IL-6 Signaling Blockade during CD40-Mediated Immune Activation Favors Antitumor Factors by Reducing TGF-β, Collagen Type I, and PD-L1/PD-1 This information is current as of September 26, 2021. Emma Eriksson, Ioanna Milenova, Jessica Wenthe, Rafael Moreno, Ramon Alemany and Angelica Loskog J Immunol 2019; 202:787-798; Prepublished online 7 January 2019; doi: 10.4049/jimmunol.1800717 Downloaded from http://www.jimmunol.org/content/202/3/787

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-6 Signaling Blockade during CD40-Mediated Immune Activation Favors Antitumor Factors by Reducing TGF-b, Collagen Type I, and PD-L1/PD-1

Emma Eriksson,* Ioanna Milenova,* Jessica Wenthe,* Rafael Moreno,† Ramon Alemany,† and Angelica Loskog*,‡

IL-6 plays a role in cancer pathogenesis via its connection to proteins involved in the formation of desmoplastic stroma and to by driving differentiation of myeloid suppressor cells together with TGF-b. Inhibition of IL-6 signaling in the tumor microenvironment may, thus, limit desmoplasia and myeloid suppressor cell differentiation. CD40 signaling can further revert myeloid cell differentiation toward antitumor active phenotypes. Hence, the simultaneous use of IL-6 blockade with CD40

stimuli may tilt the tumor microenvironment to promote antitumor immune responses. In this paper, we evaluated the mecha- Downloaded from nisms of LOAd713, an designed to block IL-6R signaling and to provide myeloid cell activation via a trimerized membrane-bound isoleucine zipper (TMZ) CD40L. LOAd713-infected cells were killed by oncolysis, whereas infection of stellate cells reduced factors involved in stroma formation, including TGF-b-1 and collagen type I. infection prevented IL-6/GM-CSF–mediated differentiation of myeloid suppressors, but not CD163 , whereas infec- tion of dendritic cells led to upregulation of maturation markers, including CD83, CD86, IL-12p70, and IFN-g. Further, IL-6R blockade prevented upregulation of programed death ligand 1 (PD-L1) and PD-1 on the stimulated dendritic cells. These results http://www.jimmunol.org/ suggest that LOAd713 can kill infected tumor cells and has the capacity to affect the tumor microenvironment by stimulating stellate cells and myeloid suppressors with TMZ-CD40L and IL-6R blockade. Gene transfer of murine TMZ-CD40L prolonged survival in an animal model. LOAd713 may be an interesting therapeutic option for cancers connected to IL-6 signaling, such as pancreatic cancer. The Journal of Immunology, 2019, 202: 787–798.

he tumor microenvironment is important for the devel- measured by upregulated a-smooth muscle actin (aSMA), is opment and progression of cancer, and it also plays a role correlated with a poor prognosis (3). in the response to treatment or lack thereof. Pancreatic The IL-6 is upregulated in pancreatic cancer, and a high

T by guest on September 26, 2021 cancer is a challenging disease highly resistant to conventional serum level of IL-6 correlates with a more-advanced disease and cancer therapeutics. The tumor microenvironment is characterized a poorer health status (4). In pancreatic cancer, type 2 tumor- by desmoplasia with stroma including fibroblasts, stellate cells, associated macrophages in the tumor stroma are the main pro- myeloid cells, and extracellular matrix (1). Less than 10–20% of ducers of IL-6, but other cell types also contribute, including stellate the cells are the malignant tumor cells. The activated stellate cells cells (5, 6). Signaling through the IL-6 pathway leads to activation produce a high amount of fibronectin and collagen type I and are, of STAT3, which in turn elevates TGF-b1 expression. TGF-b1can hence, the major contributor to the fibrosis seen in the lesions (2). activate production of collagen type I and plays a central role in Fibrosis increases the intralesion pressure, which is believed to fibrosis of the pancreas (7). TGF-b1 is also involved in immuno- reduce chemotherapy exposure. Indeed, high stromal activity, as suppression, another characteristic of pancreatic cancer, by inhibi- tion of Th1 cells and expansion of T regulatory cells (8, 9). IL-6 is used in some dendritic cell (DC) activation protocols, but it can also interfere with the differentiation and maturation of DCs because it *Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden; †L’Institut d’Investigacio´ Biome`dica promotes STAT3 activation (10, 11), and it is therefore one of the de Bellvitge–Institut Catala` d’Oncologia, L’Hospitalet de Llobregat, 08908 Barcelona, factors to promote differentiation of myeloid-derived suppressor ‡ Spain; and Lokon Pharma AB, 751 83 Uppsala, Sweden cells (MDSCs) (6). The complete role of IL-6 in tumor immunology ORCIDs: 0000-0003-1677-4230 (I.M.); 0000-0003-4385-7568 (J.W.); 0000-0001- is, therefore, obscure. Nevertheless, the presence of macrophages in 9957-3105 (R.M.); 0000-0001-8583-6138 (A.L.). the tumor and/or an increased level of circulating MDSCs correlate Received for publication May 22, 2018. Accepted for publication November 22, to a poorer prognosis (12, 13). 2018. We previously demonstrated that immunostimulatory gene This work was supported by grants to A.L. from the Swedish Cancer Society and Swedish State Support and by a contract research agreement with Lokon Pharma AB. therapy using transferring CD40L (AdCD40L) is a potent Address correspondence and reprint requests to Prof. Angelica Loskog, Department stimulator of the tumor microenvironment because of its capacity to of Immunology, Genetics, and Pathology, Uppsala University, Rudbeck Laboratory activate DCs and tilt M2 to M1 macrophages. AdCD40L showed C11, Dag Hammarskjo¨lds Va¨g 20, 751 85 Uppsala, Sweden. E-mail address: efficacy in mouse, dog, and human (14–17). In the current paper, [email protected] we explored the possibility of combining CD40L-based immune Abbreviations used in this article: BV421, Brilliant Violet 421; DC, dendritic cell; ffu, fluorescent forming unit; MDSC, myeloid-derived suppressor cell; PD-1, pro- activation with IL-6 pathway blockade using virus-mediated gene gramed death-1; PD-L1, programed death ligand-1; sIL, soluble IL; scFv, single therapy. We constructed LOAd713, an oncolytic adenovirus se- chain fragment; scFv-aIL-6R, scFv against the IL-6R; aSMA, a-smooth muscle rotype 5/35 chimera that carries a gene encoding a single chain actin; TMZ, trimerized membrane-bound isoleucine zipper. fragment (scFv) against the IL-6R in combination with a gene Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 encoding a trimerized membrane-bound isoleucine zipper (TMZ) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800717 788 CD40 STIMULI DURING IL-6 BLOCKADE IN THE TUMOR MILIEU human CD40L. In the current paper, the effect of LOAd713 in- a total volume of 100 ml. Postinfection, the infected cell suspensions were fection of pancreatic cancer cells as well as the stellate cells and diluted to 100,000 cells/ml, and 100 ml in quadruplicates was added to a myeloid immune cells that constitute most of the tumor micro- 96-well plate. After 48 and 72 h, the viability of the cells were measured by MTS CellTiter AQueous One Solution Cell Proliferation Assay kit environment were evaluated. (Promega, Madison, WI) according to the manufacturer’s instructions. The relative cell viability in percent was calculated as the absorbance for infected Materials and Methods cells divided by the absorbance for uninfected cells multiplied by 100. Primary cells and cell lines Phosphorylation of STAT3 The pancreatic cell lines BxPc3, MiaPaCa2, PaCa3, and Panc01 were a Panc01 cells were infected with 25 ffu/cell of LOAd(2), LOAd700, or kind gift from Dr. R. Heuchel (Karolinska Institute, Stockholm, Sweden). LOAd713 or left uninfected and cultured for 48 h. Supernatants were The cell origin was confirmed by short tandem repeat analysis by the harvested and added to fresh Panc01 cells. rIL-6 (20 ng/ml; BioLegend) Uppsala Genome Center, Uppsala, Sweden. BxPc3 were cultured in was then added to the cells followed by 20 min incubation at 37˚C. The RPMI 1640 supplemented with 10% FBS and 1% penicillin–strepto- level of STAT3-Y705 phosphorylation in the different groups was deter- mycin. MiaPaCa2, PaCa3, and Panc01 were cultured in DMEM sup- mined with flow cytometry according to the manufacturer’s protocol (BD plemented with 10% FBS and 1% penicillin–streptomycin. All reagents Bioscience). were purchased from Invitrogen, Carlsbad, CA. The HEK293 and A549 cells (American Type Culture Collection, Manassas, VA) were kept in Pancreatic stellate cells the DMEM or RPMI medium, respectively, as described above, except for A549, in which 1% sodium pyruvate was added to the medium. Pancreatic Human primary pancreatic stellate cells were cultured on Poly-L-Lysine– 5 stellate cells were purchased from 3H Biomedical (Uppsala, Sweden) and coated plates (3H Biomedical). 1 3 10 cells per group were infected kept in 2% FBS, 1% SteC Growth Supplements, and 1% penicillin– with 25 ffu/cell of LOAd(2), LOAd700, or LOAd713 or left uninfected streptomycin in stellate cell medium, all from 3H Biomedical. for 48 h before harvest of cells to prepare total RNA (RNeasy Minikit; Downloaded from Qiagen, Hilden, Germany) and protein lysates. For protein extraction Virus construction and production lysates, cells were lysed using RIPA buffer (50 mM Tris-HCl [pH 2 7.4], 150 nM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium Construction of LOAd adenoviral vectors LOAd( )andLOAd700has deoxycholate, 1 mM PMSF, and 10% Protease Inhibitor Cocktail from been described previously (17). LOAd713 contains TMZ-CD40L and an Thermo Fisher). The protein suspension was centrifuged to remove de- scFv against the IL-6R (scFv-aIL-6R). In short, LOAd713 was con- bris, and the lysates were then transferred to fresh tubes for storage in structed by synthesis of a pUC57-Kan plasmid containing the sequence 220˚C. Protein concentration was measured with Coomassie Plus Pro- for the CMV followed by scFv-aIL-6R, a T2A peptide, and tein Assay Reagent (Thermo Fisher Scientific) prior to analysis using a http://www.jimmunol.org/ then human TMZ-CD40L. The gene region was flanked by adenoviral 233-analyte custom multiplex array by service at Olink Proteomics AB, regions containing 59SspI and 39ScaI sites (GenScript, Piscataway (Uppsala, Sweden). Relative values are expressed as linear normalized Township, NJ). After digestion of the plasmids with SspI and 2 protein expression. Total RNA from cells was synthesized to cDNA with ScaI, the gene fragment was gel purified and inserted into LOAd( )by an iScript cDNA synthesis kit (Bio-Rad Laboratories). Gene expres- . HEK293 cells were transfected for the first sionwasthendeterminedbyquantitativePCRusingprimersfor step of adenoviral production followed by expansion in A549 cells. Virus GADPH (forward: 59-GTCAAGGCTGAGAACGGGAA-39;reverse: purification was made with cesium chloride centrifugation followed by 59-TCGCCCCACTTGATTTTGGA-39)andaSMA (forward: 59- dialysis using a Slide-A-Lyzer Cassette (Thermo Fisher Scientific, CTGTTCCAGCCATCCTTCATC-39;reverse:59-TCGCCCCACTT- Waltham, MA). Viruses were suspended in a Tris buffer (Tris-HCl GATTTTGGA-39), all purchased from Invitrogen. SYBR Green 10 mM [pH 7.9], with MgCl2 2 mM and sucrose 4%), and titer was Supermix (Bio-Rad Laboratories) was used, and amplifications were by guest on September 26, 2021 determined as fluorescent forming units (ffu) per milliliter (18). Viral 2 done by Bio-Rad CFX96. Data analysis was performed using the CFX aliquots were stored at 80˚C. Manager Software (Bio-Rad Laboratories). Detection of CD40L and scFv-aIL-6R on HEK293 cells Generation of MDSCs For detection of TMZ-CD40L and scFv-aIL-6R, HEK293 cells were PBMCs (n = 2) or CD14+ enriched cells (n = 2; MACS beads; Miltenyi transfected with 5 mg of plasmid containing only TMZ-CD40L, only scFv- Biotec, Bergisch Gladbach, Germany) from healthy blood donors were aIL-6R, TMZ-CD40L in combination with scFv-aIL-6R, or with an empty cultured as previously described to promote MDSCs differentiation (19). (mock) plasmid. The scFv-aIL-6R used in this experiment had a c-myc tag In short, cells were cultured at a density of 0.5 3 106 cells/ml in 10 ng/ml used for detection. After 48 h, transfected cells and supernatant were of IL-6 and 10 ng/ml of GM-CSF (Gentaur, Brussels, Belgium) for 7 d. harvested. The cells were incubated with 1% BSA in PBS for blocking of Supernatants from uninfected, LOAd(2), LOAd700, or LOAd713-infected unspecific binding, followed by incubation with Abs for 15 min at room A549 cells were added to the 7-d culture at a concentration of 50% of total temperature. Abs used were anti-CD40L Brilliant Violet 421 (BV421) medium. After 7 d, the cells were harvested and analyzed by flow (clone 24-31) and mouse IgG1 k BV421 (clone MOPC-21). After that, cytometry as described above. Abs used are as follows: anti-CD11b allo- the cells were washed with 0.5% BSA in PBS plus 3 mM EDTA and phycocyanin (clone ICRF44), anti-CD11b Pe/Cy7 (clone ICRF44), anti- suspended in 1% paraformaldehyde in PBS plus 3 mM EDTA before CD33 PE (clone WM53), anti-CD163 BV421 (clone GHI/61), mouse IgG1 analysis in BD FACS Canto II (BD Bioscience, San Jose, CA). Data were k BV421 (clone MOPC-21), and mouse IgG1 k PE (clone MOPC-21), all analyzed in Flow Jo (Tree Star, Ashland, OR). The supernatants were form BioLegend. added to wells coated with rIL-6R (Abbiotec, San Diego, CA) or CD19 as irrelevant control (Sino Biological, North Wales, PA). The myc tag was Maturation of DCs then detected by an anti–myc tag Ab (Abcam, Cambridge, U.K.) followed by a goat anti-rabbit IgG (Invitrogen) before TMB substrate (Millipore, CD14+ cells were sorted from healthy donor PBMCs (n = 5; CD14+ purity Billerica, MA) was added. The absorbance was evaluated at 450 nm in an mean: 98.6%, ranging from 98% to 99%) and differentiated to immature iMark Micro Plate Reader (Bio-Rad Laboratories, Hercules, CA). CD1a+ DCs by culturing for 6 d with 150 ng/ml of GM-CSF and 50 ng/ml of IL-4 (Gentaur) (CD1a+ purity: 85.2%, ranging from 82% to 87%). The Phenotype of pancreatic cell lines immature DCs were then infected with 50 ffu/ml of LOAd(2), LOAd700, or LOAd713 or left uninfected. The DCs were cultured for 48 h before Pancreatic cell lines were infected with of 25 ffu/cell of LOAd(2), being analyzed by flow cytometry as described above. Abs used are as LOAd700, or LOAd713 or left uninfected, and after 48 h, the cell phe- follows: anti-CD1a allophycocyanin (clone HI149), anti-CD70 PE (clone notype was evaluated by flow cytometry as described above. Abs used 113-16), anti-CD83 PE (clone HB15e), anti-CD86 BV421 (clone IT2.2), were anti-CD40L BV421 (clone 24-31), anti-CD40 allophycocyanin anti-CD154 BV421 (clone 24-31), anti-HLADR PerCP (clone L243), k (clone HB14), anti–IL-6R PE (clone UV4), mouse IgG1 BV421 (clone anti–IL-6R PE (clone UV4), anti–IL-10R PE (clone 3F9), mouse IgG1 k k MOPC-21), mouse IgG1 allophycocyanin (clone MOPC-21), and mouse PE (clone MOPC-21), rat IgG2a k PE (clone RTK2758), mouse IgG1 k k IgG1 PE (clone MOPC-21), all from BioLegend. BV421 (clone MOPC-21), mouse IgG2b k BV421 (clone MPC-11), and Oncolytic capacity mouse IgG2a k PerCP (clone MOPC-173), all from BioLegend. Super- natants were also collected and analyzed with three different techniques, Pancreatic cell lines were infected with 100 ffu/cell of LOAd(2), namely a custom 233-analyte multiplex proteomic array by service (Olink LOAd700, or LOAd713 or left uninfected for 2 h in serum-free medium in Proteomics AB), luminex methodology (Milliplex MAP kit Human Th17 The Journal of Immunology 789

Magnetic Bead Panel HTH17MAG-14K, Millipore, Billerica, MA, USA), intratumoral delivery of LOAd713 would likely lead to infection of and for soluble IL (sIL)-6R by ELISA (Invitrogen). stellate cells. Because our virus only infects human cells and the Animal experiment scFv-aIL-6R does not cross-react with murine IL-6R, we sought to elucidate the role of the IL-6R blockade using human stellate 3 5 Murine B16 cells (2 10 ) expressing human CD46 (kind gift cells from primary cultures. Even if replication and oncolysis from Dr. Hemmi, University of Zurich) (20) was injected in syngeneic C57BL6 mice purchased from Taconic, Denmark. At day 5, the black tumor will not occur in normal cells, the LOAd713 virus can infect cells were visible under the skin, and treatment was initiated. A virus normal cells such as stellate cells and drive expression of its expressing murine TMZ-CD40L (mLOAd700) was injected s.c. in the tu- because the expression is under control of 9 mor area (1 3 10 infectious particles per mouse in 50 ml) with or without a promiscuous CMV promoter. Hence, primary stellate cells a m coinjection of a rat anti-mouse IL-6R Ab (0.5 mg/mouse in 50 l; were infected with LOAd viruses or left uninfected. The cells BioXCell, West Lebanon, NH). Control mice were treated with physiological NaCl. Treatments were given twice per week, six times in total. were analyzed by quantitative PCR to confirm that the cells were indeed stellate cells by detection of aSMA (Fig. 3A). Further, Statistics cell lysates prepared from the cultures were analyzed using Statistical calculations were performed usingGraphPadPrism6(LaJolla,CA). Olink multiplex proteomics. Expression of CD40 was con- Kruskal–Wallis (ANOVA) with Dunn multiple comparison tests were used firmed, and CD40L was detected in the LOAd700- and when more than two groups of nonparametric data were analyzed. Data sets LOAd713-infected cells as expected (Fig. 3B). Further, both with several groups and time points were analyzed using a two-way ANOVA IL-6 and IL-6R were present in the cells. In cells infected with with Tukey multiple comparison test. Survival was analyzed by log-rank test. LOAd713, a higher IL-6 level was detected, but it did not reach

significance. Interestingly, molecules driving the hostile tumor Downloaded from Results microenvironment and/or including LAP-TGF-b1, LOAd713 efficiently induces expression of TMZ-CD40L and collagen type 1, fibroblast growth factor 5 (FGF5), hepatocyte scFv-aIL-6R and, thereafter, oncolysis of pancreatic cell lines growth factor (HGF), and TNF-like weak inducer of Gene constructs expressing a TMZ-CD40L (17) and/or an scFv- (TWEAK) were all significantly decreased by LOAd713 in- aIL-6R were produced and transfected into HEK293 cells. As fection (21–24). Virus infection [LOAd(2)] increased vascular

shown by flow cytometry, the transfected cells rapidly expressed endothelial growth factor (VEGF), but this was not seen with http://www.jimmunol.org/ TMZ-CD40L (Fig. 1A), whereas a c-myc tag enabled detection of the LOAd713 virus. Interestingly, the lymphocyte chemokines the scFv-aIL-6R by ELISA (Fig. 1B). Pancreatic lines CXCL10 and CCL20 were increased by LOAd713. Hence, BxPc3, MiaPaCa2, PaCa3, and Panc01 were infected with sero- LOAd713 infection of stellate cells reduced the pathological type 5/35 adenoviruses without transgenes [LOAd(2)], with phenotype of these cells while enhancing their capacity to alert TMZ-CD40L (LOAd700), or with a combination of scFv-aIL-6R the immune system. and TMZ-CD40L (LOAd713). CD40L expression is shown in Fig. 1C. The pancreatic cancer cell lines were not positive for LOAd viruses inhibit the differentiation of MDSCs CD40 or IL-6R (Fig. 1D), and infection with LOAd713 did not MDSCs are one of the major suppressive immune cells present in alter this expression pattern. The oncolytic capability of LOAd713 the tumor microenvironment of pancreatic cancer (25). In vitro, by guest on September 26, 2021 was tested in the four pancreatic cancer cell lines (Fig. 1E–H). All MDSCs can be differentiated from human PBMCs, or CD14+ LOAd viruses could induce tumor cell death within 48 h, and cells (e.g., monocytes), by addition of IL-6 and GM-CSF (6). We the addition of TMZ-CD40L and scFv-aIL-6R did not alter this show in this study that MDSCs defined as CD11bhighCD33+ capacity. LOAd713 showed a significant reduction of cell viability myeloid cells are indeed increased in cultures of PBMC compared with uninfected cells at both 48 and 72 h in all cell lines (Fig. 4A) or monocytes (Fig. 4B) when IL-6 and GM-CSF are tested. Hence, LOAd713 can efficiently expand in pancreatic added to the cultures obtained from two different blood donors. cancer cells to induce oncolysis. To investigate if the addition of the scFv-aIL-6R in LOAd713 can block MDSC differentiation, the PBMCs and CD14+ cells LOAd713 reduces STAT3 phosphorylation via scFv-aIL-6R stimulated with IL-6/GM-CSF were also subjected to superna- The IL-6R is often soluble binding to IL-6, thereby docking into a tants from LOAd virus–infected and uninfected A549 lung shared signaling molecule (gp130). Hence, the presence of IL-6R cancer cells. These cells produce higher amounts of scFv-aIL-6R was tested on the cells and in the supernatant using the sensitive because they are more resistant than pancreatic cancer cell lines Olink Proteomic assay. In Fig. 2A, it is indeed shown that to immediate oncolysis. When supernatants from LOAd-infected expression of IL-6R can be low on cells but found in the super- A549 cells were added to the cultures, the percentage of natant (Fig. 2A). Binding of IL-6 to IL-6R leads to phosphory- CD11bhighCD33+ cells was reduced, regardless of the virus used, lation of STAT3 at the Y705 position. To confirm the function of whereas supernatant from uninfected cells only modestly differed the scFv-aIL-6R, Panc01 and MiaPaCa2 cells were infected with from IL-6/GM-CSF stimulation alone. The CD14+ cells seemed LOAd(2), LOAd700, or LOAd713 or left uninfected. After 48 h, even less prone to differentiate into CD11bhighCD33+ cells when the supernatants with and without scFv-aIL-6R were harvested and supernatant from LOAd700-infected cells was added. The CD14+ added to fresh Panc01 and MiaPaCa2 cells stimulated with rIL-6. cells were also evaluated for the differentiation into macro- IL-6 triggered STAT3 phosphorylation in both Panc01 and Mia- phages defined as CD11b+CD163+. IL-6 and GM-CSF prevented PaCa2 cells (Fig. 2B, 2C), but cells cultured with supernatant from differentiation into CD163+ macrophages compared with mono- LOAd713-infected cells that contained scFv-aIL-6R showed sig- cytes cultured in medium only. The addition of supernatants from nificantly reduced STAT3 phosphorylation. Hence, LOAd713 can LOAd700-infected cells completely disrupted differentiation of participate to reduce IL-6–induced STAT3 activation in tumor cells. CD163 macrophages, but this was not seen when supernatants from uninfected, LOAd(2), or LOAd713-infected cells were LOAd713 reduces factors important for desmoplasia in added, suggesting that the tumor supernatant contains other stellate cells molecules that counteracted the addition of IL-6/GM-CSF by Pancreatic stellate cells play a major role in the tumor biology of promoting M2 differentiation (Fig. 4C). However, pancreatic cancer, and because of the high content in the lesions, supernatant from LOAd700-infected tumor cells reduced M2 790 CD40 STIMULI DURING IL-6 BLOCKADE IN THE TUMOR MILIEU

FIGURE 1. Infection-induced oncolysis and transgene expression. (A) Expression of CD40L on HEK293 cells (live cell gate) 48 h posttransfection with plasmids containing the transgenes. Black line demonstrates CD40L expression from the plasmid coexpressing TMZ-CD40L and scFv-aIL-6R. Dashed line shows TMZ- CD40L from a plasmid expressing only TMZ-CD40L. Gray line represents trans- fection with empty (mock) plasmid, and the filled curve represents isotype control. Plots show representative data from three Downloaded from independent experiments. (B) Detection of scFv-aIL-6R in supernatants from trans- fected HEK293 cells using ELISA for a c-myc tag on the scFv-aIL-6R. The ex- periment was run in duplicates and was repeated twice. (C) Expression of CD40L http://www.jimmunol.org/ on pancreatic tumor cell lines BxPc3, MiaPaCa2, PaCa3, and Panc01 (live cell gate) 48 h postinfection with 25 ffu/cell of LOAd viruses: LOAd713 (black line), LOAd700 (dashed line), and LOAd(2) (gray line). Filled curve represents isotype control. (D) Expression of CD40 and IL-6R 48 h postinfection with LOAd(2) (gray line), LOAd700 (black dashed line), and LOAd713 (black line). Gray dashed by guest on September 26, 2021 line represents uninfected cells. Each vertical panel corresponds to one of the four cell lines: from left, BxPc3, Mia- PaCa2, PaCa3, and Panc01. Flow cytom- etry plots are representative for three independent experiments, and mean fluo- rescence intensity (MFI) is displayed. (E–H) Relative viability, percentage of the viability of uninfected cells, is shown for infected pancreatic cell lines (E) BxPc3, (F) MiaPaCa2, (G) PaCa3, and (H) Panc01 48 h (black) and 72 h (white) postinfection with LOAd(2), LOAd700, or LOAd713. Data are shown as mean 6 SD, and sta- tistically significant differences were cal- culated using the two-way ANOVA with Tukey multiple comparison test. Statistical significance is indicated with *p , 0.05.

differentiation, which we have noted using CD40L reduced immunosuppression in the tumor. However, simulta- in animal models previously (26). These experiments suggest neous CD40 stimuli and blockade of IL-6 signaling seemed to that LOAd viruses can affect the tumor cell microenvironment allow differentiation of CD163 macrophages that also have been to reduce the differentiation of MDSCs, which could lead to a connected to immunosuppression. The Journal of Immunology 791

FIGURE 2. Effect on STAT3 phosphorylation dur- ing IL-6R blockade. (A) The level of IL-6R on Mia- PaCa2 cells or in the supernatant of cultured cells was evaluated by Olink multiplex proteomics custom panel and displayed as linear normalized protein expression

as per company instructions. The level of STAT3 Downloaded from phosphorylation was analyzed by flow cytometry 20 min after stimulation with rIL-6 in MiaPaCa2 (B) and Panc01 (C) cells (live cell gate) cultured with supernatant from the respective cell line infected with LOAd(2), LOAd700, LOAd713, or uninfected cells. Data are shown as the fold change of mean fluorescence intensity of

STAT3 Ab compared with isotype control (baseline), http://www.jimmunol.org/ and statistically significant differences were calcu- lated using the two-way ANOVA with Tukey multiple comparison test. Statistical significance is indicated with *p , 0.05. by guest on September 26, 2021

Blocking the IL-6 pathway does not alter DC maturation via uninfected DCs. The immunosuppressive cytokine IL-10 was CD40L stimulation only significantly upregulated in supernatants from DCs infected 2 The microenvironment of pancreatic cancer is known for its content with LOAd( ). These data demonstrate that LOAd713 is a po- of myeloid cells and lack of antitumor immunity. CD40L is a potent tent activator of myeloid cells, such as DCs, which is an im- portant step to activate antitumor immunity. Note that even if the activator of myeloid cells, especially DCs that drive formation of + tumor immunity. Because IL-6 blockade may prevent or accelerate CD14 monocyte purification was performed prior to DC mat- DC maturation, we sought to investigate the ability of LOAd713 to uration (mean: 98.6%, ranging from 98% to 99%), it cannot be activate immature DCs. Monocyte-derived immature DCs were excluded that other cell types can still be present in primary cell infected with LOAd713, LOAd700, or LOAd(2) or left unin- cultures, such as lymphocytes, that can participate to produce fected. Infection with LOAd(2) did not upregulate activation in these cultures. markers on DCs compared with uninfected DCs; fold change was ∼1 in all five donors. LOAd713 and LOAd700 infection led to a LOAd713 infection blocks programed death-1 and programed significant upregulation of the maturation markers CD83 and death ligand-1 expression CD86, whereas CD70 showed a significant fold change in the To further characterize the DC profile, a 233-analyte multiplex LOAd713 group only (Fig. 5A). There was no difference in the proteomics array was used (Fig. 6). DCs infected with LOAd713 expression of HLA-DR, IL-6R, and IL-10R between infected DCs demonstrated an increased expression of CD40L and CD40. In- regardless of the virus used. DC supernatants were analyzed for fection with either LOAd713 or LOAd700 led to upregulation of released cytokines (Fig. 5B). LOAd713- and LOAd700-infected many cytokines, costimulator factors, and chemokines, including DCs secreted significantly higher levels of immunostimulatory IL-12, TNF, IFN-g, 4-1BB, CXCL9, CXCL10, CXCL11, and cytokines IL-6, IL-12(p70), IFN-g, and TNF-a compared with CCL4 involved in antitumor immunity. As expected, activation 792 CD40 STIMULI DURING IL-6 BLOCKADE IN THE TUMOR MILIEU Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. CD40 stimuli and IL-6R blockade of stellate cells. Pancreatic stellate cells (n = 2) infected with LOAd(2), LOAd700, or LOAd713 or left uninfected for 48 h, after which cells were harvested for mRNA extraction and to generate protein lysates. (A)RelativeexpressionofaSMA mRNA as analyzed with quantitative PCR. (B) Protein lysates were analyzed by a 233-analyte custom Olink multiplex proteomic array. The most relevant markers are shown as median and range. Statistics were calculated with Kruskal–Wallis test (ANOVA) with Dunn multiple comparison test. Statistical significance is indicated with *p , 0.05. of DCs also upregulated programed death-1 (PD-1) and pro- blockade in vivo was attempted by administering a full-length gramed death ligand-1 (PD-L1) as seen in the LOAd700-infected anti–IL-6R Ab. The B16-hCD46 tumor cells grew rapidly in DCs. PD-L1 functions by binding to PD-1 expressed on activated mice (Fig. 7A), but repeated (n = 6) s.c. injections of mLOAd700 T cells, which induces an anergic state with an inhibition of at the tumor site significantly prolonged survival compared with antitumor responses (27). Interestingly, IL-6R blockade by control (p = 0.0145). However, combination with full-length Ab LOAd713 reduced both PD-1 and PD-L1 on the DCs. Ultimately, targeting murine IL-6R hampered the effect noted by mLOAd700 this may reduce regulatory signaling upon contact with T cells. alone, which can be due to Ab-dependent cellular of IL-6R–positive cells. The day after the third treatment, five mice TMZ-CD40L gene therapy prolongs survival in vivo per group were sacrificed, and tumors were analyzed for immune Animal models are notoriously difficult using the LOAd family of cell infiltration (Fig. 7B). mLOAd700-treated tumors had an in- viruses because adenoviruses do not replicate in mouse cells. crease of T cells. They were active as demonstrated by increased Hence, no oncolysis will occur. Further, the serotype 35 fiber on the PD-1. Interestingly, addition of IL-6R blockade tended to reduce LOAd virus enables infection of human cells only because it targets PD-1 in two mice. Further, mLOAd700 increased APCs human CD46 with no cross-reactivity to animal CD46. Finally, (CD11b+MHCII+) in the tumor. These cells had increased PD-L1, neither the TMZ-CD40L transgene nor the scFv-aIL-6R cross- but the addition of IL-6R blockade showed reduced PD-L1 on reacts with the murine counter receptors. Nevertheless, attempt- these cells. Finally, mLOAd700-treated tumors showed decreased ing to explore LOAd viruses in an in vivo model, we cloned a LOAd suppressive myeloid cells (CD11b+MHCII2) compared with mice virus expressing a murine TMZ-CD40L: mLOAd700. The murine treated with IL-6R blockade alone. B16 melanoma cell line has been engineered to express human CD46 (20) and was used in syngeneic C57BL6 mice. Because Discussion of a lack of any commercially available hybridoma expressing Pancreatic cancer is a devastating disease with rapid progression antimurine IL-6R Ab for cloning of a murine scFv, the IL-6 and high mortality. Treatment options are few and have a minor The Journal of Immunology 793

FIGURE 4. IL-6R blockade during myeloid cell stimuli. PBMCs [(A) n = 2] or CD14+ cells [(B and C) n = 2] stimulated with IL-6 and GM-CSF were cultured with supernatants from A549 cells infected by LOAd(2), LOAd700, or LOAd713 or left uninfected. Bars show CD11bhighCD33+ cells of total A B + + C cells ( and ) or CD11b CD163 cells of total cells ( ). Individual results for each donor are shown in the figure. Downloaded from impact on survival. Hence, new therapeutic strategies are warranted. (15, 17, 41, 42). To further improve therapeutic efficacy, the The microenvironment of pancreatic cancer is characterized by its LOAd713 virus carries two transgenes, an Ab scFv binding the desmoplastic stroma, which contributes to tumor progression, me- IL-6R and TMZ-CD40L. Because of the facts that LOAd viruses tastasis, and treatment resistance (28). One major contributor to the only infect human CD46+ cells, that human CD40L does not

desmoplastic microenvironment is IL-6. Upon binding to the IL-6R, cross-react with murine CD40, and that our scFv-aIL-6R cannot http://www.jimmunol.org/ STAT3 is phosphorylated and translocated into the nucleus where bind murine IL-6R, most experiments in this study needed in- it binds to STAT-responsive elements that control genes involved vestigation in human in vitro systems (17, 43). in proliferation, survival, , and immunosuppression Infection of pancreatic cancer cell lines with LOAd713 led to (29–32). For example, STAT3 induces the expression of the im- a reduction of cell viability due to oncolysis. The oncolytic munosuppressive factors TGF-b1 and IL-10 at the same time it capability of LOAd713 was similar to that of the control virus reduces the production of proinflammatory cytokines and chemo- without any transgenes, showing that addition of the two trans- kines (29). The immunosuppressive nature of the tumor microen- genes did not affect the function of the oncolytic virus. Addition vironment inhibits the infiltration of effector T cells to the cancer of scFv-aIL-6R present in the supernatant of LOAd713-infected cells, classifying pancreatic cancer as a “cold” tumor (33, 34). The cells to IL-6–stimulated Panc01 or MiaPaCa2 cells reduced by guest on September 26, 2021 few infiltrating T cells may explain the lack of effect of checkpoint STAT3 phosphorylation, showing that the scFv-aIL-6R led to an blockade Abs. Such Abs aim to block the negative signals that IL-6/IL-6R pathway blockade. inhibit effector T cells, and thus far they include Abs targeting A major player in the pathogenesis of pancreatic cancer is CTLA-4, PD-1, and PD-L1 (27). In a phase IIa eval- the stellate cell. These myofibroblast-like cells produce factors uating aCTLA-4 (ipilimumab), no effect on survival could be seen associated to proliferation, inflammation, extracellular matrix in pancreatic cancer, and in another trial using aPD-L1 (BMS- remodeling, cell motility, and invasion (44). In our analysis, the 936559), no response was noted in the 14 pancreatic cancer stellate cells expressed CD40 as well as both IL-6 and IL-6R. patients treated (35, 36). In contrast, addition of the immune ac- Stellate cells can be infected with LOAd viruses and express the tivator aCD40 to aPD-L1 treatment in a syngeneic orthotropic transgenes, but because of a lack of hyperphosphorylated reti- mouse model of pancreatic cancer increased the overall survival noblastoma in normal cells, they will not enable virus replica- and the antitumor immunity (37). Hence, stimulation of CD40 tion. Indeed, stellate cells infected with LOAd713 were viable may support the conversion of a cold tumor to an immunologically and showed a significant reduction of the immunosuppressive “hot” tumor, which enables checkpoint Ab therapy. factor TGF-b.TGF-b can suppress T lymphocytes directly by A novel therapy would benefit from simultaneously targeting the inhibiting activation and proliferation and indirectly through IL-6 signaling pathway and stimulating the tumor microenviron- differentiation of T regulatory cells (8, 9). TGF-b also plays a ment to promote antitumor immune responses via CD40. Lau et al. role in the fibrosis of the pancreas by regulating the production (38) reported that a combination of an immune stimulator with IL- of collagenase and metalloproteinases in stellate cells (7). In line 6 blockade increases apoptosis and decreases metastasis in a lung with reduced TGF-b in our experiment, collagenase type I was cancer model, and Caetano et al. (39) have shown that IL-6 also significantly reduced. Further, LOAd713-infected stellate blockade inhibits progression of lung cancer and can change the cells produced significantly reduced levels of FGF5, HGF, and tumor microenvironment toward an antitumor phenotype. In this TWEAK, factors that are connected to tumor progression and article, we report the design of a novel oncolytic adenovirus, suppression of antitumor responses (23, 24, 45–48). Neverthe- LOAd713, for the treatment of pancreatic cancer. Oncolytic less, the overall response to LOAd713 infection of stellate viruses have unique features for the with their cells seems beneficial because reduced production of tumor- capability to infect and selectively replicate in cancer cells, promoting molecules and desmoplasia may be important fac- leading to immunogenic cell death and activation of the immune tors to enhance therapeutic efficacy. Further, LOAd713-infected system (40). Adenoviruses can also be armed with therapeutic stellate cells increased expression of the chemokines CXCL10 genes to further promote antitumor responses. We have previ- and CCL20 that support T lymphocyte infiltration. ously shown that CD40L gene therapy with adenoviruses can Pancreatic cancer is characterized by the infiltration of immune- convert cold tumors to hot tumors with a high infiltration of T cells suppressive cells, especially myeloid suppressors (25). IL-6 794 CD40 STIMULI DURING IL-6 BLOCKADE IN THE TUMOR MILIEU Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. CD40 stimuli and IL-6R blockade of DCs. Immature DCs that differentiated from monocytes obtained from five healthy blood donors were infected by LOAd(2), LOAd700, or LOAd713 or left uninfected for 48 h. Expression of maturation markers on gated CD1a+ DCs was analyzed by flow cytometry (A). Data demonstrate the fold change of mean fluorescence intensity of infected cells compared with uninfected cells. Supernatants from the DCs were analyzed for release of cytokines using luminex (B) and for sIL-6R by ELISA (n = 3). Data are shown as median and range. Statistics were calculated with Kruskal–Wallis (ANOVA) with Dunn multiple comparison test for all analytes except for sIL-6R, in which unpaired t test with Welch correction were used. Statistical significance is indicated with *p , 0.05. production by stellate cells is connected to the differentiation of transgenes. If CD14+ cells were used instead of crude PBMCs, a MDSCs through the activation of STAT3 (6). When PBMCs were similar effect was seen. However, addition of supernatant from cultured with IL-6/GM-CSF, they differentiated toward a MDSC- LOAd700-infected cells had the greatest capacity to block MDSC like phenotype. To determine if virus infection of tumor cells differentiation. Hence, CD40 stimulation is an important factor to could affect myeloid differentiation, supernatants from LOAd reduce MDSC differentiation, which is in line with our previous virus–infected, or uninfected, tumor cells were added to the published results in mice (26). Simultaneous IL-6/IL-6R blockade PBMC cultures. The differentiation of MDSCs was blocked in all may revert some of that effect. LOAd-virus groups regardless of the presence of transgenes. In our previous work, TMZ-CD40L potently activated DCs, and, Hence, the differentiation blockade was connected to the overall in turn, TMZ-CD40L–activated DCs promoted expansion of Ag- inflammatory response to the adenoviral backbone and not to the specific T cells (17). IL-6, as mentioned before, can play a role in The Journal of Immunology 795 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Multiplex proteomics on DCs after CD40 stimuli during IL-6R blockade. Supernatants from DCs infected with LOAd viruses or left un- infected (n = 5) were analyzed with Olink multiplex proteomics custom panel. Data are shown as median and range. Statistical differences were calculated with Kruskal–Wallis (ANOVA) with Dunn multiple comparison test. Statistical significance is indicated with *p , 0.05. 796 CD40 STIMULI DURING IL-6 BLOCKADE IN THE TUMOR MILIEU Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Effect of LOAd viruses in vivo. (A) C57BL6/J mice (n = 10 per group) were injected with syngeneic B16 melanoma expressing human CD46 to enable virus infection. At day 5, the tumor was visible under the skin, and the mice were treated by s.c. injection in the tumor area with a LOAd virus expressing murine TMZ-CD40L (mLOAd700; 1 3 109 infection particles per mouse in 50 ml) and/or a rat anti-mouse IL-6Ra Ab (0.5 mg/mouse in 50 ml) or treated with NaCl control. The mice were treated twice a week, six injections in total. Survival was determined by log-rank test, and mLOAd700 was significantly different from control (p = 0.0145). (B) At day 13 (1 d after third treatment), five mice per group were sacrificed, and the tumors were analyzed for immune cell populations using flow cytometry. Statistical differences were calculated by Kruskal–Wallis (ANOVA) with Dunn multiple comparison test. Statistical significance is indicated with *p , 0.05. immunosuppression, but the cytokine is also linked to enhanced DC activation. Activated DCs upregulate not only costimulatory maturation of DCs and had been used in mixtures for the ex vivo molecules but also both PD-L1 and PD-1. Interestingly, maturation of DCs (49). In this article, we show that the addition LOAd713-infected DCs reduced the levels of both PD-L1 and PD- of the scFv-aIL-6R to the virus and therefore the blocking of IL-6 1. Spary et al. (50) have shown that DCs can upregulate PD-L1 signaling did not hamper the capacity of TMZ-CD40L to promote in an IL-6– and STAT3-dependent manner. Hence, IL-6/IL-6R The Journal of Immunology 797 blockade by the scFv-IL-6R in LOAd713-infected DCs may ex- reaction in pancreatic cancer: role of pancreatic stellate cells. Pancreas 29: plain this finding. LOAd713-infected DCs also had lower levels of 179–187. 3. Erkan, M., C. W. Michalski, S. Rieder, C. Reiser-Erkan, I. Abiatari, A. Kolb, sIL-6R when measured by ELISA compared with LOAd700- N. A. Giese, I. Esposito, H. Friess, and J. Kleeff. 2008. The activated stroma infected cells. It is not clear if this is due to a biological effect index is a novel and independent prognostic marker in pancreatic ductal ade- nocarcinoma. Clin. Gastroenterol. Hepatol. 6: 1155–1161. of the scFv-aIL-6R or to a steric interference to the detection Ab. 4. Miura, T., S. Mitsunaga, M. Ikeda, S. Shimizu, I. Ohno, H. Takahashi, J. Furuse, In this study, we have used DCs differentiated from monocytes M. Inagaki, S. Higashi, H. Kato, et al. 2015. Characterization of patients with obtained from five blood donors. There is some variation of the advanced pancreatic cancer and high serum interleukin-6 levels. Pancreas 44: 756–763. LOAd virus responses among the different blood donors, which 5. Lesina, M., M. U. Kurkowski, K. Ludes, S. Rose-John, M. Treiber, G. Klo¨ppel, does not seem to correlate to infectivity, but may be due to dif- A. Yoshimura, W. Reindl, B. Sipos, S. Akira, et al. 2011. Stat3/Socs3 activation ferent base levels of biomarkers on DCs and also to the genetic/ by IL-6 transsignaling promotes progression of pancreatic intraepithelial neo- plasia and development of pancreatic cancer. Cancer Cell 19: 456–469. proteomic makeup variations among individuals. However, the 6. Mace, T. A., Z. Ameen, A. Collins, S. Wojcik, M. Mair, G. S. Young, J. R. Fuchs, pattern of which biomarkers are upregulated on DCs seems similar T. D. Eubank, W. L. Frankel, T. Bekaii-Saab, et al. 2013. Pancreatic cancer- even if the magnitude can be different. It may be of great interest associated stellate cells promote differentiation of myeloid-derived suppressor cells in a STAT3-dependent manner. Cancer Res. 73: 3007–3018. to investigate patient DC responsiveness to LOAd virus in corre- 7. Nagashio, Y., H. Ueno, M. Imamura, H. Asaumi, S. Watanabe, T. Yamaguchi, lation to treatment responses in later clinical trials. M. Taguchi, M. Tashiro, and M. Otsuki. 2004. Inhibition of transforming growth There are no available animal models that can be used to fully factor beta decreases pancreatic fibrosis and protects the pancreas against chronic injury in mice. Lab. Invest. 84: 1610–1618. evaluate the LOAd713 virus in vivo. Adenoviruses do not replicate 8. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, and in mouse cells but can show some oncolysis in Syrian hamster. S. M. Wahl. 2003. Conversion of peripheral CD4+CD25- naive T cells to CD4 +CD25+ regulatory T cells by TGF-beta induction of factor Foxp3. Downloaded from Nevertheless, the LOAd family of viruses has a serotype 35 fiber J. Exp. Med. 198: 1875–1886. that targets the virus infection to human cells expressing CD46. 9. Pickup, M., S. Novitskiy, and H. L. Moses. 2013. The roles of TGFb in the This receptor is not similar across species, but the human CD46 tumour microenvironment. Nat. Rev. Cancer 13: 788–799. 10. Park, S. J., T. Nakagawa, H. Kitamura, T. Atsumi, H. Kamon, S. Sawa, receptor can be cloned into murine cells such as B16 melanoma. D. Kamimura, N. Ueda, Y. Iwakura, K. Ishihara, et al. 2004. IL-6 regulates Hence, we used B16-hCD46 cells in syngeneic mice and treated in vivo dendritic cell differentiation through STAT3 activation. J. Immunol. 173: them with a LOAd virus expressing a murine version of TMZ- 3844–3854. 11. Bharadwaj, U., M. Li, R. Zhang, C. Chen, and Q. Yao. 2007. Elevated CD40L. mLOAd700 increased T cell infiltration in the treated interleukin-6 and G-CSF in human pancreatic cancer cell conditioned medium http://www.jimmunol.org/ tumors and reduced growth rate, thereby increasing the survival suppress dendritic cell differentiation and activation. Cancer Res. 67: rates of the mice compared with controls. Unfortunately, there are 5479–5488. 12. Gabitass, R. F., N. E. Annels, D. D. Stocken, H. A. Pandha, and no commercially available hybridomas that can be used to clone an G. W. Middleton. 2011. Elevated myeloid-derived suppressor cells in pancreatic, anti-mouse IL-6R scFv, but we attempted to block IL-6 signaling esophageal and gastric cancer are an independent prognostic factor and are as- by injecting a full-length Ab targeting the murine IL-6R. However, sociated with significant elevation of the Th2 cytokine interleukin-13. Cancer Immunol. Immunother. 60: 1419–1430. combining mLOAd700 with this full-length Ab showed worse 13. Kurahara, H., H. Shinchi, Y. Mataki, K. Maemura, H. Noma, F. Kubo, survival compared with mLOAd700 alone. This can be due to Ab- M. Sakoda, S. Ueno, S. Natsugoe, and S. Takao. 2011. Significance of M2- dependent cellular cytotoxicity causing death of cells expressing polarized tumor-associated macrophage in pancreatic cancer. J. Surg. Res. 167: e211–e219. by guest on September 26, 2021 IL-6R because full-length Abs can be used for depleting cells as 14. Loskog, A., A. Maleka, S. Mangsbo, E. Svensson, C. Lundberg, A. Nilsson, well as for signaling blockade. IL-6 in the tumor microenvironment J. Krause, M. Agnarsdo´ttir, A. Sundin, H. Ahlstro¨m, et al. 2016. Immunosti- mulatory AdCD40L gene therapy combined with low-dose cyclophosphamide in is driving tumor growth and causing fibrosis, but systemically it metastatic melanoma patients. Br. J. Cancer 114: 872–880. may have a beneficial effect on the immune system. s.c. injections 15. Westberg, S., A. Sadeghi, E. Svensson, T. Segall, M. Dimopoulou, O. Korsgren, will unfortunately also spread systemically because the tumors are A. Hemminki, A. S. Loskog, T. H. To¨tterman, and H. von Euler. 2013. Treatment efficacy and immune stimulation by AdCD40L gene therapy of spontaneous too small to contain the injection volume. Our model can only show canine malignant melanoma. J. Immunother. 36: 350–358. convincing benefits of TMZ-CD40L gene transfer via LOAd virus, 16. Liljenfeldt, L., K. Gkirtzimanaki, D. Vyrla, E. Svensson, A. S. 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