The local application of Toll-Like Receptor (TLR) agonists

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1 ORIGINAL ARTICLE Regression of Established AB1 Murine Mesothelioma Induced by Peritumoral Injections of CpG Oligodeoxynucleotide Either Alone or in Combination with Poly(I:C) and CD40 Ligand Plasmid DNA Geoffrey W. Stone, PhD,* Suzanne Barzee, BS,* Victoria Snarsky, MSc,* Camila Santucci, RVT,* Brian Tran, BS,* and Richard S. Kornbluth, MD, PhD* Introduction: Stimulation of the CD40 receptor using an agonistic anti-cd40 antibody can slow the growth of AB1 tumors. Stimulation of the GITR receptor may also have antitumor activity by countering the immunosuppressive effects of regulatory CD4 T cells. Similarly, agonists for Toll-Like Receptors (TLR) such as CpG oligodeoxynucleotides (TLR9 agonist) have activity against AB1 tumors. Combinations of CpG with CD40 ligand and polyinosinic-polycytidylic acid (poly(i:c), TLR3 agonist) may be even stronger than CpG alone. The synergistic effects of these combinations have been tested in other tumor types but not in mesothelioma. Methods: Established AB1 mesothelioma tumors were injected with either plasmid DNA encoding a novel 4-trimer form of murine CD40 ligand (psp-d-cd40l), GITR ligand (GITRL), or control plasmid DNA. In addition, CpG with or without poly(i:c) was also injected intratumorally. Results: Plasmid injections of psp-d-cd40l or psp-d-gitrl, had no significant antitumor effect, possibly reflecting the difficulty of administering DNA injections into this very dense tissue. However, the injection of CpG with or without poly(i:c) strongly suppressed tumor growth and led to long-term tumor-free survival. The response to a triple combination of psp-d-cd40l CpG poly(i:c) was demonstrated by an increase in intratumoral CD8 T cells and a dramatic increase in F4/80 macrophages. Conclusions: Intratumoral injections of plasmid DNAs encoding highly active forms of either CD40 ligand or GITR ligand had no significant antitumor effects in this model, although improved DNA delivery techniques could possibly improve this strategy. In contrast, intratumoral CpG injections had significant antitumor effects and *Department of Medicine, University of California San Diego, La Jolla, California, and VA San Diego Healthcare System, San Diego, California; and Multimeric Biotherapeutics Inc., San Diego, California. Disclosure: Geoffrey W. Stone and Richard S. Kornbluth are listed as inventors on patent applications related to combinations of CD40L and TLR agonists filed by the University of California, San Diego. Address for correspondence: Richard S. Kornbluth, MD, PhD, Department of Medicine, University of California San Diego, 9500 Gilman Drive No. 0679, La Jolla, CA rkornbluth@ucsd.edu Geoffrey W. Stone is currently at Miller School of Medicine, University of Miami, 1580 NW 10th Ave., Miami, Florida. Copyright 2009 by the International Association for the Study of Lung Cancer ISSN: /09/ there were indications that CpG plus poly(i:c) was even more effective. Taken together, these data confirm previous reports that immune stimulants, especially CpG TLR9 agonists, have potential as a treatment for mesothelioma. Key Words: Tumor immunity, Vaccination, Dendritic cells, Monocyte/ macrophages, Mesothelioma, CD40, GITR, TLR3, TLR9. (J Thorac Oncol. 2009;4: ) The local application of Toll-Like Receptor (TLR) agonists may have antitumor effects. 1,2 Equally promising in mice but difficult to apply in humans, is the use of CD40 stimulation. Numerous studies have shown that agonistic antibody to CD40 can have major antitumor effects either on its own 3,4 or when combined with TLR agonists. 5,6 However, agonistic anti-cd40 antibody can be toxic, especially if used repeatedly. 3,7 The van Mierlo group noted death of 50% of mice following a second intravenous injection of anti-cd40 antibody. Intratumoral injections of an adenovirus expressing CD40L has also been shown to be effective against mesothelioma in mice. 8 From an immunologic point of view, most of these immunostimulants activate dendritic cells (DCs), shifting them from their resting, tolerogenic state to that of fully effective antigen presenting cells. These immunostimulants counteract the deactivating effects of tumors on the DCs in their environment. 9 For IL-12p70 production, for example, Napolitani et al. 10 found that the combination of two TLR agonists (e.g., poly(i:c) for TLR3 and R-848 for TLR7/8) was markedly synergistic, and 10-fold more IL-12p70 was produced when CD40L was added to the cultures. In vivo, these findings have correlates in reports that CD40 stimulation combined with TLR agonists is capable of inducing strong antitumor CD8 T cell activity. 5,6 This study examined the effects on established AB1 mesothelioma of combinations of TLR agonists along with a new form of CD40L (4-trimer SP-D-CD40L) that dramatically enhanced CD8 T cell responses in a murine DNA vaccine model. 11 In addition, a 4-trimer form of GITR ligand (psp-d-gitrl) was tested, based on the concept that stim-

2 Immunotherapy of Murine Mesothelioma with CpG and CD40L ulation of the GITR receptor can antagonize the immunosuppressive effects of regulatory CD4 T cells found in many tumors. 12 CpG and poly(i:c) showed activity against AB1 tumors when injected directly into established tumors every other day 5. In addition, synergies between these two TLR agonists and between psp-d-cd40l and TLR agonists were indicated in some experiments, suggesting that these combinations should be studied further. MATERIALS AND METHODS Plasmids CD40L and GITRL plasmids were previously described. 11,13 Plasmids tested include: psp-d-cd40l; psp-d- GITRL; pifn (porf5-mifng, InVivoGen, San Diego, CA); and pcdna3.1( ) (Invitrogen, Carlsbad, CA) as vector control. Plasmid Preparation Plasmids were isolated by anion-exchange chromatography resin (EndoFree Plasmid MaxiKit, QIAgen, Inc, Valencia, CA). An additional purification step using Triton X-114 detergent extraction was used to further purify plasmid. 14 Before injection, plasmid DNA was diluted in Dulbecco calcium- and magnesium-free phosphate buffered saline (PBS) to 1 mg/ml (50 g per 50 l injection). TLR Agonists TLR agonist compounds were prepared following their manufacturer s instructions. TLR3 agonist poly(i:c) (Amersham, Piscataway, NJ) was administered at 25 g per injection in PBS. TLR9 agonist CpG TGACTGTGAACGTTC- GAGATGA-3 (all DNA linkages phosphorthioate) was a gift of Dr. Eyal Raz 15 or was purchased from Trilink Biotechnologies (San Diego, CA). CpG was administered at 25 g per injection in PBS. All solutions were endotoxin free. Tumor Cell Lines The AB1 malignant mesothelioma tumor cell line was derived by asbestos treatment of BALB/c mice by Dr. Bruce Robinson, 16 and was obtained through Dr. Steven Albelda, University of Pennsylvania. The A20 tumor cell line was obtained from the ATCC. All cell lines were cultured in RPMI 1640, 2 mm L-glutamine, and 10% FBS. Tumor Challenge and Immunotherapy Mice studies were approved by the Institutional Animal Care and Use Committee of the VA San Diego Healthcare System. Tumor cells were detached using trypsin/edta followed by neutralization with cold RPMI 10% FBS and pelleting at 300 g for 6 minutes. Cells were washed three times with cold PBS and resuspended at cells/ml in PBS. To initiate tumors, a total of cells (0.1 ml) were injected subcutaneously into the abdomen of 6 to 8 week old female BALB/cByJ mice (Jackson Labs, Bar Harbor, ME). Five mice/group were used for each condition. When tumors became palpable, they were measured on two orthogonal axes using an electronic caliper and the diameter was recorded as the mean of the two measurements. Once the tumors measured 4 mm in mean diameter, a total of 50 l of test substance was injected into or around the tumor using a 0.5 ml insulin syringe. The peritumoral injections were repeated every other day for a total of five injections. Mice were euthanized when tumors became 15 mm in mean diameter or ulcerated. Tumor Rechallenge Sixty days after initial AB1 injection, tumor-free survivors were rechallenged s.c. either with AB1 cells or A20 lymphoma cells. Tumor growth was measured from day 12 and every other day until all mice that became tumor-bearing were euthanized. Tumor Histology Mice with established tumors (as detailed above) at 7 mm diameter were given peritumoral injections of immunostimulatory compounds every other day 5 and euthanized 2 days after the final dose. The tumors were then excised and divided. Half was fixed in 1% paraformaldehyde and embedded in paraffin, and the other half was flash frozen in OCT compound. Serial sections (10 m in thickness) were obtained. For immunostaining the following primary antibodies were used: CD11c clone HL3 (BD Pharmingen, San Diego, CA); CD8 clone (BD Pharmingen); and F4/80 clone BM8 (ebioscience, San Diego, CA). Slides were examined with a Zeiss Axioskop microscope and images were recorded using an Optronics CCD camera. Duplicate tumors were evaluated for each condition. Statistics Geometric mean tumor sizes were compared using Student t test (Prism 4.0 Software, GraphPad Systems, San Diego, CA). A log-rank test was used to determine the significance of the differences between groups in Kaplan-Meier survival plots. A p value of 0.05 was considered significant. RESULTS Construction of 4-Trimer Soluble CD40L Fused with the Body of Surfactant Protein D In a previous vaccine study we found that plasmids encoding soluble forms of CD40L had adjuvant activity when combined with plasmids for HIV antigens. The most active form of CD40L in those studies was a 4-trimer form produced as a fusion protein with the body of pulmonary surfactant protein D (SP-D) 11,17 (Figure 1). FIGURE 1. Depiction of SP-D-CD40L. Using recombinant DNA techniques, the N-terminal portion of murine pulmonary surfactant protein D (minus the carbohydrate recognition domain) was fused to the entire extracellular domain of murine CD40L. The resulting molecule is expected to form a 4-trimer cruciate homododecamer as shown. Copyright 2009 by the International Association for the Study of Lung Cancer 803

3 Stone et al. For Established AB1 Mesothelioma, psp-d- CD40L, and psp-d-gitrl Alone were Inactive but a Triple Combination of psp-d-cd40l CpG poly(i:c) Cured a Majority of Treated Mice To determine if psp-d-cd40l or psp-d-gitrl had antitumor effects for mesothelioma, AB1 tumor cells were injected subcutaneously into BALB/c mice and treated FIGURE 2. Combinations of psp-d-cd40l, CpG, and poly(i:c) showed strong antitumor effects against established AB1 mesothelioma. AB1 cells were injected s.c. in BALB/c mice. When the tumors were 4 mm in diameter, they were injected every other day 5 with 50 g of plasmid DNA or 25 g of TLR agonists or their combinations. Panel A, For AB1 mesothelioma, neither psp-d-cd40l nor psp-d-gitrl had an effect on tumor growth. However, the combination of psp-d-cd40l CpG or the triple combination of psp-d-cd40l CpG poly(i:c) significantly reduced tumor growth compared with phosphate buffered saline or pcdna3.1 (p 0.05 for psp-d-cd40l CpG and p 0.01 for psp-d-cd40l CpG Poly(I:C) from day 20 by Student t test). In those cases, many of the established tumors regressed entirely following treatment, an effect never seen under other conditions. Panel B, Combinations that included CpG significantly enhanced survival and cured mice of established AB1 mesothelioma. Consistent with the tumor growth data, there was no survival benefit using psp- D-CD40L or psp-d-gitrl alone. However, the two combinations that included CpG had significant effects on survival. Remarkably, the triple combination of psp-d-cd40l CpG poly(i:c) cured 5/6 mice which remained tumor free for more than 4 months. FIGURE 3. CpG alone or combinations that included CpG showed strong antitumor effects on established AB1 mesothelioma. As in Figure 2, AB1 tumors were established and treated with injections of test substances every other day 5 beginning when the tumors were 4 mm in diameter. For these experiments, the plasmid DNA was extracted with Triton X-114 to remove adventitious immunostimulatory compounds. Panel A, CpG or combinations that included CpG significantly slowed tumor growth. As before, psp-d-cd40l did not slow AB1 mesothelioma growth. However, both CpG and CpG poly(i:c) had significant antitumor effects compared with phosphate buffered saline from day 18 (p 0.05 by Student t test). The double combination of TLR agonists CpG poly(i:c) slowed tumor growth, but this was not significantly greater than using CpG alone in this experiment. For combinations that included psp-d-cd40l, this molecule made a significant difference for two combinations when compared with pcdna3.1 as a control: psp-d-cd40l CpG (p 0.01 from day 16); and psp-d-cd40l CpG poly(i:c) (p 0.05 from day 16). However, the substitution of control plasmid pcdna3.1 for psp-d-cd40l in the triple combination with CpG poly(i:c) did not significantly reduce the antitumor effect. Because of the restricted time axis, this graph does not indicate the complete regression of individual tumors produced by CpG-containing treatments, as reflected in the tumor-free survival data in Panel B. Panel B, CpG or combinations that included CpG led to long term tumor-free survival. As before, psp-d-cd40l did not lead to long term tumor free survival. For CpG alone, 60% of mice were long term tumor-free survivors in this experiment. Similarly, the combination of CpG with either psp-d-cd40l or control pcdna3.1 led to long term tumor-free survivors, and this was statistically significant when compared with pcdna3.1 alone (p 0.01). This indicated that CpG was the dominant factor in the antitumor effects observed. 804 Copyright 2009 by the International Association for the Study of Lung Cancer

4 Immunotherapy of Murine Mesothelioma with CpG and CD40L with various combinations. Although psp-d-cd40l and psp-d-gitrl produced significant antitumor effects in two other tumor systems, B16F10 melanoma and A20 lymphoma, respectively (data not shown), they had no effect on the growth of established AB1 mesothelioma or the survival of mice bearing this tumor (Figure 2). However, given the strong synergy reported for CD40 stimulation combined with TLR stimulation, 5,10 the combination of psp-d-cd40l with CpG oligodeoxynucleotide (ODN) and the combination of CpG ODN and poly(i:c) were also tested. As shown in an initial experiment, combinations of psp-d-cd40l with one or more TLR agonists led to marked antitumor responses. Remarkably, the triple combination of psp-d-cd40l, CpG ODN, and poly(i:c) cured a significant number of mice (Figure 2). It was also noted that mice receiving treatments containing SP-D-CD40L or GITRL displayed no signs of toxicity up to 1 year after vaccination, consistent with previous DNA vaccine studies. 11 For Established AB1 Mesothelioma, CpG was the Most Active Component of the Triple Therapy Regimen Given the promising results shown in Figure 2, a number of additional controls were tested. As shown in Figure 3, psp-d-cd40l alone again had no significant antitumor effect. The triple combination of a plasmid CpG poly(i:c) again showed strong antitumor effects, which were just as effective if the plasmid was control pcdna3.1 rather than psp-d-cd40l. Importantly, however, CpG alone was remarkably effective as an antitumor agent under these conditions. The Combination of Plasmids for CD40L and IFN- was not Effective Against AB1 Mesothelioma Although CD40L is an important stimulus for IL-12p70 production, it is not effective without a second stimulus such as a TLR agonist or IFN-. 18,21,22 Consequently, psp-d- CD40L was tested in combination with plasmid DNA for IFN- and injected into established AB1 tumors as previously described. As shown in Figure 4, the combination of psp-d- CD40L pifn- had no significant antitumor effects under the same conditions where combinations containing CpG were highly effective, as judged by either tumor growth (Panel A) or survival (Panel B). Mice Cured of AB1 Mesothelioma Tumors were Resistant to Rechallenge with the Same Tumor Type Mice cured of AB1 mesotheliomas for 2 months were rechallenged with the same or an unrelated tumor cell line. As shown in Figure 5, these mice were protected from challenge with the homologous AB1 tumor, but they remained suscep- FIGURE 4. The combination of plasmids for CD40L and IFN- had no effect on AB1 mesothelioma. Because IFN- has been reported to synergize with CD40 stimulation, the antitumor effects of psp-d-cd40l pifn- were studied. However, there were no antitumor effects from these plasmids either alone or in combination, as judged by tumor growth measurements (Panel A) or survival (Panel B). As before, the triple combination of psp-d-cd40l CpG poly(i:c) led to tumor regressions and long term tumor-free survival (p 0.01 compared with pcdna3.1). FIGURE 5. Mice cured of AB1 mesothelioma resist homologous tumor challenge. Mice that were cured of established AB1 mesothelioma following treatment with the triple combination of psp-d-cd40l CpG poly(i:c) were rested for 2 months and then challenged with either AB1 mesothelioma or A20 B cell lymphoma, an unrelated tumor of BALB/c mice. As determined by tumor growth, the mice cured of AB1 mesothelioma tumors completely suppressed the growth of the homologous tumor cell line. This was specific, however, as the unrelated A20 B cell lymphoma tumors grew in an unrestrained manner in the AB1-cured mice. All of the AB1-cured mice that were rechallenged with AB1 tumors survived until the end of the experiment 40 days later, whereas none of the mice in the other groups survived. Copyright 2009 by the International Association for the Study of Lung Cancer 805

5 Stone et al. tible to heterologous A20 tumor cells (an unrelated B cell lymphoma). In a repeat study, mice cured of AB1 for greater than 90 days were also protected from rechallenge with the homologous tumor cell line (data not shown). These data are consistent with immunologic memory as the basis for protection. Regressing Tumors were Marked by an Infiltration of CD8 T Cells and F4/80 Macrophages Given the possible role of adaptive immunity in tumor eradication, tumor sections were analyzed for the presence or absence of immune cells, including DCs, CD8 T cells, and macrophages. Palpable AB1 tumors were treated with psp-d- CD40L CpG poly(i:c) every other day times five, followed by tumor dissection 2 days later. As shown by hematoxylin and eosin staining (Figure 6A), AB1 tumors treated with psp-d-cd40l CpG poly(i:c) were much less hypercellular, as an indication of cell loss. To identify immune cells within the tumors, tumor sections were stained for the presence of CD11c DCs, CD8 T cells, and F4/80 macrophages (Figure 6B, C, and D, respectively). Tumors treated with psp-d-cd40l CpG poly(i:c) showed an overall decrease in the density of intratumoral DCs (Figure 6B). This may have resulted from the activation of these cells followed by their migration to the tumordraining lymph nodes as previously reported. 23 At the same time point, there was an increase in intratumoral CD8 T cells (Figure 6C), suggesting a role for CD8 T cell-mediated activity in the antitumor effects observed. Surprisingly, the number of F4/80 macrophages increased to become the majority of cells within the regressing tumor (Figure 6D). DISCUSSION Numerous studies have shown that strong CD40 stimulation can lead to the eradication of established tumors. 3,24,25 FIGURE 6. Tumor-dependent differences in the immunohistology of induced tumor regression. Tumors were injected every other day 5 with phosphate buffered saline as a control or with psp-d-cd40l CpG poly(i:c). As shown in Figures 2 through 4, this triple combination often eradicated established AB1 tumors. To investigate the mechanism for this effect, tumor tissue was dissected out 2 days after the final treatment injection and examined by histology. In each case, the images shown are representative fields from two similarly-treated mice. Panel A, Paraffin sections of control and treated tumors. Tumor sections were stained with hematoxylin and eosin. AB1 tumors treated with phosphate buffered saline (left panel) were hypercellular and consisted of sheaf-like arrangements of fusiform cells with plump cytoplasm and no areas of necrosis. In contrast, AB1 tumors treated with the triple combination (right panel) showed reduced cellularity and replacement of tumor cells by collections of infiltrating cells with less condensed nuclei, which were found to correlate with F4/80 macrophages (see Panel D). Panel B, Immunofluorescence analysis of intratumoral CD11c dendritic cells. In the AB1 tumors injected with phosphate buffered saline as a control, numerous CD11c dendritic cells were seen, often in clusters as shown in the figure (left panel). After treatment with the triple combination, fewer DCs were identified and no clusters of DCs were seen (right panel). Panel C, Immunofluorescence of intratumoral CD8 T cells. For tumors injected with phosphate buffered saline as a control, relatively few CD8 T cells were seen (left panel). However, after injections with the triple combination, there was a marked increase in intratumoral CD8 T cells in all tumor sections examined (right panel). Panel D, Immunofluorescence of intratumoral F4/80 macrophages. AB1 tumors injected with phosphate buffered saline as a control contained many F4/80 intratumoral macrophages (left panel). However, after treatment with the triple combination, a massive infiltration of macrophages was apparent in the regressing tumors (right panel). 806 Copyright 2009 by the International Association for the Study of Lung Cancer

6 Immunotherapy of Murine Mesothelioma with CpG and CD40L Particularly noteworthy was the demonstration by van Mierlo et al. 3 showing that agonistic CD40 antibody could lead to the rejection of tumors formed by malignant cells that themselves lack the CD40 receptor, indicating that the primary effect of this treatment was on the host immune response and not on the tumor cells themselves. The most likely mechanism for this effect is the known ability of CD40 stimulation to activate DCs, which in turn promote effective CD8 T cell responses. 26,27 Consequently, it was disappointing that intratumoral injections of psp-d-cd40l were ineffective against AB1 tumors in this mesothelioma model. One explanation is the relative inefficiency of gene expression from plasmids injected into tumors, as opposed to intramuscular DNA injections used in our previous study. 28 Further studies are needed to determine if polymer delivery agents 29 or electroporation 30 will have beneficial effects on the antitumor activities of psp-d- CD40L and psp-d-gitrl. In addition, the combination of effective CD40 stimulation with TLR agonists deserves further study. 5,10,20,31 TLR stimuli were effective and have already been shown to have antitumor effects, as shown by a recent study by Currie et al. 32 Combinations of two TLR agonists may be even more effective, given the synergistic effects on dendritic cell function by the combination of a MyD88 pathway TLR agonist like CpG and a TRIF pathway TLR agonist like poly(i:c). 10,33,34 The immunohistological studies shown in Figure 6 are consistent with current concepts on how an intratumoral treatment might lead to tumor rejection. The apparent decrease in intratumoral DCs following treatment in AB1 mesothelioma models (Figure 6B) is consistent with DC activation, a shift to CCR7 expression, and chemotaxis through lymphatics to the tumor-draining lymph nodes. 1 The appearance of CD8 T cells in the tumors (Figure 6C) is consistent with the recognized effects of these cytotoxic cells on mesothelioma. 8,35 The influx of macrophages into regressing mesothelioma tumors (Figure 6D) is remarkable, although it is not clear if the macrophages are killing the tumor cells or simply removing dead tissue. Buhtoiarov et al. 36 found that CD40 stimulation using an agonistic anti-cd40 antibody combined with systemic CpG slowed the growth of B16 melanoma tumors even in SCID/beige mice lacking T cells and NK cells, and this effect was abrogated when silica was given to deplete macrophages. That study suggests that macrophages should be considered as one possible effector cell for the antitumor effects of CD40L/TLR agonist combinations. In conclusion, CpG demonstrated impressive antitumor effects on established AB1 mesotheliomas, leading to tumor regression and long term tumor-free survival in the majority of cases. Intratumoral injections of plasmid DNAs for SP-D- CD40L, psp-d-cd40l plus IFN-, or psp-d-gitrl were ineffective, possibly reflecting the difficulty of injecting the very dense tissue formed by AB1 tumors. Further studies are needed to determine if this difficulty can be circumvented using polymers or electroporation to improve DNA delivery and transgene expression. No toxic effects of this treatment were apparent, however, suggesting further studies of immunotherapy as a treatment for mesothelioma are warranted. ACKNOWLEDGMENTS Supported by the Mesothelioma Applied Research Foundation, NIH grants R21AI (to R.S.K.), K22AI (to G.W.S.), and R21AI (to G.W.S.), American Foundation for AIDS Research (amfar) grant RGV (to R.S.K.), the UCSD Center for AIDS Research (NIH P30AI036214, Douglas D. Richman), and the Research Center on AIDS and HIV Infection of the VA San Diego Healthcare System. Supported by an NIH AIDS Training Grant to UCSD (T32AI007384) (to G.W.S). The authors thank Kendra McCafferty and Dr. Mari V. Bray (VA San Diego Veterinary Medical Unit) for training in veterinary procedures. Dr. Nissi Varki (UCSD Cancer Center) for assistance with the immunofluorescence studies. Dr. Robert Hancock provided ideas used in designing the AB1/ A20 tumor rechallenge experiment. Dr. Eyal Raz provided CpG 1018 for initial studies. REFERENCES 1. Vicari AP, Chiodoni C, Vaure C, et al. 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