Intratumoral Coinjection of Two Adenoviral Vectors Expressing

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Intratumoral coinjection of two adenoviral vectors expressing functional interleukin-18 and inducible protein-10, respectively, synergizes to facilitate regression of established tumors Yongqing Liu,1,2 Hui Huang,1,2 Anurag Saxena,3 and Jim Xiang1,2 1Research Unit, Saskatchewan Cancer Agency, University of Saskatchewan, Saskatoon, Saskatchewan Canada S7N 4H4; Departments of 2Oncology; and 3Pathology, University of Saskatchewan, Saskatoon, Saskatchewan Canada S7N 0W0.

We have constructed two recombinant adenoviral vectors AdVIP-10 and AdVIL-18 expressing the functional chemokine IFN- inducible protein (IP)-10 and cytokine interleukin (IL)-18, respectively. Injection of either AdVIP-10 or AdVIL-18 subcutaneously into tumor nodules derived from the J558 murine myeloma cell line delayed some tumor growth but it was not curative in all cases. Coinjection of these two vectors at the same tumor nodule not only significantly suppressed the tumor growth, but also cured established tumors in 8 of 10 (80% tumor free) mice. The latter treatment stimulated T-cell infiltration into tumors in association with tumor necrosis formation, induced a type 1 immune response and induced the activation of J558 tumor–specific cytotoxic T lymphocytes. Moreover, the antitumor activity of IP-10 and IL-18 combined gene therapy was significantly diminished in mice with depletion of either CD4 + (50% tumor free) or CD8 + (40% tumor free) T cells, and completely lost (0% tumor free) in T cell– deficient nude and IFN- knockout mice, indicating the critical roles of T cells and IFN- in this therapeutical model. Taken together, the findings of this study demonstrate that the combined use of two adenoviral vectors expressing IP-10 and IL-18, respectively, synergize to facilitate regression of established tumors. These observations also suggest the potential use of double- recombinant adenoviral vectors expressing chemokines and immunomodulatory cytokines in cancer gene therapy. Cancer Gene Therapy (2002) 9, 533–542 doi:10.1038/sj.cgt.7700466 Keywords: adenoviral vector; IL-18; IP-10; cancer gene therapy

nterleukin (IL)-18 is a cytokine that was originally IL-18 can induce antitumor immune responses that are Idesignated as gamma interferon (IFN- )–inducing factor mediated by both CD4 + and CD8 + T cells and associated (IGIF).1 This cytokine strongly induces IFN- production with IFN- production.8,9 in spleen cells and enhances the expression and function of IFN- inducible protein-10 (IP-10)10 is a chemokine Fas ligand on T cells.2,3 Furthermore, it induces the that belongs to the CXC chemokine family known to production of granulocyte–macrophage colony-stimulating stimulate the IP-10 receptor CXCR3.11 IP-10, which is factor (GM-CSF).4 The in vitro expression of IL-2 receptor produced by activated monocytes, fibroblasts, endothelial by T cells is enhanced by IL-18, as is the production of IL-2. cells, and keratinocytes can induce chemotaxis of activated T IL-18 also enhances the production of Th1-type cytokines, cells and inhibit angiogenesis.12 IP-10 binds to a seven- which are associated with antitumor cytotoxic T-cell transmembrane G protein–coupled receptor, CXCR3, responses,5 and plays some roles in synergy with IL-12, expressed on activated T cells, leading to chemotaxis.11 particularly in the induction of IFN- .1,6 The use of IL-18 Stably transfected tumor cell lines expressing IP-10 were in the treatment of cancer has not been thoroughly rejected through an immune system–mediated mecha- investigated. Recently, Osaki et al demonstrated that nism.13 Furthermore, it has been recently shown that IP-10 administration of IL-18 before and after challenge with also displays its ability to impair tumoral angiogenesis.14 mouse melanoma cells significantly suppressed tumor Therefore, IP-10 has been involved in the antitumor immune growth and reduced the number of mice developing tumors responses by recruitment of T cells to the malignancies and from 60% to 20%.7 More recently, it has been shown that the nonimmune responses by its antiangiogenesis effect. These data suggest that both IL-18 and IP-10 have potent antitumor effects and are potent candidates for transgene Received November 11, 2001 therapy of cancer. Address correspondence and reprint requests to: Dr Jim Xiang, Research Adenoviral vectors have been extensively used for Unit, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, transimmunogene delivery into tumors in cancer gene 15 - 17 Saskatchewan, Canada S7N 4H4. E-mail: [email protected] therapy. In this study, we constructed two adenoviral Combination IL-18 and IP-10 gene therapy of tumor YLiuet al 534 vectors AdVIL-18 and AdVIP-10 expressing IL-18 and AdVLacZ expressing the E. coli -galactosidase and IP-10 molecules, respectively, and investigated the anti- AdVpLpA (i.e., with no gene insert) used as the marker tumor effects by intratumoral coinjection of these two and the control adenovirus, respectively, in this study were adenoviral vectors into J558 tumors established in BALB/c previously constructed in our laboratory.17 These E1- mice. deleted replication-deficient recombinant adenoviruses under the control of the cytomegalovirus (CMV) early/ immediate promoter/enhancer were amplified in 293 cell Materials and methods line (adenoviral E1 transformed human embryonic kidney cells), purified by cesium chloride ultracentrifugation and Antibodies, cell lines, and mice stored at À808C. Rat monoclonal antibodies Gk1.5 (anti-CD4) and 3.155 (anti-CD8) were purified by affinity chromatography Adenoviral transfection in vitro and in vivo from the respective hybridoma ascites. These two hybrid- To test the susceptibility of J558 tumor cells to adenoviral oma cell lines were purchased from the American Type infection, serial dilutions of AdVLacZ stock (2Â1010 pfu/ Culture Collection (ATCC, Rockville, MD). The biotin- d d mL) were added to J558 cells seeded in triplicate in 96- conjugated anti-mouse H-2K ,Ia, CD3, CD4, CD8, well plates (1Â105 cells/well) to form different multi- CD25, CD28, CD40L, CD69, Fas, and FasL antibodies as plicities of infection (MOIs). The cells were incubated with well as the phycoerythrin (PE)-conjugated anti-mouse the adenovirus in 293 serum-free medium (GIBCO, IL-4, IFN- , and perforin antibodies were obtained from Gaithersburg, MD) for 2 hours at 378C, then the media Pharmingen (Mississauga, Ontario, Canada). The fluores- was replaced with DMEM/10% FCS and the cells cein isothiocyanate (FITC)-conjugated avidin was incubated for an additional 24 hours at 378C. To assess obtained from Bio/Can Scientific (Mississauga, Ontario, -galactosidase expression16 the cells were fixed in form- Canada). J558 and SP2/0 are poorly immunogenic mouse aldehyde/glutaraldehyde, then stained and counterstained myeloma cell lines obtained from ATCC, and were with X-gal and nuclear fast red, respectively. The maintained in Dulbecco’s modified Eagle’s medium proportions of positive (i.e., blue-staining) cells were (DMEM) containing 10% fetal calf serum (FCS), determined from triplicate wells and taken as the percentage penicillin, and streptomycin (100 g/mL). Female of transduction. Control J558 cells transfected with BALB/c mice and T cell–deficient nude as well as AdVpLpA did not exhibit any intrinsic -galactosidase IFN- knockout (KO) mice on BALB/c background activity or false-positive staining. We observed a dose- (4–6 weeks old) were obtained from Charles River (St. dependent response to the infecting dose of adenovirus, Constant, Quebec, Canada) and The Jackson Laboratory with maximal staining (70%) at an MOI of 200. (Bar Harbor, ME), respectively, and maintained in the Therefore, an MOI of 200 was selected for transfection of animal facility at the Saskatoon Cancer Center. J558 cells with AdVIP-10 and AdVIL-18 in this study. For transfection of J558 cells with AdVIP-10 and AdVIL-18 at Recombinant adenoviral vectors 200 MOI, following viral adsorption for 1 hour at 378Cin A 1-kb cDNA fragment coding for the full open reading 24-well culture plates, the culture medium was replaced frame of mouse IP-10 gene was cloned by reverse with DMEM/10% FCS and the cells (0.1Â106 cells/well) transcription–polymerase chain reaction (RT-PCR) from incubated for another 24 hours at 378C. The J558 tumor a cDNA library of monocytes using the TaqI polymerase. cells transfected with AdVIP-10 (termed J558/AdVIP-10) The monocytes were collected by peritoneal lavage of mice and AdVIL-18 (termed J558/AdVIL-18) and their culture given an intraperitoneal (i.p.) injection of 1.5 mL of supernatants were then harvested for examination of IP-10 thioglycollate broth (Becton Dickinson, San Jose, CA) and IL-18 expression using the reverse transcription 4 days before harvest.17 Two primers specific for the mouse polymerase chain reaction (RT-PCR) and functional IP-10 gene were used, namely, the sense primer (50 - analysis of secreted IP-10 and IL-18, respectively. ATGAA CCCAA GTGCT GCCGT C-30 ) and the The ability of AdVLacZ to infect J558 tumors in vivo was antisense primer (50 -TTAAG GAGCC CTTTT AGACC also investigated. Briefly, 0.5Â106 J558 cells were subcuta- TTT-30 ). The cloned cDNA fragment was ligated into the neously (s.c.) injected into each athymic nude mouse. pCR2.1 vector (Invitrogene, Carlsbed, CA) to form Twelve days later, when the J558 tumors were 8mmin pCR2.1-IP-10. The IP-10 sequence was verified by the diameter, bolus 50-l aliquots of AdVLacZ virus (2Â109 dideoxy nucleotide sequencing method. The cDNA frag- plaque-forming units [PFU] or 5-fold dilutions thereof) ment of IP-10 (XbaI/HindIII) from the pCR2.1-IP-10 were injected into the J558 tumors. One day after viral vector was further ligated into the pLpA plasmid to injection, each J558 tumor was removed and cut into three form the adenoviral vector pLpA-IP-10. The cDNA clone approximately equal sections. These tumor tissues were encoding IL-18 was generously provided by Dr Hua Yu, mounted in tissue-freezing medium (Triangle Biomedical University of South Florida. The IL-18 cDNA insert (SalI Sciences, Durham, NC) and snap-frozen by immersion in 2- and XbaI) was ligated into the plasmid pLpA to form methylbutane (JT Baker, Phillipsburg, NJ), that had been the adenoviral vector pLpA-IL-18. The construction of chilled over liquid nitrogen. Frozen 6-m sections were recombinant adenoviruses AdVIP-10 and AdVIL-18 obtained for the analysis of -galactosidase expression.17 To from pLpA-IP-10/pLpA-IL-18 and pJM17 vectors was accomplish this, the tissue sections of transfected J558 conducted as previously described.17 The adenoviruses tumors were fixed in phosphate-buffered saline (PBS)

Cancer Gene Therapy Combination IL-18 and IP-10 gene therapy of tumor YLiuet al 535 containing 37% formaldehyde and 25% glutaraldehyde, Functional activity of IL-18 stained with X-gal, and then counterstained with nuclear fast 6 To test the functional activity of IL-18, 3Â10 splenocytes in red (Poly Scientific, Bay Shore, NY). The mean proportion 1 mL DMEM containing 10% FCS, anti-CD3 antibody of blue-staining J558 cells from triplicate wells or from three (2 g/mL) were added to each well of a 24-well plate, tumor sections were taken as the percentage of transfection. which contained 1 mL of culture supernatant from J558/ We observed a dose-dependent response to the adenovirus, AdVpLpA and J558/AdV-IL-18 cells, respectively. The with maximum staining ( 40% of cells) at 2 109 PFU Â cells were incubated at 378C for 24 hours. Quantitation of AdVLacZ. Thereafter, 2 109 PFU was selected as the Â secreted IFN- from cultured splenocytes was carried out in optimal dose for delivery of AdVIP-10/AdVIL-18 to an enzyme-linked immunosorbent assay (ELISA) by using tumors in vivo. the IFN- ELISA kit (Endogene, Woburn, MA). The results were normalized to the known standard curves. RT-PCR analysis Naı¨ve splenic T lymphocytes were nylon wool purified from Animal studies 18 BALB/c mice as previously described (>99% purity as BALB/c mice (10 per group) were inoculated s.c. in their determined using the anti-CD3 antibody by flow cytometry, right thighs with 0.5Â106 viable J558 tumor cells. Ten days data not shown). T cells were activated in DMEM, 10% later, when tumors reached 4–5 mm in diameter, different FCS, IL-2 (20 U/mL), and Con A (2 g/mL) for 4 days. recombinant adenoviruses (AdVIP-10, AdVIL-18, Total RNA was obtained from the activated T lymphocytes, AdVpLpA, or a mixture of AdVIP-10 and AdVIL-18) at the wild-type J558, and the transfected J558/AdVIP-10 and 2Â109 PFU consisting of 1Â109 PFU of each virus were J558/AdVIL-18 cells. The first-strand cDNA synthesis for intratumorally injected in a volume of 10–20 l. Tumor the RT-PCR was performed with 5 g of RNA using a growth was monitored once every 2 days by measuring two commercial kit (Stratagene, La Jolla, CA), following the perpendicular tumor diameters using a precision caliper. manufacturer’s instructions. The PCR primers were specific 0 Animal showing severe distress or with tumors that exceeded for IP-10 (the sense primer, 5 -ATGAA CCCAA GTGCT 1.5 cm in diameter were sacrificed for ethical reasons GCCGT C-30; the antisense primer 50 -TTAAG GAGCC 0 0 according to the institutional guidelines. Animals with tumor CTTTT AGACC TTT-3 ), IL-18 (the sense primer, 5 - regression were monitored for a total of 60 days. To induce CGTTG ACAAA AGACA GCGTG-30; the antisense 6 0 0 bilateral tumors, 0.5Â10 viable J558 tumor cells were primer, 5 -CGTTG ACAAA AGACA GCGTG-3 ), chemo- injected s.c. into BALB/c mice (six per group) at both right kine (IP-10) receptor CXCR3 (the sense primer, 5’- and left thighs. To study the protective immunity, mice with CCTAC GATTA TGGG AAAC GAG-3’; the antisense tumor regression were rechallenged with s.c. injection of primer, 5’-TGATT CTCTC CGTGA AGATG ACG-3’, 6 11 0.5Â10 J558 or SP2/0 tumor cells 2 months subsequent to Ref. ), and glyceraldehyde phosphate dehydrogenase the tumor regression. (GAPDH: sense primer, 5’-CAGGT TGTCT CCTGC To study the immune mechanisms involved in J558 GACTT-3’; antisense primer, 5’-CTTGC TCAGT GTCCT tumor regression with adenovirus-mediated gene transfer, TGCTG-3’). The PCR conditions comprised 1 cycle at we investigated tumor growth or tumor regression in T-cell 948C (5 minutes), 548C (1 minute), and 728C(1minute) subset depleted, T cell–deficient nude and IFN- – and 25 cycles at 948C (1 minute), 558C (1 minute), and deficient KO mice, respectively. T-cell subset depletion 728C (1 minute). All PCR reaction products were resolved experiments were performed in vivo as described.19 Briefly, on 1% agarose gels with ethidium bromide staining. BALB/c mice (eight per group) were i.p. injected with anti-CD4 and anti-CD8 antibodies, respectively, at 0.5 mg Chemotaxis assay per mouse 2 days before, on the day of and 3 and 6 days subsequent to the intratumoral injection of adenovirus. A The chemotactic response of activated T lymphocytes to IP- 12 total of four injections was given to each mouse. As a 10 was examined using modified Boyden microchemo- control, one group of mice was injected with the same taxis chambers (Neuroprobe, Gaithersburg, MD) and amount of irrelevant isotype-matched rat antibodies, polyvinyl pyrrolidone-free 5-m pore-size polycarbonate + + 18 respectively. The depletion of CD4 and CD8 subsets membranes, essentially as described. Recombinant IP-10 of T cells in spleens were >95% by flow cytometric (R&D Systems, Minneapolis, MN), diluted in DMEM/ analysis as previously described9 (data not shown). Mice 0.1% bovine serum albumin (BSA) to 0.1–1000 ng/mL, 5 were monitored for tumor progression or regression as was added to triplicate lower chambers of the wells, and 10 described above. activated T lymphocytes in DMEM/BSA were added to the upper chambers. After incubation for 2 hours at 378C, the cells that had not migrated into the membranes were wiped Histology from the upper surfaces of the membranes, which were then Tumor nodules or tissues at the injection sites were removed fixed in 70% methanol and stained using a Diff-Quik kit for histological analysis 1, 3, and 7 days subsequent to the (American Scientific Products, McGraw Hill, IL). The coinjection of AdVIP-10 and AdVIL-18. J558 tumor lymphocytes that were associated with the membranes were nodules or tissues at the tumor inoculation site were fixed enumerated by direct counting in a blinded fashion of at least in 10% formaldehyde and embedded in paraffin. Sections of nine 40Â objective fields per well. The results are expressed 6- to 7-m thickness were stained with hematoxylin–eosin as the mean number of cells/40Â field ( ±SEM). according to the standard procedures.

Cancer Gene Therapy Combination IL-18 and IP-10 gene therapy of tumor YLiuet al 536 Phenotypic characterization of activated T and J558 tumor (Fig 1B). We then assessed the activated T-cell chemotactic cells properties of recombinant IP-10, as well that of the culture supernatants of J558/AdVIP-10 tumor cells. Our data Mouse spleens were removed for preparation of single-cell suspensions 2 weeks after treatment of AdVIP-10 and confirmed that both the recombinant IP-10 and the J558/ AdVIP-10 supernatant (but not the control J558/AdVpLpA AdVIL-18. Red cells were lysed using 0.84% ammonium 6 5 one) were able to chemoattract the activated T cells in a chloride. Splenocytes (5Â10 ) were cocultured with 2Â10 dose-dependent fashion (Fig 2). irradiated J558 cells in each well of a 24-well plate. Four days subsequently, the activated lymphocytes were purified Functional IL-18 secreted by transfected J558/AdVIL-18 from the cultures using Ficoll-Paque density gradient tumor cells centrifugation and analyzed by flow cytometry. Briefly, these T cells were incubated for 1 hour on ice with the To examine IL-18 expression of transfected J558/AdVIL- biotin-conjugated rat anti-mouse CD3, CD25, CD28, 18 cells, RNA extracted from J558/AdVIL-18 cells was CD40L, CD69 and FasL antibodies (5 g/mL), washed subjected to RT-PCR analysis. As shown in Figure 1A, IL- with PBS, then incubated for another 1 hour on ice with 18 expression was found in AdVIL-18–transfected J558/ FITC-conjugated avidin. After another three washes with AdVIL-18, but not in the control adenovirus-transfected PBS, the cells were analyzed by flow cytometry. Isotype- J558/AdVpLpA cells. To evaluate whether the IL-18 is matched irrelevant antibodies were used as controls. To functional, the culture supernatant of J558/AdVIL-18 was examine the intracellular expression of cytokines, these added to the splenocyte culture. One day following activated T cells were processed using a commercial kit incubation, the IFN- production in splenocyte culture with (Cytofix/CytoPerm Plus with GolgiPlug; Pharmingen), J558/AdVIL-18 supernatant was estimated to be 80 U/mL stained with RE-conjugated anti–IL-4, IFN- and perforin compared to the 20 U/mL seen in splenocyte culture with antibodies according to the manufacturer’s protocols and J558/AdVpLpA supernatant. These data indicate that IL-18 analyzed by flow cytometry. For phenotypic characterization secreted by J558/AdVIL-18 cells is functional and able to of SP2/0 tumor cells, these tumor cells were stained with the significantly enhance IFN- production of splenocytes in anti–H-2Kd,Iad, and Fas antibodies and analyzed by flow vitro. cytometry as described above.

Cytotoxicity assay The activated T cells were further used as effector cells in chromium release assays. Target cells included J558 and SP2/0 tumor cells, which were radiolabeled with [ 51Cr]chromate. Ten thousand labeled target cells per well were mixed in triplicate with effector cells at various effector/target cell ratios, either as is or with anti–H-2Kd and anti-Iad antibodies (15 g/mL) added, respectively, and were incubated for 6 hours. Percentage of specific lysis was calculated as: 100Â[(experimental CPMÀspontaneous CPM)/(maximal CPMÀspontaneous CPM)]. Spontaneous count per minute (CPM) released in the absence of effector cells was less than 10% of specific lysis. The maximal CPM was released by adding 1% Triton X-100 to wells in the experiment.

Results Functional IP-10 secreted by transfected J558/AdVIP-10 tumor cells To examine IP-10 expression of transfected J558/AdVIP- 10 cells, RNA extracted from J558/AdVIP-10 cells was subjected to RT-PCR analysis. As shown in Figure 1A, IP- 10 expression was found in AdVIP-10–transfected J558/ AdVIP-10, but not in the control adenovirus-transfected Figure 1 Expression of chemokine IP-10, IL-18, and chemokine J558/AdVpLpA cells. To evaluate whether the secreted IP- receptor CXCR3. A: RNA was obtained from wild-type J558 and the 10 is functional, the culture supernatant of J558/AdVIP-10, transfected J558/AdVIP-10 or J558/AdVIL-18 cells for examination of IP-10 or IL-18 expression. B: RNA was obtained from naı¨ve and which contained IP-10 was subjected to chemotaxis activated T cells for examination of CXCR4 expression. The first- analysis. To validate the IP-10–mediated T-cell response, strand cDNA was synthesized from RNA using the reverse tran- we first demonstrated using RT-PCR that highly purified scriptase, and the PCRs were conducted using sets of primers for mouse activated T cells expressed substantial levels of the IP-10, CXCR4, and the control ‘‘housekeeping’’ gene GAPDH, IP-10 receptor CXCR3 whereas J558 tumor cells did not respectively.

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Figure 3 Combination of intratumoral AdVIP-10 and AdVIL-18. Figure 2 IP-10 is a specific chemoattractant for activated T cells. A: Groups of mice bearing J558 tumor nodules inoculated 10 days Chemotaxis assays were performed using naı¨ve and activated T cells before treatment onset were intratumorally injected with the indicated in response to recombinant IP-10 (0–1000 ng/mL). After a 2-hour adenovirus (2Â109 PFU). Individual evolution of the tumor diameter chemoattraction period, the number of activated T lymphocytes that is shown, and the fraction of mice completely rejecting their tumors is were associated with the membranes was determined by direct indicated. One representative experiment of two is shown. We counting and the results expressed as the mean number of cells/40Â performed a statistic analysis on the combined data from two field ( ±SEM). Chemotaxis assays were also performed using independent experiments by using the 2 (chi-squared) test. We activated T cells in response to aqueous supernatants of J558/ found that the mouse survival rates (tumor-free mice/total treated AdVpLpA and J558/AdVIP-10 cells. One representative experiment mice) in CD4 + and CD8 + T cell–depleted groups are significantly of two is shown. lower than that in the immune competent mouse group (P<.05).

Intratumoral coinjection of AdVIP-10 and AdVIL-18 displayed synergistic antitumor effects transgenes in the same tumor nodule for induction of the synergistic effects, we conducted experiments in which two To explore whether AdVIP-10 or AdVIL-18 had ther- tumor nodules were simultaneously generated by s.c. apeutic effects against tumors, J558 tumor cells were s.c. inoculation of J558 tumor cells into opposite thighs of injected into syngeneic BALB/c mice. On day 10, tumor the same mouse. As shown in Table 1, the combination nodules ranging 4–5 mm in diameter were injected with 9 treatment of AdVIP-10 and AdVIL-18 led to tumor nodule 2Â10 PFU of AdVIP-10, AdVIL-18, and the control adenovirus AdVpLpA. As shown in Figure 3, all tumors regression at the treated sites in five of six mice. However, treatment of mice with AdVIP-10 and AdVIL-18 at each were lethal in the control group of mice. Tumor-bearing thigh of the same mouse, respectively, did not cure any mice injected with AdVpLpA died within 4 weeks after mice although the tumor growth in these mice was also tumor inoculation. Although either AdVIP-10 or AdVIL- delayed compared with that in mice treated with AdVpLpA 18 treatment alone did not cure any mice, a significant delay of tumor growth was noted. However, no significant difference was found between these two treatment groups. To study the potent synergistic antitumor effects derived Table 1 Tumor regression of bilateral s.c. J558 tumors upon AdVIP- from a combinational treatment of AdVIP-10 and AdVIL- 10 and AdVIL-18 intratumoral treatment 18, we then conducted coinjection of AdVIP-10 (1Â109 9 PFU) and AdVIL-18 (1Â10 PFU) into tumors simulta- Intratumoral treatment Tumor regression* neously. As shown in Figure 3, the combinational treatment of AdVIP-10 with AdVIL-18 resulted in complete Right tumor Left tumor Right Left tumor regression in 8 of 10 (80%) mice. The tumor AdVpLpA AdVpLpA 0/6 0/6 growth in the other 20% of mice was dramatically retarded. AdVIP-10 AdVIL-18 0/6 0/6 These data clearly indicate the synergistic antitumor effects AdVIP-10/AdVIL-18y –5/6– derived from the combination treatment of AdVIP-10 and AdVIL-18. Mice bilaterally injected with J558 tumors received the indicated treatment in each thigh 10 days after tumor inoculation and were monitored for 60 days for complete tumor regression. A total of 6 The synergistic effect required coinjection of AdVIP-10 and 0.5Â10 J558 tumor cells were injected s.c. into each thigh of the AdVIL-18 in the same tumor nodule mice. Mean size, 4–5 mm in diameter. *Fraction of mice with undetectable tumor 60 days after treatment. To ascertain as to whether adenoviral gene transfer of IP- yA total of 0.5Â106 J558 tumor cells were injected s.c. into only the 10 and IL-18 required expression of the therapeutic right thigh of the mice.

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Figure 4 Lymphocyte infiltration into J558 tumors after coinjection of AdVIP-10 and AdVIL-18. BALB/c mice were s.c. injected with J558 tumor cells. Ten days later, when tumors reached 4–5 mm in diameter, each tumor was treated by coinjection of AdVIP-10 and AdVIL-18. At varying times thereafter injection site biopsies were processed for histologic assessment of the tissue responses. A: Section from a 10-day J558 tumor growing in a BALB/c mouse, showing brisk mitotic activity (arrows) without lymphocyte infiltration. B–E:Sections taken from tumors at 6 and 12 hours or 2 and 6 days subsequent to the coinjection of AdVIP-10 and AdVIL-18 into J558 tumors. B: Six hours subsequent to the treatment, J558 tumors displayed significant lymphocyte infiltration (arrows) and isolated apoptotic tumor cells (arrow heads). C: Twelve hours subsequent to the treatment, there were small foci of necrosis (center) associated with many lymphocytes (arrows). D: Areas of significant necrosis (arrows) appeared on day 2. E: Six days subsequent to the adenovirus injection, tumor cells were no longer detectable, with the injections sites comprising only lymphocytes, macrophages and fibroblasts. Sections were stained with hematoxylin and eosin. Magnification: Â240 for (A), (B), (C), and (E) and Â120 for (D).

injection. Their tumor growth curves are similar to those of IFN- in tumor regression derived from the treatment of shown in Figure 3 (data not shown). These results indicate AdVIP-10 and AdVIL-18. the requirement of coexpression of IP-10 and IL-18 in the same tumor nodule for induction of the synergistic Both CD4 + and CD8 + T cells were involved in the antitumor effects. antitumor efficacy of combined administration of AdVIP- 10 and AdVIL-18 IFN- played a critical role in the antitumor effect of To study the cellular immune responses, tumor nodules or combinational treatment tissues at the injection sites were removed for histological Because the antitumor immunity of both IP-10 and IL-18 is analysis 12 hours or 2 and 6 days subsequent to the dependent on the induction of IFN- production, we further coinjection of AdVIP-10 and AdVIL-18. As shown in conducted an experiment to study the potent role of IFN- Figure 4A, the wild-type J558 tumors grew aggressively using the IFN- KO mice in this therapeutic tumor model. with many mitosis. Six hours postinjection, a few areas of As shown in Figure 5 intratumoral treatment of AdVIP-10 tumor displayed a significant amount of lymphocyte and AdVIL-18 did not cure any tumor-bearing IFN- KO infiltration associated with some isolated apoptotic tumor mice. Tumors grew aggressively. All eight mice died within cells (Fig 4B). Twelve hours subsequent to the treatment, 18 days after adenovirus injection, indicating the critical role small foci of necrosis were observed in association with

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Figure 7 Phenotypic analysis of the wild-type J558 tumor cells. J558 tumor cells were stained with anti–H-2Kd,Iad and Fas antibodies (solid lines) or isotype-matched irrelevant control antibodies (dotted lines), followed by FITC-anti-mouse antibody and analyzed by flow cytometry.

phages, and fibroblasts were present (Fig 4E). These data suggest that T cells may be involved in the tumor necrosis formation leading to the complete tumor regression derived Figure 5 T-cell and IFN- requirement for tumor rejection with from the coinjection of AdVIP-10 and IL-18. AdVIP-10 and AdVIL-18. J558 tumor cells were s.c. injected to To confirm the potent involvement of T cells in the groups of mice depleted of CD4 + and CD8 + T cells, and to groups of antitumor immune responses, we further conducted the T cell–deficient nude mice and IFN- KO mice. When J558 tumor + + nodules reached 4–5 mm in diameter, tumor-bearing mice were therapeutic experiments in CD4 and CD8 T cell–depleted intratumorally injected with the indicated adenovirus (2Â109 PFU). or T cell–deficient mice. We found that 50% and 40% of + + Individual evolution of the tumor diameter is shown, and the fraction of CD4 and CD8 T cell–depleted mice became tumor free mice completely rejecting their tumors is indicated. A group of mice after intratumoral coinjection of AdVIP-10 and AdVIL-18 receiving the polyclonal rat IgG rejected their tumor on treatment of (Fig 5). The mouse survival rates in CD4 + and CD8 + T AdVIP-10 and IL-18 as fast as those in mice not receiving antibody cell–depleted groups are significantly lower than that in the treatment (data not shown). One representative experiment of two is immune competent mouse group after the treatment shown. (P<.05). These data suggest that both CD4 + and CD8 + T cells are involved in mediating the antitumor immune many lymphocytes in tumors (Fig 4C). Two days post- responses of combined treatment of AdVIP-10 and injection, multiple areas of necrosis occurred in the tumors AdVIL-18. Furthermore, in T cell–deficient BALB/c nude (Fig 4D). By 6 days postinjection the tumor cells were no mice, intratumoral treatment resulted in lethal tumor longer detectable, although numerous lymphocytes, macro- progression in all eight nude mice (Fig 5), further confirming the absolute T-cell requirement.

Protective immunity was against the wild-type J558 tumors To examine whether the tumor regression of combination treatment results in the induction of protective immunity against the wild-type J558 tumor, eight mice that experi- enced tumor regression were rechallenged in a similar fashion using the parental J558 or SP2/0 tumor cells. We found that no J558 tumor growth was found in mice with tumor regression after the combination treatment, although the tumor growth of an irrelevant SP2/0 tumor cell line occurred in all these immunized mice (data not shown). These data illustrate that mice with tumor regression after the combination treatment of AdVIP-10 and AdVIL-18 induced a Figure 6 Phenotypic analysis of J558 tumor–specific T cells. Upper protective immune response specifically against the parental panel: Splenic lymphocytes from mice with tumor regression after treatment of AdVIP-10 and AdVIL-18 were stimulated in vitro for J558 tumor, but not against the irrelevant SP2/0 tumor. 4 days with irradiated J558 cells (activated T cells), then immunos- tained for assessment of multiple markers by flow cytometry using Intratumoral coinjection of AdVIP-10 and AdVIL-18 specific antibodies. Unstimulated T cells from naı¨ve mice were used induced type 1 antitumor immune responses as negative control cells (naı¨ve T cells). Cell surface CD3, CD25, To elucidate the immune mechanism involved in tumor CD28, CD40L, CD69, and FasL were detected using marker specific regression, the activated T cells were prepared by coculturing primary antibodies (solid lines) or isotype-matched irrelevant control antibodies (dotted lines) and FITC-conjugated secondary anti- splenocytes of the mice with tumor regression with irradiated bodies. Lower panel: The activated T cells were permeabilized J558 tumor cells for 4 days. These T cells were purified with and stained for intracellular IL-4, IFN- , and perforin using a Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, Swe- commercial kit and PE-conjugated antibodies (solid lines). Unper- den), and then subjected to phenotypic characterization by meablized T cells were used as negative control cells (dotted lines). flow cytometry. As shown in Figure 6 (upper panel),

Cancer Gene Therapy Combination IL-18 and IP-10 gene therapy of tumor YLiuet al 540 essentially all of these cells were CD3 + and CD28 + ; they cells. As shown in Figure 7, the wild-type J558 tumor cells uniformly displayed high-level expression of the two T-cell displayed major histocompatibility complex (MHC) class I activation markers, CD25 (IL-2R) and CD69, compared to (H-2Kd ) antigen, but neither MHC class II (Iad ) antigen nor low-level expression of these molecules in naı¨ve T cells, Fas molecule. We then conducted chromium release assays indicating they were indeed activated T cells. In addition, using activated T and 51Cr-labeled J558 tumor cells as these activated T cells also displayed the costimulatory effector and target cells, respectively. As shown in Figure 8, molecule CD40L and high level of FasL, compared to T cells the activated T cells from mice with tumor regression from naı¨ve mice. Furthermore, following in vitro J558 cell showed a specific killing activity (48% specific killing at an challenge, these T cells stained positively for intracellular effector:target cell ratio of 50) for J558 tumor cells, but not IFN- , but not for IL-4 (Fig 6, lower panel), indicating that for the irrelevant SP2/0 tumor cells (0% specific killing at an the intratumoral treatment of AdVIP-10 and AdVIL-18 effector:target cell ratio of 50). Nearly 80% of CTL- induced type 1 antitumor immune responses. In addition, mediated killing could be blocked using the anti–H-2Kd, these activated T cells also expressed significant amounts of but not the anti-Iad, antibodies (data not shown). On the intracellular perforin. contrary, T cells from mice inoculated with irradiated J558 tumor cells showed little killing activity (4% specific killing at an effector:target cell ratio of 50) for J558 tumor cells. Cytotoxic T lymphocytes mediated the antitumor immune These results indicate that mice with complete tumor responses regression after the treatment developed a strong J558- Because the phenotypic markers associated with these specific immune response mediated by CD8 + CTLs. activated T cells were consistent with cytotoxic T lymphocytes (CTLs), we tested their cytolytic activities. We first examined the phenotypic characterization of J558 tumor Discussion

In cancer therapy, the successful manipulation of the immune system requires a deep understanding of the endogenous immune response to neoplastic cells and, more specifically, comprehending why natural antitumor responses are rela- tively ineffective. The current paradigm holds that tumor cells are poorly immunogenic. Bearing this in mind, much work has been directed at enhancing the immunogenicity of cancer cells. Adenoviral vectors have been extensively used in cancer gene therapy for transgene delivery into tumors to increase the immunogenicity of tumors.15 - 17 Although these approaches are effective in induction of protective immunity against certain tumors, their therapeutic benefit is still limited because they can only either delay tumor growth or regress some of the tumors in early stages. To improve the therapeutic efficacy, the combinational use of two immunogene-expressing adenoviral vectors was reported. Putzer et al have demonstrated that IL-12 and B7-1 costimulatory molecule expressed by an adenovirus vector acted synergistically to facilitate tumor regression in a transgenic PyMidT animal model.20 Recently, Emtage et al have shown that adenoviral vectors expressing lymphotactin and IL-12 enhanced tumor regression in murine breast cancer models.21 More recently, Narvaiza et al have reported that intratumoral coinjection of two adenoviruses, one encoding the chemokine IP-10 and another encoding cytokine IL-12, resulted in marked antitumor synergy.22 In this study, we constructed two recombinant adenoviral vectors AdVIL-18 and AdVIP-10 expressing the functional Figure 8 Cytotoxicity assay. The activated T cells (black squares), IL-18 and IP-10 molecules, respectively, and investigated as described in Figure 6, were used as effector cells in chromium the antitumor effects by intratumoral injection of these two 51 release assays, in which Cr-labeled J558 tumor cells were used as adenoviral vectors into established J558 tumors in animal target cells. To confirm that T-cell cytotoxicity was J558 tumor– models. Our data showed that treatment of either AdVIP-10 specific, we also included SP2/0 cells (black circles) as a target control. Activated T cells from mice inoculated with irradiated J558 or AdVIL-18 alone cannot cure any established tumors tumor cells were used as a effector control (open circles) in chromium though the delay of tumor growth in these treated mice was release assays, in which 51Cr-labeled J558 tumor cells were used as noted. Interestingly, treatment of coinjection of AdVIP-10 target cells. Each point represents the mean of triplicates. One and AdVIL-18 not only significantly suppressed the tumor representative experiment of two is shown. growth, but also cured established tumors in 8 of 10 (80%)

Cancer Gene Therapy Combination IL-18 and IP-10 gene therapy of tumor YLiuet al 541 tumor-bearing mice, indicating a synergized antitumor activation of CTLs,30 natural killer cells,31 and macro- immunity. However, the synergy of chemokine IP-10 and phages32,33 and to induce/enhance the expression of class II cytokine IL-18 only took place when both recombinant MHC antigens.34 Through these effects, IFN- plays a adenoviruses were given to the same nodule, but not when central role in promoting innate and adaptive mechanisms identical doses were injected into distant tumor nodules. This of host defense including tumor immunity.35 In fact, this is in agreement with the so-called ‘‘attraction and activa- notion was supported recently by the observations that IL- tion’’ hypothesis by Paillard,23 which predicts the necessity 12–induced tumor regression is blocked by neutralizing of colocalization of immunostimulatory and chemoattractant IFN- produced after IL-12 injections.36,37 Tumor rejection factors as previously seen for chemokine lymphotactin and involves a number of processes: sensitization/activation of cytokine IL-2.24 effector T cells with tumor antigens in lymphoid organs; In this study, we also showed that the coinjection of migration of T cells together with other effector cells to AdVIP-10 and AdVIL-18 induced activation of tumor- tumor masses; and tumor cell attack by these tumor- specific T cells with a dominant expression of IFN- ;this infiltrating effectors. To study the role of IFN- , we grew pattern of cytokine expression is consistent with the type 1 J558 tumors in IFN- KO mice and treated these tumors immune response usually associated with antitumor immun- with intratumoral coinjection of AdVIP-10 and AdVIL-18. ity.25 These activated T cells displayed efficient tumor- Our data showed that all tumors grew aggressively in these specific killing activity (48% specific killing at an effector: IFN- KO mice after the treatment, indicating the critical target cell ratio of 50) for J558 tumor cells in vitro. CTLs can role of IFN- in this antitumor therapeutic model. Previous directly eradicate tumor cells through cognate interactions studies38,39 have suggested that IFN- plays a role in some that may involve either perforin or Fas-mediated lytic of the processes such as an intratumoral antitumor effector mechanisms.26 In the present study, the lymphotoxin mechanism. More recently, Nakajima et al have demon- perforin but not Fas/FasL interaction mediates the CTL strated another critical role of IFN- in the process of activity because these activated T cells uniformly displayed inducing T-cell migration into tumors.40 In their report, T high level expression of FasL and perforin, whereas the cells with capacity to reject tumor cells failed to migrate to wild-type J558 tumor cells do not express Fas molecule. tumor sites in IFN- –deficient mice. Therefore, IFN- may IP-10 has two potential antitumor effects, antiangio- play two roles in this therapeutic model, the recruitment of genesis and immunodulation, similar to what has been CTLs into tumor sites and the local antitumor effect. described for IL-12.27 Recently, IP-10 was reported Taken together, the findings of this study demonstrate that necessary for effector T-cell trafficking and function and the combined use of two adenoviral vectors expressing IP- host survival in Toxoplasma gondii infection.28 Neutraliza- 10 and IL-18, respectively, synergize to facilitate regression tion of IP-10 inhibited the influx of T cells into spleens and of established tumors. These observations also suggest the livers of mice infected by T. gondii, resulting in a potential use of double-recombinant adenoviral vectors simultaneous 3-log increase in the parasite burden and a expressing chemokines and immunomodulatory cytokines in significant decrease in survival. In the present study, we cancer gene therapy. demonstrated that significant amounts of lymphocytes infiltrated into tumors in association with tumor necrosis formation leading to complete tumor regression after the combined treatment of AdVIP-10 and AdVIL-18. We also Acknowledgments demonstrated that the number of tumor-free mice was decreased to 50% and 40% in CD4 + and CD8 + T cell– This study was supported by a research grant (ROP-15151) depleted mice, respectively, after the treatment. The mouse from the Canadian Institute of Health Research. We thank Dr survival rates in CD4 + and CD8 + T cell–depleted groups H Tabel for his useful comments on this manuscript and Mr are significantly lower than that in the immune competent X Bi for his technical support in this study. mouse group after the treatment (P<.05). The above results indicate that both CD4 + and CD8 + T cells were involved in the antitumor efficacy of combined administration of References AdVIP-10 and AdVIL-18. 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