Adenovirus-Mediated Gene Transfer of Pathogen-Associated Molecular

ORIGINAL ARTICLE Adenovirus-mediated gene transfer of pathogen-associated molecular patterns for cancer immunotherapy C Tosch1, M Geist1, C Ledoux1, C Ziller-Remi1, S Paul1, P Erbs1, N Corvaia2, P Von Hoegen3,4, J-M Balloul1 and H Haegel1 1Molecular Immunology Department, Transgene, Strasbourg, France; 2Centre d’Immunologie Pierre Fabre, Saint Julien en Genevoix, France and 3Euraccine, Lasne, Belgium

The delivery of stimulatory signals to dendritic cells (DCs) in the tumor microenvironment could be an effective means to break tumor- induced tolerance. The work presented here evaluates the immunostimulatory properties of pathogen-associated molecular patterns (PAMPs), microbial molecules which bind Toll-like receptors and deliver activating signals to immune cells, when expressed in tumor cells using adenoviral (Ad) vectors. In vitro, transduction of A549 tumor cells with Ad vectors expressing either flagellin from Listeria monocytogenes or P40 protein from Klebsiella pneumoniae induced the maturation of human monocyte-derived DCs in co-cultures. In mixed lymphocyte reactions (MLRs), Ad-flagellin and Ad-P40 transduction of tumor cells stimulated lymphocyte proliferation and the secretion of IFN-g. In vivo, these vectors were used either as stand-alone immunoadjuvants injected intratumorally or as vaccine adjuvants combined with a tumor antigen-expressing vector. When Ad-PAMPs were administered intratumorally to mice bearing subcutaneous syngeneic B16F0-CAR (cocksackie-adenovirus receptor) melanomas, tumor progression was transiently inhibited by Ad- P40. In a therapeutic vaccine setting, the combination of Ad-MUC1 and Ad-PAMP vectors injected subcutaneously delayed the growth of implanted RenCa-MUC1 tumors and improved tumor rejection when compared with vaccination with Ad-MUC1 alone. These results suggest that Ad-PAMPs could be effective immunoadjuvants for cancer immunotherapy. Cancer Gene Therapy (2009) 16, 310–319; doi:10.1038/cgt.2008.85; published online 24 October 2008 Keywords: pathogen-associated molecular pattern; adenoviral vector; cancer immunotherapy

Introduction activated or mature DCs can stimulate lymphocyte proliferation and Th1-oriented antitumor responses.1–8 One mechanism by which tumors induce immunosuppres- Current knowledge suggests that successful immu- sion is the functional inhibition of antigen-presenting cells, notherapy of cancer should involve breaking of tumor- resulting in inefficient stimulation of the cytotoxic T-cell induced tolerance. This requires powerful adjuvants to responses required for tumor cell lysis. Dendritic cells (DCs) adjunct cancer vaccines. The delivery of stimulatory are professional antigen-presenting cells that play an signals to DCs directly in the tumor microenvironment important role in immune resistance to tumors.1 Cancer could be an effective means to stimulate a tumor-specific patients present defects in the myeloid cell compartment immune response. Indeed, many reports have documented with an accumulation of immature myeloid cells and the presence of DCs in human tumors, including reduced numbers of differentiated DCs.2 Moreover, in melanoma, breast, lung, head and neck, and prostate 2 cancer patients as in tumor-bearing mice, DCs are found in cancers. Harnessing this population offers some advan- an immature state characterized by low expression of tages as all potential tumor antigens are available at this costimulatory molecules and weak antigen presentation site and if proper activation of the tumor-infiltrating DCs capacity.2–4 This impaired DC function may be a local or a is provided, they have the potential to stimulate T cells 9,10 systemic phenomenon.2 Although immature myeloid cells and tumor rejection. With this in mind, intratumoral or DCs induce T-cell unresponsiveness and tolerance administration of ligands for Toll-like receptors (TLRs) is notably through the development of regulatory T cells, being explored as a means to deliver activating signals to immune cells9,11–13 and has been shown to act favorably for the host in the context of cancer immunotherapy.14,15 Correspondence: Dr H Haegel, Immunopharmacology, Transgene, The evolutionary conserved family of TLR mediates 11 rue de Molsheim, Strasbourg Cedex 67082, France. 16 E-mail: [email protected] signals regulating innate and adaptive immunity. At 4Current address: The Weinberg Group LLC, Brussels, Belgium. least 11 different TLRs in human or mouse have been Received 5 May 2008; revised 22 July 2008; accepted 24 August identified with varying expression patterns on DCs, 2008; published online 24 October 2008 macrophages, other APCs and lymphocytes. Adenovirus-PAMPs for cancer immunotherapy C Tosch et al 311 Microbial products termed pathogen-associated mole- Materials and methods cular patterns (PAMPs), which may be proteins, lipids Adenoviral vectors and/or glycans, activate TLRs thereby triggering innate All recombinant viral genomes were derived from an Ad immunity. The binding of PAMPS to TLRs induces the serotype 5 constructed by homologous recombination in expression of co-stimulatory molecules on DCs and the Escherichia coli as described earlier.31 Ad serotype 5 were production of cytokines, which may drive Th1- or Th2- 14 deleted in E1 (nucleotides 459–3327) and E3 regions type responses. When expressed on the surface of tumor (nucleotides 28 592–30 470). Expression cassettes were cells, PAMPs may be recognized as ‘danger signals’ by 17 inserted in place of the E1 region, in sense orientation. tumor-resident DCs and other TLR-expressing cells and For the generation of viruses, the viral genomes were trigger a potent immune response. released from the respective recombinant plasmids by The stimulatory properties of the P40 protein from PacI digestion and transfected into 293 cells. Virus stock Klebsiella pneumoniae (outer membrane protein A, amplification, purification and titration of infectious units KpOmpA) have been demonstrated on human and 32 18,19 (i.u.) were carried out as described earlier. The particle/ murine antigen-presenting cells. P40 binds TLR2 on i.u. ratio was approximately 30 for all vector prepara- the surface of antigen-presenting cells (DCs and macro- tions. Purified virus was stored in viral storage buffer phages) and induces the phenotypic maturation of containing 1 M sucrose, 10 mM Tris-HCl (pH 8.5), 1 mM DCs,18–20 secretion of interleukin (IL)-12 and the MgCl2, 150 mM NaCl and 0.005% Tween 80. inflammatory cytokines IL-6, IL-8 and tumor necrosis All transgenes were under the control of the CMV factor (TNF)-a. P40 mixed with a tumor antigen peptide promoter and flanked by a chimeric bglobin-IgG intron was shown to elicit tumor-specific CTL responses and to and a SV40 polyadenylation signal. The coding sequence significantly delay the growth of B16F10 melanoma 21 of L. monocytogenes flagellin was cloned by PCR from tumors implanted at the time of immunization. Listeria DNA (kindly provided by Professor F Bromba- Flagellin is the main component of the bacterial flagella cher, University of Cape Town, Cape Town, South and induces DC maturation and chemokine secretion by Africa). The gene encoding the P40 from K. pneumoniae activating NF-kB through their interaction with 22,23 was provided by the Centre d’Immunologie Pierre Fabre TLR5. Recombinant flagellin from Salmonella typhi- (CIPF, Saint Julien en Genevois, France).18–21 The murium induces maturation of human DCs and causes 23 human CD40L cDNA was purchased from InvivoGen secretion of chemokines. Salmonella flagellin may act as (Cayla, Toulouse, France). The gene encoding the HN of a carrier of epitopes for vaccination against bacteria or the NDV was derived from plasmid p36/7-HN.26 The viruses24 and has been shown to enhance immunogenicity 25 cDNA of the MUC1 tumor antigen was derived from of plasmid vaccination. plasmid pHMG-ETAtm (MUC-1) for cloning into Ad Immunostimulatory properties of the hemagglutinin- vector.33 Ad vectors were diluted just before use in the neuraminidase (HN) of the Newcastle disease virus viral storage buffer described above. (NDV) have been demonstrated in vitro.26,27 Mouse fibroblasts transfected with HN or infected with NDV have been shown to stimulate specific T-cell proliferation Tumor cell lines with greater efficacy than untreated fibroblasts.26 A major A549 human lung carcinoma cells (American Type effect of NDV infection is the induction of IFN-a/b Culture Collection (ATCC; CCL 185), LGC Standards, secretion, which can be mediated either through TLRs or Molshein, France) were cultured in Dulbecco’s modified 28,29 by RIG-I-dependent signaling. NDV-infected auto- Eagle’s medium supplemented with 1 mM sodium pyr- logous tumor cells as vaccines have lead to improvement uvate (Sigma, St Quentin Fallavier, France), 40 mgmlÀ1 of patient survival for different cancer types.30 It is still gentamycine (Schering-Plough, Levallois Perret, France), unclear whether NDV HN has a direct effect on non-essential amino acids (Gibco, Invitrogen, Cergy- immunostimulatory DCs. Pontoise, France) and 2 mML-glutamine (Sigma), referred In this study, we first used Ad vectors to deliver PAMP- to as ‘complete medium’, with 10% fetal calf serum (FCS; encoding genes to tumor cells. The genes encoding P40 JRH Biosciences, Sigma). from K. pneumoniae or the flagellin from Listeria The CT26 murine colon carcinoma cell line was monocytogenes were cloned in replication-deficient Ad obtained from ATCC (CRL 2638). The TC1 murine lung vectors. Their immunostimulatory properties were first tumor cell line was obtained from Dr TC Wu (John investigated in vitro, using co-culture models studying the Hopkins University, Baltimore, MD). The B16F0-CAR effects of Ad-transduced tumor cells on the maturation of cell line was derived from the B16F0 murine melanoma human monocyte-derived DCs (moDCs) and on the cell line (ATCC; CRL 6322) stably transfected with an allogeneic proliferation of lymphocytes. In parallel to expression plasmid expressing the human cocksackie and Ad-PAMPs, we tested the effect of an Ad vector encoding adenovirus receptor (CAR), a high-affinity receptor for the HN of NDV. Ad-PAMPs and Ad-HN were evaluated adenovirus.34 Cytofluorometric analysis was used to in vivo in tumor models as stand-alone treatments, by demonstrate CAR expression on the surface of B16F0- injecting them intratumorally. The properties of Ad- CAR cells by staining with the RmcB monoclonal PAMPs were further studied in a tumor vaccine setting, antibody.35 The expression of CAR by B16F0-CAR cells by administering them subcutaneously as adjuvants to an increased their susceptibility to adenovirus infection Ad vector encoding a tumor-associated antigen. approximately 100-fold (unpublished data).

Cancer Gene Therapy Adenovirus-PAMPs for cancer immunotherapy C Tosch et al 312 RenCa-MUC1 cells were obtained by stable transfec- resuspended by gentle scraping and pelleted. Clarified tion of the murine renal cell carcinoma cell line RenCa, supernatants were collected and frozen until used for obtained from Dr Galli Hillmann (Barbara Ann cytokine analysis using ELISA. The ELISA kits for Karmanos Cancer Institute at Wayne State University human IL-12 (p75), human TNF-a and human IL-10 School of Medicine and Harper Hospital, Detroit, MI, (R&D Systems, Lille, France) were used for cytokine USA), with plasmid pHMG-ETAtm (MUC-1).33 titrations in culture supernatants as indicated by the manufacturer. Transduction of tumor cell lines in vitro Harvested cells were stained with fluorescein isothio- For transduction with Ad vectors, A549 cells were cyanate-conjugated anti-CD86, anti-CD83 or control trypsinized, washed in phosphate-buffered saline and IgG1 (references BD Pharmingen 555657, ImmunoTech resuspended in phosphate-buffered saline containing 2410 and BD Pharmingen 555748, respectively) and 100 mg lÀ1 sodium acetate, 100 mg lÀ1 calcium chloride analyzed by FACS. and 2% FCS (100 ml for 106 cells). Infections were done by the addition of Ad vectors at multiplicities of infection Mixed lymphocyte reactions (MOI) of 5–50 and incubation at 37 1C for 30 min. Cells A549 cells were transduced by Ad vectors at an MOI of 20 were then resuspended in complete medium with 10% and plated in duplicates at 2 Â 105 cells per well in 24-well FCS and plated in tissue culture dishes. plates in 1 ml of complete Dulbecco’s modified Eagle’s Expression of transgene-encoded product on the sur- medium containing 5% FCS. In the next day, medium face of Ad-transduced A549 cells or tumor cell lines was was changed to complete Dulbecco’s modified Eagle’s verified by immunostaining using a biotinylated anti-L. medium containing 5% human serum (1 ml per well) and monocytogenes antiserum (Accurate, Westbury, NY) for cultures were incubated for 48 h. DCs were differentiated flagellin detection or a biotinylated anti-Klebsiella from monocytes as described above. Peripheral blood antiserum (Accurate) for detection of the P40, both leukocytes (PBLs) enriched in lymphocytes were obtained followed by phycoerythrine-conjugated streptavidin (BD by elutriation of cytapheresis from healthy donors at the Pharmingen, Le Pont de Claix, France). Staining with an EFS). DCs and allogeneic PBLs at ratios 1:5 or 1:10 (105 anti-NDV-HN monoclonal antibody provided by V moDCs per well and 5 Â 105 or 106 PBLs per well) in 1 ml Schirrmacher (DKFZ, Heidelberg, Germany), an anti- of complete RPMI medium containing 5% human serum human CD40L mAb (BD Pharmingen) or an anti- were added onto A549 cell monolayers. Co-cultures were 33 MUC1mAb (H23) was detected by an fluorescein incubated for 24 h at 37 1C and 5% CO2. Cells from each isothiocyanate-conjugated anti-mouse immunoglobulin well were resuspended by gentle pipeting to harvest PBLs antibody (BD Pharmingen) and analyzed on a FACScan and DCs but to avoid collecting the A549 adherent flow cytometer using the CellQuest Pro software (Becton monolayer. The cell suspension was centrifuged. Clarified Dickinson, Le Pont de Claix, France). supernatants were collected and frozen before cytokine analysis by ELISA. The cell pellet was resuspended in 2 ml Co-cultures of human moDCs and Ad-transduced A549 RPMI containing 10% human serum. Cell suspension cells (200 ml) was seeded in triplicate in round-bottomed 96- Human DCs were derived from elutriated monocytes well plates. Cultures were incubated for 4 days at 37 1C collected at the Etablissement Franc¸ais du Sang-Alsace and 5% CO2 before the addition of 3H thymidine (1 mCi (EFS, Strasbourg, France) from healthy blood donors. per well). After 18 h, cells were harvested and thymidine Cells were cultured in RPMI 1640 medium supplemented incorporation was measured in a b-counter (TOPCOUNT À1 with 1 mM sodium pyruvate (Sigma), 40 mgml genta- NTX; Packard, Gmi, Ramsey, Minnesota). mycine (Schering-Plough), non-essential amino acids (Gibco), 2 mML-glutamine (Sigma), 10% FCS and Mouse tumor models containing 20 ng mlÀ1 human recombinant granulocyte C57Bl/6N, B6D2F1 and BALB/c female mice were macrophage-CSF and 10 ng mlÀ1 IL-4 (PeproTech, Le- supplied by Charles River (Les Oncins, France) and were vallois-Perret, France) at 37 1C and 5% CO2. The acclimatized in a specific pathogen-free animal facility for phenotype of immature moDCs obtained on days 5 or 6 1 week before experiments were started. They were of culture was verified by flow cytometry analysis of cell approximately 7 weeks old at the time of the experiments. surface markers (CD1a þ , CD14À, HLA-DR þ , Specific pathogen-free status was checked by regular CD40 þ , CD80 þ , CD83À and CD86 þ ). serological and bacterial control on sentinel animals. This For the co-culture experiments, A549 cells were study was conducted in compliance with CEE directive transduced with Ad vectors at an MOI of 5 and plated 86/609 of 24 November 1986 relating to the protection of in six-well tissue culture plates in complete medium animals used for experimental or other scientific pur- containing 5% FCS at a cell density of 4 Â 105 cells per poses, and also in compliance with the French law, de´ cret well. In the next day, medium was removed and replaced no. 87.848 of 19 October 1987 modified by de´ crets no. by 1 ml complete culture medium containing 2% FCS. 2001-464 and 2001-486. After 40 h, 5–7 Â 105 immature human moDCs at day 5 of To evaluate the PAMP-expressing adenoviruses as differentiation adjusted to 1 ml of their culture medium stand-alone antitumor products, 15 mice were included were added on the infected A549 cells. Co-cultures were per experimental group. Here, 3 Â 105 B16F0-CAR or incubated for 24 h at 37 1C and 5% CO2. Cells were RenCa cells in 100 ml of phosphate-buffered saline were

Cancer Gene Therapy Adenovirus-PAMPs for cancer immunotherapy C Tosch et al 313 implanted subcutaneously on the flank of B6D2 mice. Here, 3 Â 105 CT26 cells were implanted in BALB/c mice and 2 Â 105 TC1 cells in C57Bl/6 mice. For intratumoral treatment, 5 Â 108 i.u. of Ad vectors were injected into palpable tumors (around 9 mm3) in 100 ml formulation buffer. Four administrations were performed within 1 week. For therapeutic vaccination, B6D2 mice carrying RenCa-MUC1 tumors (3 Â 105 cells implanted subcutaneously) were injected subcutaneously on the contralateral flank on days 4, 11 and 18 post-tumor implantation with 2 Â 108 i.u. of Ad vectors (108 i.u. of each vector when used in combination) in 100 ml viral storage buffer. Tumor volumes and survival were monitored twice a week. For ethical reasons, mice were killed when the tumor mass reached 10% of body weight of the animal at time of inoculation.

Results Surface expression of PAMPs mediated by transduction of tumor cells with Ad vectors Genes encoding the flagellin from L. monocytogenes, P40 from K. pneumoniae or HN of NDV were cloned in non- replicative Ad vectors in CMV promoter-driven expression cassettes. We first verified that transgenes were correctly expressed following transduction of tumor cells with the recombinant Ad vectors in vitro. To this aim, A549 human lung carcinoma cells were transduced and cultured for 48 h before analysis by immunochemistry and flow cytometry. Cell surface immunostaining with anti- bodies specific to the transgene products were compared with that of A549 cells transduced with an empty Ad vector (Figures 1a–c). Flagellin, P40 or NDV HN was detected on the surface of A549 cells transduced with the corresponding Ad vector. MOIs varying from 5 to 50 were tested. Figure 1 shows the results obtained at MOIs giving for each transgene the maximal signal intensity. Flagellin expression was best detected by flow cytometry on the surface of A549 cells transduced at an MOI 50 (Figure 1a), but its expression could also be detected by western blot at MOIs 1, 5, 10 and 50 (result not shown). The level of cell surface expression may vary depending on the transgene. However, bacterial PAMPs such as flagellin or the P40, as well as the viral HN, are efficiently expressed through Ad vectors and found on the surface of transduced tumor cells. Figure 1 Fluorescence-activated cell sorting (FACS) analysis of transgene expression on the cell surface of A549 cells, 48 h after in vitro transduction with Ad vectors. (a) A549 cells were transduced at Effects of Ad-PAMPs on the maturation of DCs multiplicity of infection (MOI) of 50 with Ad-flagellin (bold line) or Ad- co-cultured with transduced A549 cells empty (thin line), stained with an anti-Listeria monocytogenes We investigated whether PAMPs expressed by Ad- antiserum. (b) A549 cells were transduced at an MOI of 5 with Ad- transduced tumor cells could induce the maturation of P40 (bold line) or Ad-empty (thin line), stained with an anti-Klebsiella neighboring DCs. Human moDCs are known to express a antiserum. (c) A549 cells were transduced at an MOI of 50 with Ad- wide range of TLRs, including the P40-binding TLR2 and HN (bold line) or Ad-empty (thin line), stained with anti-HN antibody. the flagellin-binding TLR5.36 We set up an in vitro model (d) A549 cells were transduced at an MOI of 20 with Ad-CD40L, in which human moDCs were cultured in the presence of stained with an anti-CD40L MAb (bold line) or with an isotype control A549 cells transduced at an MOI of 5 with Ad-flagellin, (thin line). Ad-P40 or Ad-HN. Ad-CD40L was used as a positive control, as CD40L is a powerful inducer of DC

Cancer Gene Therapy Adenovirus-PAMPs for cancer immunotherapy C Tosch et al 314 maturation through its interaction with the CD40 the maturation marker CD83 was also observed, but molecule on the DC surface.37 The human CD40L was varied less in terms of MFI increase (not shown). The expressed on the surface of A549 cells transduced with increase in CD86 and CD83 expression induced with Ad- Ad-CD40L (Figure 1d). CD40L was reproducibly stronger than that observed The effect of Ad-transduced A549 cells was studied with Ad-PAMPs. Ad-flagellin was a stronger inducer of using moDCs derived from three different blood donors. DC maturation than Ad-P40 in moDCs from the three Upregulation of the CD86 co-stimulatory molecule, donors (mean values of CD86 increase: 49±10% for Ad- measured by flow cytometry, was used to evaluate DC flagellin versus 25±11% for Ad-P40). Ad-HN produced maturation after 24 h of co-culture. For each Ad vector only a marginal effect on moDCs with a reproducible tested, the induction of CD86 on DCs was expressed as a 15±1% induction of CD86. percentage of the CD86 mean fluorescence intensity (MFI) increase obtained with Ad-CD40L when compared Cytokine secretion by moDCs in culture with with an empty Ad vector. The following formula was Ad-PAMP-transduced A549 cells used: % CD86 induction ¼ (MFIAd testÀMFIAd-empty)/ TNF-a was titrated in culture supernatants of human (MFIAd-CD40LÀMFIAd-empty) Â 100. moDCs with Ad-transduced A549 cells (Figure 2b). For The data in Figure 2a show that for all three donors, three different donors, Ad-flagellin induced only a minor co-culture of moDCs with A549 cells transduced with Ad- increase in TNF-a secretion (mean 76±19 versus PAMPs upregulated CD86 expression. Upregulation of 49±9pgmlÀ1 with Ad-empty) when compared with that induced by Ad-CD40L (above 1000 pg mlÀ1). Neither Ad-P40 nor Ad-HN induced a detectable increase of TNF-a secretion. The P40 has been reported to increase IL-12 p75 secretion by human and murine macrophages.20 We therefore investigated the presence of IL-12 in co-culture supernatants of human or murine DCs with Ad-transduced A549 cells. Low levels of IL-12 were found in co-cultures with Ad-PAMPs, Ad-HN or Ad-empty. In contrast, IL-12 p75 secretion observed in the presence of Ad-CD40L was increased by more than 100-fold (data not shown). In co-culture experiments, moDCs secreted high amounts of IL-10 in the presence of Ad-CD40L (data not shown). In contrast, basal levels of IL-10 produced by DCs were not significantly increased in the presence of Ad-PAMP-transduced A549 cells. Transduced A549 cells alone did not secrete any IL-10 or IL-12. Altogether, these results show that in this system, expression of the flagellin, P40 or the NDV HN on A549 cells does not affect the secretion of the cytokines examined. In contrast, CD40L expression by Ad-transduced tumor cells induces strong secretion of TNF-a, IL-10 and IL-12 by moDCs.

Stimulation of T-cell proliferation and cytokine secretion in allogeneic MLR by Ad-PAMP transduction of A549 cells As shown above, co-stimulatory molecule expression by human moDCs was augmented following culture in the Figure 2 Effects of Ad-PAMP (pathogen-associated molecular presence of tumor cells transduced with Ad-PAMP when pattern)-transduced A549 cells on CD86 expression by human compared to cells transduced with Ad-empty. To see monocyte-derived DCs (moDCs) in co-cultures. (a) DCs derived from whether this correlated with an increased immunostimu- monocytes obtained from three different blood donors were co- latory function of DCs, we performed mixed lymphocyte cultured with A549 cells transduced with Ad-empty, Ad-hCD40L reaction (MLR) experiments in which human moDCs and (human), Ad-PAMPs or Ad-HN at a multiplicity of infection (MOI) of 5. PBLs were co-cultivated in the presence of A549 cells CD86 expression was measured by flow cytometry after 24 h co- transduced with Ad-PAMPs at an MOI of 20. Culture culture. Histograms represent the mean increase in CD86 MFI supernatants were sampled for the determination of ± ( s.d.) obtained with each Ad vector in moDCs derived from the IFN-g, and cell proliferation was measured by the incor- three donors, expressed as % CD86 induction and calculated by poration of [3H]thymidine. Minimal proliferation was (MFIAd testÀMFIAdÀempty)/(MFIAd-CD40LÀMFIAd-empty) Â 100. (b) Pro- duction of tumor necrosis factor (TNF)-a in 24 h supernatants from observed in cultures of Ad-transduced A549 cells with or human moDCs co-cultured with A549 cells transduced with Ad- without moDCs, as expected (data not shown). In MLRs, empty, Ad-CD40L, Ad-HN or Ad-PAMPs (mean±s.d. of three lymphocyte proliferation was low in the presence of A549 experiments using moDCs from three different blood donors). cells transduced with Ad-empty, but very intense in the

Cancer Gene Therapy Adenovirus-PAMPs for cancer immunotherapy C Tosch et al 315 increased 3-fold with Ad-P40, 5-fold with Ad-flagellin and 10-fold with Ad-CD40L when compared with the proliferation observed with Ad-empty. IFN-g secretion in MLR was correlated with cell proliferation. It was markedly induced in co-cultures of PBLs with A549/Ad-PAMPs (Figure 3b), as well as on addition of allogeneic moDCs (data not shown). IFN-g was not detected in MLR with non-transduced A549 cells (not shown), and hardly detected with Ad-empty-transduced A549 cells (Figure 3b). IFN-g secretion was lower with Ad-PAMPs than with Ad-CD40L; Ad-flagellin transduction of A549 cells resulted in higher IFN-g secretion by co-culturedPBLswhencomparedwithAd-P40.

Intratumoral injection of Ad-PAMPs alone in immunocompetent mouse models provides weak therapeutic effects Immunocompetent mouse tumor models were used to evaluate the effect of Ad-PAMPs injected directly into established tumors. It was expected that the innate immune response might be stimulated locally by the expression of PAMPs within the tumor, favoring the development of a specific immune response and tumor regression. The following syngeneic subcutaneous tumor models were used: B16F0-CAR (melanoma), RenCa (renal carcinoma), CT26 (colon carcinoma) or TC1 (lung carcinoma). Three injections were performed into palpable tumors at 2–3 day intervals with Ad-PAMPs, Ad- HN (which, as shown above, did not efficiently stimulate DC maturation in vitro) or Ad-empty. In most of these models (RenCa, TC1 and CT26), treatment with Ad-PAMPs did not provide significant effect on tumor growth or mouse survival (results not shown). However, significant results were obtained in the Figure 3 Stimulation of lymphocyte proliferation and interferon B16F0-CAR melanoma tumor model. B16F0-CAR is a (IFN)-g secretion in mixed lymphocyte reaction (MLR) with Ad-PAMP tumor cell line derived from B16F0 melanoma cells stably 34 (pathogen-associated molecular pattern)-transduced tumor cells. transfectedwiththeCAR. Expression of CAR increases A549 cells were transduced with Ad-PAMPs, Ad-empty or Ad- the efficacy of transduction by Ad vectors, which otherwise CD40L and cultured with monocyte-derived dendritic cells (moDCs), infect murine cells with low efficacy as compared with allogeneic peripheral blood leukocytes (PBLs), or moD- human cells (PE, unpublished results). In mice bearing Cs þ allogeneic PBLs, as indicated on the graphs. (a) Incorporation 3 B16F0-CAR tumors, treatment with Ad-P40 transiently of [ H]thymidine in cellular DNA, measured in triplicate and inhibited tumor growth between days 30–40 (days 20–30 expressed as counts per minute (c.p.m.), was used to assess lymphocyte proliferation. Upper panel: MLR using 105 moD- after Ad injection) (Figure 4a). Survival was also transiently Cs þ allogeneic PBLs (DC/PBL ratios 1:5 or 1:10) cultured with Ad- but significantly increased (log-rank test P ¼ 0.018) in the transduced A549 cells; lower panel: PBLs (5 Â 105 or 106 per well) mouse group that received this vector when compared with were cultured with Ad-transduced A549 cells in the absence of the group injected with Ad-empty (Figure 4b). At day 49, moDCs. (b) IFN-g concentration in the culture medium was titrated whereas 13, 17 and 20% of mice survived in the groups by ELISA after 24 h incubation of PBLs (5 Â 105 or 106 per well) co- injected with Ad-flagellin, Ad-HN or Ad-empty, respec- cultured with A549 cells transduced with Ad-empty, Ad-CD40L or Ad- tively, 47% of mice treated with Ad-P40 were still alive. PAMPs, in the absence of moDCs. Combination of Ad-PAMPs with Ad-MUC1 improves presence of A549 cells transduced with Ad-CD40L therapeutic vaccination against RenCa-MUC1 tumors (Figure 3a). In co-cultures of A549 cells transduced with The immunoadjuvant effects of Ad-PAMPs were also Ad-flagellin or Ad-P40, lymphocyte proliferation was tested in a model of therapeutic vaccination against increased when compared with MLRs performed in the RenCa-MUC1 tumors. B6D2 mice were inoculated with presence of Ad-empty-transduced A549 cells. This was RenCa renal carcinoma cells stably transfected to express observed whether or not moDCs were added to the PBLs, the tumor-associated antigen MUC1 (RenCa-MUC1).33 which also contained non-lymphocytic cells from periph- Ad-MUC1 was combined with Ad-PAMPs, Ad-HN or eral blood. In conditions using 106 responder PBLs, in the Ad-empty for subcutaneous administration to tumor- absence of additional moDCs, cell proliferation was bearing mice. At days 20–30 after tumor inoculation,

Cancer Gene Therapy Adenovirus-PAMPs for cancer immunotherapy C Tosch et al 316

Figure 5 Effect of Ad-PAMPs (pathogen-associated molecular Figure 4 Effect of Ad-P40 injected intratumorally in C57Bl/6 mice pattern) co-administered with Ad-MUC1 in the RenCa-MUC1 model bearing B16F0-CAR (cocksackie-adenovirus receptor) tumors. (a) of therapeutic vaccination. (a) Tumor volumes are transiently Transient inhibition of tumor growth by intratumoral administration of controlled in B6D2 mice treated with Ad-flagellin or Ad-P40 þ Ad- Ad-P40. Here, 3 105 B16F0-CAR cells were implanted subcuta- MUC1 as compared to mice treated with Ad-empty or Ad-HN þ Ad- Â 5 neously on the flank of B6D2 mice and 5 108 i.u. of Ad vectors were MUC1, at days 20–30 following inoculation of 3 Â 10 RenCa-MUC1 Â 8 injected into palpable tumors on days 10, 12, 14 and 16 following cells. Here, 10 i.u. of each vector used in combination was their implantation. (b) Survival was transiently but significantly administered on the contralateral flank on days 4, 11 and 18 after increased in the mouse group treated with intratumoral Ad-P40 as tumor implantation. (b) Increased survival of B6D2 mice bearing compared with Ad-empty (*log-rank test P ¼ 0.018). Mice remaining RenCa-MUC1 tumors and vaccinated with Ad-PAMPs þ Ad-MUC1 at day 70 were tumor-free. as compared with mice treated with Ad-empty or Ad-HN þ Ad-MUC1 (*log-rank test P ¼ 0.039). tumors were smaller in mice treated with Ad-flagellin or Ad-P40 combined with Ad-MUC1 as compared with all cells, induce the phenotypic and functional maturation of other groups, including those treated with Ad-emp- human moDCs. DC maturation was indeed stimulated by ty þ Ad-MUC1 or Ad-HN þ Ad-MUC1 (Figure 5a). tumor cell transduction with Ad-flagellin and Ad-P40, but These results are consistent with survival rates at day not as efficiently as with Ad-CD40L. These results 110 after tumor implantation, with 20 and 28% survival correlate with the stimulation of lymphocyte proliferation for groups co-treated with Ad-P40 and Ad-flagellin, and IFN-g secretion in MLR experiments performed in respectively, versus 7 and 0% in mice co-treated with the presence of Ad-PAMP- or Ad-CD40L-transduced Ad-empty or Ad-HN (Figure 5b). The difference between A549 cells. Ad-flagellin and Ad-P40 were then tested in the Ad-flagellin or Ad-P40 þ Ad-MUC1-treated mice and syngeneic mouse tumor models, first by direct injection those treated with Ad-empty or Ad-HN þ Ad-MUC1 is into established tumors. Ad-P40 produced a weak statistically significant (log-rank test P ¼ 0.039). therapeutic effect on tumor sizes and survival in B16F0- CAR tumor-bearing mice, whereas Ad-flagellin did not produce any effect in this model. No effect was observed in three other syngeneic tumor models (RenCa, TC1 and Discussion CT26) for any of the viruses tested. However, in a therapeutic vaccination protocol, where Ad-PAMPs were In this study, we first used in vitro models to find out used as adjuvants in combination with an Ad-MUC1 whether PAMPs, when expressed by transduced tumor tumor antigen vaccine administered subcutaneously to

Cancer Gene Therapy Adenovirus-PAMPs for cancer immunotherapy C Tosch et al 317 RenCa-MUC1 tumor-bearing mice, Ad-P40 and Ad- treatment with Ad-P40, with continuing repeated injec- flagellin reduced tumor sizes and improved survival. tions, improves its therapeutic benefits. DCs cultivated for 24 h with Ad-flagellin- or Ad-P40- When expression of the P40 protein following Ad transduced A549 cells may be described as ‘semi-mature’: transduction was investigated in the murine tumor cell expression of the co-stimulatory molecule CD86 was lines, the level of expression was found to vary between upregulated, but not as much as following culture with cell lines (data not shown). Expression of the P40 could Ad-CD40L. Using an Ad vector encoding the HN of not be detected in the CT26 cell line in vitro, which may NDV, which has not been characterized as a PAMP, explain the lack of effect of Ad-P40 in the CT26 tumor transduction of A549 cells did not result in significant model. Low or no expression may result from inefficient modulation of the DC maturation state. In correlation cell transduction by Ad vectors. The CT26 cell line is with this lack of DC stimulatory capacity in vitro, Ad-HN sensitive to NDV virus infection41 but is poorly sensitive did not show any therapeutic effects in the tumor to Ad infection, as tested using an Ad-GFP (green models tested. This favors the idea that a viral infection fluorescence protein) vector. Infectivity studies were leading to interferon induction is key for the effect seen performed on TC1, RenCa, CT26 and B16F0-CAR cells with NDV. transduced with varying MOIs of Ad-GFP. TC1 and In the DC/tumor cell co-culture model, secretion of the B16F0-CAR cells were transduced with high efficiency, pro-inflammatory cytokine TNF-a by moDCs was both giving 52±4% GFP-positive cells 24 h after dramatically enhanced by Ad-CD40L, whereas it transduction at an MOI of 1 and 96 and 80±4%, was only weakly induced by Ad-flagellin. No effect was respectively, at an MOI of 10. RenCa cells were slightly observed with Ad-HN or Ad-P40. The latter result more resistant to infection, with only 11±1% GFP- was unexpected because the P40 has been reported to positive cells at an MOI of 1, but 76±1% at an MOI of stimulate TNF-a secretion by human moDCs or mono- 10. In contrast, CT26 cells revealed very resistant to cytes.18,23,25 Secretion of IL-12 was not induced by transduction by Ad-GFP, giving only 1% positive cells at transduction with Ad-PAMPs, whereas Ad-CD40L po- an MOI of 1 and 19±3% at an MOI of 100 (unpublished tently upregulated this important Th1-inducing cytokine. results). This raises the possibility that some transgenes It has already been shown that flagellin treatment of cannot be expressed through Ad vectors in certain types human DCs does not stimulate IL-12 secretion.23 In the of tumor cells, which may represent an obstacle to Ad- case of P40, macrophages treated with the purified protein based therapy of cancers using intratumoral administra- have been reported to secrete IL-12, but only in low tion. amounts.20 A recent study indicates that only the IL-12 For Ad-flagellin, no beneficial effect on tumor progres- p40 subunit can be secreted by TLR-stimulated DCs, sion was observed in any of the models tested using whereas IFN-g or DC stimulation by activated T cells is intratumoral administration. Recently published data42 required for the production of IL-12 p75.38 show that purified flagellin administered peritumorally Ad-PAMPs did not cause the secretion of the ‘im- 8–10 days after tumor implantation did not affect the munosuppressive’ cytokine IL-10 by DCs. In contrast, growth rate of a poorly immunogenic tumor, whereas it Ad-CD40L induced high levels of this cytokine, which is significantly inhibited the growth of an antigenic human believed not to be beneficial for cancer immunotherapy as ErB-2-transfected tumor variant. Low immunogenicity of notably shown in a recent study.39 B16F0-CAR cells might explain the lack of effect The induction of DC maturation by A549 cells observed for Ad-flagellin. transduced with Ad-flagellin or Ad-P40 was correlated Cancer-induced immunosuppression has been charac- by an increased stimulation of lymphocyte proliferation in terized notably by the presence of tumor-derived im- MLR. In this assay, Ad-flagellin appeared to have the munosuppressive cytokines, such as IL-10 or TGFb strongest effect on lymphocyte proliferation, maybe due which, among other effects, can inhibit DC maturation to the fact that T cells express TLR5 and are directly and functions.1,4,5,13 Thus, in the C26 model, it has been stimulated by flagellin.40 IFN-g expression is crucial for shown that intratumoral administration of CpG induces immune response to cancer.13 The secretion of IFN-g was tumor regression only when combined with anti-IL-10R induced by Ad-PAMPs, with a superior effect of mAb treatment.9 Our in vitro results showed that DCs Ad-flagellin as compared with Ad-P40. cultured in the presence of Ad-PAMP-transduced tumor In mouse models where Ad-PAMPs were administered cells did not secrete IL-12, whereas they did when intratumorally, only marginal or no effects were observed stimulated with Ad-CD40L. Basal levels of IL-10 present in the increase in tumor volumes or in survival. However, in our co-culture system may contribute to inhibit IL-12 Ad-P40 showed some efficacy in the B16F0-CAR model production on TLR stimulation. Similarly, in the tumor for both of these criteria. The higher susceptibility to models studied, IL-10 and other cytokines may contribute transduction by Ad conferred by the expression CAR on to maintain unresponsiveness to TLR signals delivered B16F0 melanoma cells may contribute to this effect of intratumorally. Ad-P40. However, the therapeutic effect of Ad-P40 on In the RenCa-MUC1 model of therapeutic vaccination, tumor growth was only transient, which may be due to the Ad-PAMPs combined with Ad-MUC1 and administered negative selection of P40-expressing tumor cells, poten- subcutaneously improved mouse survival and inhibited tially more immunogenic than their non-transduced early tumor growth. Purified P40 has previously been counterparts. It should be tested whether a prolonged found to stimulate the immune response against a TRP-2

Cancer Gene Therapy Adenovirus-PAMPs for cancer immunotherapy C Tosch et al 318 peptide antigen present on B16F10 tumors and to confer 11 Furumoto K, Soares L, Engleman EG, Merad M. Induction protection against these tumors when administered with of potent antitumor immunity by in situ targeting of the peptide at the time of tumor implantation.21 In our intratumoral DCs. J Clin Invest 2004; 113: 774–783. RenCa-MUC1 model, Ad-flagellin combined with Ad- 12 Acres B, Paul S, Haegel-Kronenberger H, Calmels B, MUC1 produced the highest survival rates when com- Squiban P. Therapeutic cancer vaccines. Curr Opin Mol pared with Ad-P40 or Ad NDV HN. Ad-flagellin also Ther 2004; 6: 40–47. 13 Smyth MJ, Godfrey DI, Trapani JA. A fresh look at tumor showed the strongest effects in vitro on the maturation of immunosurveillance and immunotherapy. Nat Immunol human moDCs and on lymphocyte proliferation in MLR. 2001; 2: 293–299. This PAMP has recently been reported to function as an 25 14 Seya T, Akazawa T, Uehori J, Matsumoto M, Azuma I, adjuvant when expressed by a plasmid. Our results Toyoshima K. Role of toll-like receptors and their adaptors suggest that it may also be an interesting adjuvant for a in adjuvant immunotherapy for cancer. Anticancer Res 2003; tumor-associated antigen vaccine when expressed from an 23: 4369–4376. Ad vector. Further studies are needed to dissect the 15 Sharma P, Old LJ, Allison JP. Immunotherapeutic strategies properties of Ad-PAMPs as vaccination adjuvants. Our for high-risk bladder cancer. Semin Oncol 2007; 34: 165–172. results show that Ad-PAMPs have immunostimulatory 16 Iwasaki A, Medzhitov R. Toll-like receptor control of the properties and could be effective adjuvants for cancer adaptive immune responses. Nat Immunol 2004; 5: 987–995. immunotherapy using tumor antigen vaccines. 17 Matzinger P. The danger model: a renewed sense of self. Science 2002; 296: 301–305. 18 Jeannin P, Renno T, Goetsch L, Miconnet I, Aubry J-P, Delneste Y et al. OmpA targets dendritic cells, induces their Acknowledgements maturation and delivers antigen into the MHC class I presentation pathway. Nat Immunol 2000; 1: 502–509. We thank Professor Frank Brombacher for the gift of L. 19 Jeannin P, Magistrelli G, Goetsch L, Haeuw J-F, Thieble- monocytogenes DNA, Professor Volker Schirrmacher for mont N, Bonnefoy J-Y et al. Outer membrane protein A the gift of NDV-HN monoclonal antibody, Dr Ronald (OmpA): a new pathogen-associated molecular pattern that Rooke and Dr Joerg Schneider for critical review of this interacts with antigen-presenting cells—impact on vaccine paper. strategies. Vaccine 2002; 20(Suppl. 4): A23–A27. 20 Soulas C, Baussant T, Aubry J-P, Delneste Y, Barillat N, Caron G et al. Outer membrane protein A (OmpA) binds to and activates human macrophages. J Immunol 2000; 165: 2335–2340. 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