Vaccine 27 (2009) 3401–3404

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Ampligen: A potential toll-like 3 receptor adjuvant for immunotherapy of cancer B. Jasani ∗ , H. Navabi, M. Adams Departments of Pathology, School of Medicine, Cardiff University, Cardiff, Wales, UKVelindre Cancer Centre, Velindre NHS Trust, Cardiff, Wales, UK

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Article history: Available online 5 February 2009 Keywords: Toll-like 3 Poly (I:C12 U) Cancer Immunotherapy

a b s t r a c t Therapeutic vaccination against cancer-associated antigens represents an attractive option for cancer therapy in view of its potential efficacy, the possibility of long-lasting immunity against the cancer, and safety profile. Cancer patients are however usually immunosuppressed and most cancer-associated antigens are ‘self-antigens’, making it a significant challenge to vaccinate patients against a cancer-associated antigen. Various immunostimulant adjuvants are therefore under investigation in an effort to boost the immune system to overcome the tolerance to tumour associated self-antigens and the ambient immunosuppressory influence of cytokines and regulatory T-cells (Tregs). Many adjuvants appear to be specific ligands for toll-like receptors (TLR) which are potent activators of innate immune responses [Kwissa M, Kasturi SP, Pulendran B. Expert Rev Vaccines. The Science of Adjuvants 2007 6(October(5)):673–84] [1], activating dendritic cell (DC) maturation and inflammatory cytokine secretion, inducing an increase in the cross talk between the innate and adaptive immune systems, and thereby driving the expansion of CTLs that can destroy cancer cells. However, recently some TLR agonists such as CpG have been shown to generate parallel immunosuppressive and inflammatory responses in innate immune cells capable of induction and expansion of Tregs [Conroy H, Marshall NA, Mills KH. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 2008 27(January 7(2)):168–80 [2]; Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 2008;27(2):218–24] [3]. In this context it is noteworthy that poly(I:C), a synthetic double-stranded RNA polymer and a TLR3 agonist, has been shown in mouse models to be capable of enhancing the T cell response to nonimmunogenic self-antigen linked dendritic cell based vaccine strategy, and stimulating diabetic insulitis in the presence of Tregs [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65–72] [4]. Ampligen poly(I:C12 U) is a GMP-grade synthetic analogue of poly(I:C) and therefore suitable for human use. Its effects in a preclinical setting have been recently tested to be similar to poly(I:C) [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65–72]. In particular, it potently induces in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. In addition human monocyte derived DC primed with tumour lysate and matured with Ampligen are capable of generating Th1 specific anti-cancer responses in peripheral blood T-cells derived from cancer patients in the presence of ascites medium containing immunosuppressory cytokines. In this review the key findings are summarised and discussed with a view to offering a rationale for the use of Ampligen as a potentially safe adjuvant capable of overcoming the tumour-related immune tolerance mechanisms in a clinical setting. © 2009 Published by Elsevier Ltd.

1. Introduction To generate an effective anti-tumour T cell response, it is necessary to develop vaccines capable of reversing tumour antigenspecific T cell tolerance [5]. This would require reversal of cancer-associated immunosuppressive influences, such as IL-10 which is produced by advanced tumours [6]. In vitro data sup-

∗ Corresponding author. Tel.: +44 2920 742700; fax: +44 2920 742701. E-mail address: [email protected] (B. Jasani). 0264-410X/$ – see front matter © 2009 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2009.01.071

port that IL-12-producing mature DC are capable of breaking immunological tolerance [7]. Supernatant from activated monocytes (monocyte condition medium), is capable of inducing a stable maturation state of DC in vitro, but its composition is not readily quality controlled [8]. PolyI:C (polyribosinic:polyribocytidylic acid), a synthetic double stranded RNA (dsRNA), has been found to induce a stable mature phenotype (lasting for up to 48 h), with production of heterodimeric IL-12 p70, the bioactive form of IL-12 [9]. Unfortunately, polyI:C may produce toxic side effects in vivo, including shock, renal failure, coagulopathies and hypersensitivity reactions [10].

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Modification of polyI:C by the introduction of unpaired bases (uracil and guanine) results in unique “mismatched” dsRNAs capable of undergoing accelerated hydrolysis, and is associated with reduced toxicity in humans [11], without reduction of pharmacological activity. Poly(I:C12 U) is one such synthetic dsRNA containing regularly occurring regions of mismatching (non-hydrogen bonding), along the helical dsRNA chain. No evidence of dose-limiting organ toxicity, including haematological, liver or renal toxicity, has been observed following intravenous administration to HIVpositive patients [12]. Poly(I:C12 U) (Ampligen® ) has been produced under GMP conditions for clinical use for intravenous infusion [13]. Ampligen has been evaluated as a non-toxic agent for in vitro human DC maturation, prior to their use as an adjuvant in anti-cancer immunotherapy [14]. This study showed that human monocyte-derived DC treated with Ampligen in vitro, express a mature phenotype with high levels of bioactive IL-12 production and significantly less IL-10 compared to those treated with the parent compound, polyI:C. In a recent study, Navabi et al. [15] have used in vitro immunological assays to demonstrate that Ampligen matured DC, have an increased capacity to activate antigen-specific cytotoxic T cell responses, and also the differentiation of CD4+ T cells towards IFN ␥, producing Th1 response, when compared to that of immature DC. These findings are reviewed in the light of relevant literature, to develop a rationale for the use of Ampligen as a non-toxic, clinical grade adjuvant for stimulating effective anti-cancer responses. 2. Ovarian cancer ascites: an in vitro model for study of anti-tumour adjuvant effect of Ampligen Peripheral blood and ascites fluid obtained from the patients undergoing paracentesis, for advanced ovarian cancer (FIGO stage III–IV) have been used as readily accessible source of blood monocytes to derive immature dendritic cells (iDC) and tumour specific T-cells and matched autologous tumour cells, respectively, to study the adjuvant effect of Ampligen in a preclinical setting. 2.1. Ampligen-induced maturation and activation of iDC In brief iDC were generated from peripheral blood monocytes (PBMC) according to an established standard method [16]. They were harvested on Day 5 with >90% cells depicting an immature DC phenotype as confirmed using flow cytometry. Ampligen (poly[I:C12 U]) (Bioclones Ltd., Sandton, S.A.) was applied at the optimal concentration of 100 ␮g/ml, with a high stimulatory capacity and no toxicity. It was compared with optimal concentrations of poly I:C (10 ␮g/ml), LPS (0.5 ␮g/ml) (Sigma Ltd.), and a cytokine mixture (CM) prepared according to [17] containing 10 ng/ml IL1␤, 10 ng/ml TNF-␣ (R&D Systems), 1000 U/ml IL-6 (Peprotech Ltd.) and 1 ␮g/ml PGE2 (Sigma–Aldrich, UK). Poly(I:C12 U) matured DC (m(DC) were >70% viable (determined by trypan blue exclusion) similar to untreated DC after 24–48 h in culture, and had reduced antigen uptake. At 48 h these exhibited typical mDC morphology, with a dramatic increase in dendritic projections from the cell surface and an up-regulation of CD83, CCR7 and MHC molecules [18]. Poly(I:C12 U)-induced mDC obtained from cancer patients were phenotypically comparable to those derived from healthy donors. These cells also demonstrated up-regulation of MHC Class II, CD 40 and CD 86, three markers of DC activation, following poly(I:C12 U) treatment. Similar up-regulation was observed with the other adjuvants. 2.2. Ampligen-induced Th1 cytokine release Using cytokine ELISA Assay poly(I:C12 U) and polyI:C-treated DC were shown to induce IL-12p70 release with similar kinetics.

Cytokine secretion was sustained for up to 48 h. Neither polyI:C nor poly(I:C12 U) produced any detectable IL-12 up to 4 h of culture. Subsequently, they both produced significant and equally high levels of IL-12 up to 19 h of incubation. The IL-12 level began to decrease by 27 h and subsequently further by 48 h. Similar results were observed from a panel of healthy volunteers and cancer patients donors. Over the time-course studied, the untreated DC did not produce any IL-12. Poly(I:C12 U) was compared with other agents for their ability to induce the production from DC of a spectrum of immunomodulatory cytokines such as IL-6, IL-12p70 and IL-10. There was an increase in the secretion of IL-6, following adjuvant treatment, irrespective of the type of adjuvant used, producing similar levels of IL-6 (mean concentration of 1200 pg/ml produced by 5 × 105 DC). Untreated DC produced no detectable IL-6. The IL12p70 production induced by the poly(I:C12 U) and polyI:C-treated DC was considerably greater compared to LPS-treated DC. In contrast, IL-10 production by poly(I:C12 U)-treated DC was significantly lower than that induced in poly I:C and LPS-treated DC (200, 1000 and 2500 pg/ml, respectively). The supernatants derived from untreated iDC and poly(I:C12 U) matured DC, when tested on purified CD4+ T cells, in vitro autologous system showed that after 48 hrs of culture in the presence of iDC supernatant (negative for IL-12 and IL-4) a negligible amount of IFN-␥ was produced, whilst in the CD4+ priming cultures containing poly(I:C12 U) matured DC supernatant, substantial amounts of IFN-␥ were produced. When Th1 medium was supplemented with exogenous recombinant human IL-4, undetectable levels of IFN-␥ were observed using ELISA assay. Similar results were confirmed at mRNA level. The studies directed at expression of specific transcription factors T-bet for Th1, and GATA-3 for Th2, showed that compared to the non-polarised CD4+ T cells, a substantial amount of mRNA for both T-bet and GATA-3 was accumulated in the CD4+ T cells cultured in the presence of supernatant of poly(I:C12 U) matured DCs (Th1 priming conditions). Whilst in the Th1 priming cultures supplemented with IL-4 (Th2 priming conditions) T-bet expression is greatly down-regulated, and GATA-3 levels were comparatively elevated. 2.3. Ampligen-induced cytotoxic activity of CD8+ T cell lines Specific cytototoxic activity of DC-stimulated CD8+ T cell responses in tumour associated lymphocytes were conducted after 3 weeks, following in vitro separation of pure populations (98%) of CD8+ T cells derived ascites fluid of 3 ovarian cancer patients. CD8+ T cell cytotoxicity against autologous tumour cell targets was demonstrated for the 3 patients, at 3 different effector target cell ratios. Autologous ovarian tumour cells were derived from ascites fluid using the method described by Santin et al. [19]. Efficient lysis of autologous tumour cells was demonstrated for all 3 patients when CD8+ T-cells were primed with tumour antigen loaded DC matured with poly(I:C12 U). In the cultures primed with DC-treated poly(I:C12 U) without tumour antigen, there are much lower levels of tumour cell specific lysis in all 3 patients. Whilst CD8+ T-cells alone, in the absence of antigen and poly (I:C12 U), fail to induce tumour specific lysis in all patients. Negligible cytotoxicity was observed against autologous BLCLs and K562, the natural killer cell sensitive cell line. 3. Discussion Large numbers of functionally effective tumour antigen specific CD8+ cytotoxic T-cells (CTLs), delivered either through adoptive cell transfer, or via active in vivo induction, have been recognised to be important in the control or eradication of established tumours [18,20]. For generation of an adequate effector function of these

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CTLs, tumour antigen presentation by mature activated DC to CD8+ T-cells, in the presence of tumour specific CD4+ T-helper cells (Th), appears to be essential [21]. For CD8+ T-cell priming, differentiation and memory development, there is a need for DC to generate a third signal (i.e. bioactive IL-12 release) in co-ordination with antigen presentation (signal 1) and co-stimulation (signal 2) [22,23]. This requires the preparation of optimally mature and activated DCs capable of generating functionally effective CTLs in large numbers. The in vivo production of the third signal has been found to be effective in animal models, through the adjuvant effect of Tolllike receptor ligands, such as CPG (TLR9), lipopolysaccharide (LPS) [TLR4], and polyI:C (TLR3). This has led to the development of clinical grade analogues of these ligands [22,23]. Over the past few years we have investigated the efficacy of a clinical grade analogue of polyI:C, poly(I:C12 U) to appropriately mature and activate DCs for in vitro and in vivo use for cancer immunotherapy [24]. Poly(I:C12 U) is a clinically safe adjuvant capable of appropriately maturing and activating DCs prepared in vitro from peripheral blood mononuclear cells (PBMCs) of healthy human volunteers as well as cancer patients. Poly I:C prepared as a synthetic analogue of viral double stranded RNA, was introduced clinically for its ability to stimulate type I interferons. Thereafter, its effect on myeloid DC and memory T cells was recognised, which led to investigation of its potential therapeutic role in leukaemia and HIV. However, it induced severe toxic side effects in some patients, including shock, renal failure, coagulopathies, and hypersensitivity reactions [10]. These were found to be related largely to poly I:C’s relatively long pharmacokinetic half-life. Modification of the poly I:C structure by the introduction of unpaired uracil and guanine bases resulted in a unique dsRNA, poly (I:C12 U), capable of undergoing accelerated hydrolysis. This polymer, produced under GMP conditions for intravenous administration (Ampligen® ), has since been tested clinically, and found to be associated with reduced toxicity in humans [11]. Gowen et al. found that poly I:C is a ligand for TLR3 as well as other cytosolic sensors (e.g. mda-5), whereas poly(I:C12 U) is only TLR3 restricted. This is especially important considering the known toxicity of poly(I:C), which they suggest may be linked to the combined signalling from mda-5 and TLR3. The findings reviewed here show that poly(I:C12 U)-treated DC satisfy the criteria for phenotypic maturation [25], through consistent up-regulation of cell surface MHC molecules, CD40, and CD86 molecules in all healthy volunteers, as well as in cancer patient DC preparations. It is important to note that these phenotypic alterations were also achieved with polyI:C and LPS applied in parallel experiments. Poly(I:C12 U), similarly to poly I:C and LPS, also has the capacity to cause functional maturation of DC, in that antigen uptake is reduced in poly(I:C12 U)-treated DC, indicating a switch from antigen processing to antigen presentation mode. In addition, the appropriate functional activation of DCs, defined according to the criteria of [23], is also adequately achieved by poly(I:C12 U) through its capacity to cause sustained release of bioactive IL-12 from DCs starting after 4 h of treatment, and lasting at least up to 48 h without further adjuvant exposure. The sustained mode of IL-12 release by DCs has been shown to be necessary for proliferation, clonal expansion as well as functional activation of CTLs [22,23]. Activation of DC with a microbial stimulus (e.g. CpG oligonucleotides) and a range of bacterial stimuli, in the absence of a T-cell derived signal, appears to be sufficient to release significant levels of IL-12 [26]. Under these conditions DC up-regulate CD40 expression, and subsequent cross-linking of CD40 can result in further enhanced IL-12 production [26]. The notion that optimal production of IL-12 by DC involves synergy between CD40 crosslinking and microbial stimulation, is compatible with human in vitro studies, that demonstrate that the interaction between T-cells

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and antigen-presenting cells is not sufficient to induce high levels of IL-12 production, unless microbial stimuli and/or cytokines are used as the adjuvant. Alternatively IL-12 production is induced by the interaction of the DC with the T-cells [27,28]. Whether the use of the poly (I:C12 U) in soluble form, as applied in the present study, represents the most efficient approach for its in vivo adjuvant action, remains to be determined. Analogous to viral dsRNA, the mode of action of poly (I:C12 U) on DCs may occur via the Toll-like receptor 3 (TLR3), which resides within the intracellular compartment. Poly(I:C12 U) is a macromolecule, a polymer of average molecular weight of more 100,000 Da, and unlike the virus associated dsRNA, it lacks a natural mechanism to enter the cell. The mechanism by which its soluble form interacts with the DC is uncertain. Under the conditions used in this study, it appears to enter the DC in sufficient quantity to produce DC maturation and activation effects. Recent studies using deliberate cellular association or intracellular introduction of polyI:C or poly(I:C12 U), have shown DC maturation and activation [29]. However IL-12 production was shown to be consistently less efficient compared to their respective soluble forms [29,30]. The synergistic effect of TNFa/IL-1␤ + polyI:C + IFN␣ DCs in yielding DCs (␣DC1), with the ability to produce high levels of IL-12, has been well demonstrated by [26], and the contribution of IL-12 to high CTL inducing ability of these DCs in vitro. They also point to the need for a clinical trial to evaluate their effectiveness as a cellular vaccine. Due to its ability to induce IL-12 production and promote Th1 effect, poly(I:C12 U) as a clinical grade adjuvant, has the potential to be used systemically for immunotherapy. The dose and the route of administration of poly(I:C12 U) also seem to be important in producing its optimal effects. In a recent in vitro study, a lower dose of 10 ␮g/ml [31] (compared to 100 ␮g/ml used in this study) was found to be sub-optimal. Also, for in vivo use, the sub-cutaneous route [31] appears to be less effective compared to i.p. or i.v. routes [30,32]. Another study [33] which tested the adjuvant capacity of poly (I:C12 U), was found to be efficient at promoting both specific CTL expansion and Tc1 differentiation in HHD2 mice, when injected i.v. at the dosage of 250 ␮g. In summary, poly (I:C12 U) appears to have potential for use as a clinical grade adjuvant both for adoptive transfer or for active immunisation in cell-mediated cancer immunotherapy. Acknowledgements Grant support: EU FP5 funded proposal QLRT-2001-00093 and Cancer Research Wales References [1] Kwissa M, Kasturi SP, Pulendran B. Expert Rev Vaccines. The Science of Adjuvants 2007;6(October (5)):673–84. [2] Conroy H, Marshall NA, Mills KH. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 2008;27(January 7 (2)):168–80. [3] Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H. TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 2008;27(2):218–24. [4] Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65–72. [5] Mehta-Damani A, Markowicz S, Engleman EG. Generation of antigen-specific CD8+ CTLs from naive precursors. J Immunol 1994;153(3):1003–996. [6] Steinbrink K, Jonuleit H, Muller G, Schuler G, Knop J, Enk AH. Interleukin10-treated human dendritic cells induce a melanoma-antigen-specific anergy in CD8(+) T cells resulting in a failure to lyse tumor cells. Blood 1999;93(5): 1634–42. [7] Xu S, Koski GK, Faries M, Bedrosian I, Mick R, Maeurer M, et al. Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12dependent mechanism. J Immunol 2003;171(5):2251–61. [8] Bender A, Sapp M, Schuler G, Steinman RM, Bhardwaj N. Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. J Immunol Methods 1996;196(2):121–35.

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