2.cmv.mtr.hsvtk nd pe3.1.ef1.spd1-ig were digested with DrIII/ PflMI, ligted with AdenoQuick13.1, nd then trnsfected into HEK293 cells. For Ad5EF1.sPD1, pe3.1.ef1.spd1-ig nd pe1.2 empty vector were digested with DrIII/PflMI nd then ligted with AdenoQuick13.1, followed y trnsfection into HEK293 cells s descried for.spd1. Construction of ws performed s previously descried. 1,2 In vitro GCV uptke nd cell cytotoxicity ssy. HSVtk enzyme ctivity ws determined y mesuring ccumulted phosphorylted GCV in cells. 1 A cell prolifertion ssy (Dojindo Lortories, Rockville, MD) ws performed to evlute denovirus cytotoxicity using the stndrd protocol, with some modifictions. Briefly, cells (3 1 3 ) were seeded in 96-well pltes nd incuted overnight t 37 C. Cells were exposed to denovirus t the indicted MOI. One dy lter, GCV ws dded to finl concentrtion of 1 µmol/l nd then cell prolifertion ws mesured on dy 3. All experiments were performed in triplicte. Antiodies nd regents. To confirm spd1-ig protein expression from Ad5CMV.mTR.sPD1, HEK293 cells (4 1 5 ) were infected with denovirus t n MOI of 1. Twenty-four hour postinfection, culture superntnts nd cell lystes (8 µg) were nlyzed y immunolot using n nti-pdcd-1 ntiody (Snt Cruz Biotechnology, Snt Cruz, CA). The concentrtion of spd1-ig in the culture superntnt ws t lest 58 ng/ml, which ws clculted from the purified spd1-ig using protein G ffinity purifiction. The yield ws 5.23 μg out of 9 ml of culture superntnt. For CD8 T-cell depletion, 5 µg of 2.43 nti-cd8 ntiody ws injected intrperitonelly 2 dys efore denovirus tretment nd then every 5 dys therefter for 15 dys. CD8 T cell/dc coculture ssy for IFN-γ production. Drining lymph node derived DCs were enriched using 17.5% Nycodenze grdient nd then further purified on MACS column using nti-cd11c microeds (Miltenyi Biotec, Auurn, CA). Nive CD8 T cells from OT-1 mice were isolted using nti-cd8 microeds. Lymph node derived DCs (5 1 4 ) were cultured together with nive CD8 T cells (1 1 5 ) in 96 well-pltes for 72 hours. Culture superntnts were collected fter 72 hours nd the mount of IFN-γ ws mesured y ELISA (ebioscience, Sn Diego, CA). In vitro OT-1 T-cell ctivtion ssy. MC38/OVA cells, which re murine colorectl cncer cells (MC38, B6 ckground) stly expressing OVA, were prepred on 6-well pltes. spd1-ig ws otined from culture superntnts of HEK293 cells infected with denovirus t n MOI of 1 for 24 hours nd then incuted with MC38/OVA cells (1 1 4 ) plus OT-1 CD8 T lymphocytes (1 1 6 ) for 72 hours. Nive CD8+ T lymphocytes from OT-1 mice were isolted using MACS column s descried ove. IFN-γ produced y CD8 T lymphocytes ws mesured using mouse IFN-γ CBA ssy (BD ioscience, Sn Jose, CA). In vivo niml studies nd ex vivo nlysis. Femle BALB/c mice (6-weeksold) were inoculted sucutneously with CT26 cells. When tumors were plple (round dy 7), plque forming units of denovirus were injected intrtumorlly nd GCV (75 mg/kg) ws dministered intrvenously twice dy for 14 dys. The denovirus ws dministered twice 7 dys prt, unless indicted otherwise. Tumor volume ws determined using the following formul: length width Similr procedures for sucutneous tumor formtion nd virus injection were performed in the CD8 T cell depletion experiment. E.G7 cells (C57/BL6 ckground; ) were injected into Rg1 / or C57/BL6 mice, nd denovirus nd GCV tretment ws performed s descried for CT26 cells. For ex vivo nlysis, E.G7 tumors in Rg1 / mice were treted with.spd1 long with intrvenous infusion of CD8 T cells isolted from n OT-1 mouse. Eightdy postinfection, peripherl lood mononucler cells were hrvested from the lood nd nlyzed y flow cytometry. SUPPLEMENTARY MATERIAL Figure S1. Schemtic digrm of the mouse TERT-trgeting trnssplicing riozyme (mtert-tr)-hsvtk construct. Figure S2. PD-L1 expression on the surfce of mouse cncer cells. Figure S3. Inhiition of secondry tumors y.spd1 tretment. ACKNOWLEDGMENTS This work ws supported y grnts from the Innovtive Reserch Institute for Cell Therpy (A6226) nd the Glol Core Reserch Center grnt (No )), Repulic of Kore vol. 21 no. 3 mr. 213

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