CD8+ tissue-resident memory T cells are expanded in primary Sjögren's disease and can be therapeutically targeted by CD103 blockade.

OBJECTIVE: Tissue-resident memory cells (Trm) are a subset of T cells residing persistently and long-term within specific tissues that contribute to persistent inflammation and tissue damage. We characterised the phenotype and function of Trm and the role of CD103 in primary Sjogren's syndrome (pSS). METHODS: In both pSS and non-pSS sicca syndrome patients, we examined Trm frequency, cytokine production in salivary glands (SG) and peripheral blood (PB). We also analysed Trm-related gene expression in SG biopsies through bulk and single-cell RNA sequencing (scRNAseq). Additionally, we investigated Trm properties in an immunisation-induced animal model of pSS (experimental SS, ESS) mouse model and assessed the effects of Trm inhibition via intraglandular anti-CD103 monoclonal antibody administration. RESULTS: Transcriptomic pSS SG showed an upregulation of genes associated with tissue recruitment and long-term survival of Trm cells, confirmed by a higher frequency of CD8+CD103+CD69+ cells in pSS SG, compared with non-specific sialadenitis (nSS). In SG, CD8+ CD103+ Trm contributed to the secretion of granzyme-B and interferon-γ, CD8+ Trm cells were localised within inflammatory infiltrates, where PD1+CD8+ T cells were also increased compared with nSS and MALT lymphoma. scRNAseq of PB and pSS SG T cells confirmed expression of CD69, ITGAE, GZMB, GZMK and HLA-DRB1 among CD3+CD8+ SG T cells. In the SG of ESS, CD8+CD69+CD103+ Trm producing Granzyme B progressively expanded. However, intraglandular blockade of CD103 in ESS reduced Trm, reduced glandular damage and improved salivary flow. CONCLUSIONS: CD103+CD8+Trm cells are expanded in the SG of pSS and ESS, participate in tissue inflammation and can be therapeutically targeted.

Therapeutic ISCOMATRIX™ adjuvant vaccine elicits effective anti-tumor immunity in the TRAMP-C1 mouse model of prostate cancer.

Cancer vaccine development has proven challenging with the exception of some virally induced cancers for which prophylactic vaccines exist. Currently, there is only one FDA approved vaccine for the treatment of prostate cancer and as such prostate cancer continues to present a significant unmet medical need. In this study, we examine the effectiveness of a therapeutic cancer vaccine that combines the ISCOMATRIX™ adjuvant (ISCOMATRIX) with the Toll-like receptor 3 agonist, polyinosinic-polycytidylic acid (Poly I:C), and Flt3L, FMS-like tyrosine kinase 3 ligand. We employed the TRAMP-C1 (transgenic adenocarcinoma of the mouse prostate) model of prostate cancer and the self-protein mPAP (prostatic acid phosphatase) as the tumor antigen. ISCOMATRIX™-mPAP-Poly I:C-Flt3L was delivered in a therapeutic prime-boost regime that was consistently able to achieve complete tumor regression in 60% of animals treated and these tumor-free animals were protected upon rechallenge. Investigations into the underlying immunological mechanisms contributing to the effectiveness of this vaccine identified that both innate and adaptive responses are elicited and required. NK cells, CD4+ T cells and interferon-γ were all found to be critical for tumor control while tumor infiltrating CD8+ T cells became disabled by an immunosuppressive microenvironment. There is potential for broader application of this cancer vaccine, as we have been able to demonstrate effectiveness in two additional cancer models; melanoma (B16-OVA) and a model of B cell lymphoma (Eµ-myc-GFP-OVA).

Results of a randomized, double-blind phase II clinical trial of NY-ESO-1 vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in participants with high-risk resected melanoma.

BACKGROUND: To compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence. METHODS: Participants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity. RESULTS: The ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4+ and CD8+ responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)+/Human Leukocyte Antigen (HLA) class I+ double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants. CONCLUSIONS: The vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse.

Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells.

Gamma delta (γδ) T cells are essential to protective immunity. In humans, most γδ T cells express Vγ9Vδ2+ T cell receptors (TCRs) that respond to phosphoantigens (pAgs) produced by cellular pathogens and overexpressed by cancers. However, the molecular targets recognized by these γδTCRs are unknown. Here, we identify butyrophilin 2A1 (BTN2A1) as a key ligand that binds to the Vγ9+ TCR γ chain. BTN2A1 associates with another butyrophilin, BTN3A1, and these act together to initiate responses to pAg. Furthermore, binding of a second ligand, possibly BTN3A1, to a separate TCR domain incorporating Vδ2 is also required. This distinctive mode of Ag-dependent T cell activation advances our understanding of diseases involving pAg recognition and creates opportunities for the development of γδ T cell-based immunotherapies.

Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products.

The immunogenicity of protein therapeutics is an important safety and efficacy concern during drug development and regulation. Strategies to identify individuals and subpopulations at risk for an undesirable immune response represent an important unmet need. The major histocompatibility complex (MHC)-associated peptide proteomics (MAPPs) assay directly identifies the presence of peptides derived from a specific protein therapeutic on a donor's MHC class II (MHC-II) proteins. We applied this technique to address several questions related to the use of factor VIII (FVIII) replacement therapy in the treatment of hemophilia A (HA). Although >12 FVIII therapeutics are marketed, most fall into 3 categories: (i) human plasma-derived FVIII (pdFVIII), (ii) full-length (FL)-recombinant FVIII (rFVIII; FL-rFVIII), and (iii) B-domain-deleted rFVIII. Here, we investigated whether there are differences between the FVIII peptides found on the MHC-II proteins of the same individual when incubated with these 3 classes. Based on several observational studies and a prospective, randomized, clinical trial showing that the originally approved rFVIII products may be more immunogenic than the pdFVIII products containing von Willebrand factor (VWF) in molar excess, it has been hypothesized that the pdFVIII molecules yield/present fewer peptides (ie, potential T-cell epitopes). We have experimentally tested this hypothesis and found that dendritic cells from HA patients and healthy donors present fewer FVIII peptides when administered pdFVIII vs FL-rFVIII, despite both containing the same molar VWF excess. Our results support the hypothesis that synthesis of pdFVIII under physiological conditions could result in reduced heterogeneity and/or subtle differences in structure/conformation which, in turn, may result in reduced FVIII proteolytic processing relative to FL-rFVIII.

A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo.

Dendritic cell activation of CD4 T cells in the lymph node draining a site of infection or vaccination is widely considered the central event in initiating adaptive immunity. The accepted dogma is that this occurs by stimulating local activation and antigen acquisition by dendritic cells, with subsequent lymph node migration, however the generalizability of this mechanism is unclear. Here we show that in some circumstances antigen can bypass the injection site inflammatory response, draining freely and rapidly to the lymph nodes where it interacts with subcapsular sinus (SCS) macrophages resulting in their death. Debris from these dying SCS macrophages is internalized by monocytes recruited from the circulation. This coordinated response leads to antigen presentation by monocytes and interactions with naïve CD4 T cells that can drive the initiation of T cell and B cell responses. These studies demonstrate an entirely novel pathway leading to initiation of adaptive immune responses in vivo.

A pilot study of peripheral blood BDCA-1 (CD1c) positive dendritic cells pulsed with NY-ESO-1 ISCOMATRIX™ adjuvant.

AIM: Pilot clinical trial of NY-ESO-1 (ESO) protein in ISCOMATRIX™ adjuvant pulsed onto peripheral blood dendritic cells (PBDC), to ascertain feasibility, evaluate toxicity and assess induction of ESO-specific immune responses. PATIENTS & METHODS: Eligible participants had resected cancers expressing ESO or LAGE-1 and were at high risk of relapse. PBDC were produced using CliniMACS®plus, with initial depletion of CD1c+ B cells followed by positive selection of CD1c+ PBDC. Patients received three intradermal vaccinations of ESO/IMX-pulsed PBDC at 4-week intervals. RESULTS: The process was feasible and safe. No vaccine-induced immune responses were detected. Assays of immunomodulatory cells did not correlate with outcomes. One patient had a long lasting complete remission. CONCLUSION: This method was feasible and safe but was minimally immunogenic.

A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus.

To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.

Low-dose cyclophosphamide enhances antigen-specific CD4(+) T cell responses to NY-ESO-1/ISCOMATRIX™ vaccine in patients with advanced melanoma.

Clinical outcomes from cancer vaccine trials in patients with advanced melanoma have so far been disappointing. This appears at least partially due to a state of immunosuppression in these patients induced by an expansion of regulatory cell populations including regulatory T cells (Tregs). We have previously demonstrated potent immunogenicity of the NY-ESO-1/ISCOMATRIX™ vaccine in patients with resected melanoma (study LUD99-08); however, the same vaccine induced only a few vaccine antigen-specific immune responses in patients with advanced disease (study LUD2002-013). Pre-clinical models suggest that the alkylating agent cyclophosphamide can enhance immune responses by depleting Tregs. Therefore, we have enrolled a second cohort of patients with advanced melanoma in the clinical trial LUD2002-013 to investigate whether pre-treatment with cyclophosphamide could improve the immunogenicity of the NY-ESO-1/ISCOMATRIX™ vaccine. The combination treatment led to a significant increase in vaccine-induced NY-ESO-1-specific CD4(+) T cell responses compared with the first trial cohort treated with vaccine alone. We could not detect a significant decline in regulatory T cells in peripheral blood of patients 14 days after cyclophosphamide administration, although a decline at an earlier time point cannot be excluded. Our observations support the inclusion of cyclophosphamide in combination trials with vaccines and other immune-modulatory agents.

The combination of ISCOMATRIX adjuvant and TLR agonists induces regression of established solid tumors in vivo.

The development of therapeutic vaccines for treatment of established cancer has proven challenging. Cancer vaccines not only need to induce a robust tumor Ag-specific immune response but also need to overcome the tolerogenic and immunosuppressive microenvironments that exist within many solid cancers. ISCOMATRIX adjuvant (ISCOMATRIX) is able to induce both tumor Ag-specific cellular and Ab responses to protect mice against tumor challenge, but this is insufficient to result in regression of established solid tumors. In the current study, we have used B16-OVA melanoma, Panc-OVA pancreatic, and TRAMP-C1 prostate cancer mouse tumor models to test therapeutic efficacy of ISCOMATRIX vaccines combined with other immune modulators. The coadministration of an ISCOMATRIX vaccine with the TLR3 agonist, polyinosinic-polycytidylic acid, and TLR9 agonist, CpG, reduced tumor growth in all tumor models and the presence of ISCOMATRIX in the formulation was critical for the therapeutic efficacy of the vaccine. This vaccine combination induced a robust and multifunctional CD8(+) T cell response. Therapeutic protection required IFN-γ and CD8(+) T cells, whereas NK and CD4(+) T cells were found to be redundant. ISCOMATRIX vaccines combined with TLR3 and TLR9 agonists represent a promising cancer immunotherapy strategy.

Optimal effector functions in human natural killer cells rely upon autocrine bone morphogenetic protein signaling.

Natural killer (NK) cells are critical for innate tumor immunity due to their specialized ability to recognize and kill neoplastically transformed cells. However, NK cells require a specific set of cytokine-mediated signals to achieve optimal effector function. Th1-associated cytokines promote effector functions that are inhibited by the prototypic Th2 cytokine IL4 and the TGFß superfamily members TGFß1 and activin-A. Interestingly, the largest subgroup of the TGFß superfamily are the bone morphogenetic proteins (BMP), but the effects of BMP signaling on NK cell effector functions have not been evaluated. Here, we demonstrate that blood-circulating NK cells express type I and II BMP receptors, BMP-2 and BMP-6 ligands, and phosphorylated isoforms of Smad-1/-5/-8, which mediate BMP family member signaling. In opposition to the inhibitory effects of TGFß1 or activin-A, autocrine BMP signaling was supportive to NK cell function. Mechanistic investigations in cytokine and TLR-L-activated NK cells revealed that BMP signaling optimized IFNγ and global cytokine and chemokine production, phenotypic activation and proliferation, and autologous dendritic cell activation and target cytotoxicity. Collectively, our findings identify a novel auto-activatory pathway that is essential for optimal NK cell effector function, one that might be therapeutically manipulated to help eradicate tumors. Cancer Res; 74(18); 5019-31. ©2014 AACR.

Inflammasome-dependent and -independent IL-18 production mediates immunity to the ISCOMATRIX adjuvant.

Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1ß and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.

Combination of adjuvants: the future of vaccine design.

It is thought that the development of vaccines for the treatment of infectious diseases and cancer is likely to be achieved in the coming decades. This is partially due to a better understanding of the regulatory networks connecting innate with adaptive immune responses. The innate immune response is triggered by the recognition of conserved pathogen-associated molecular patterns by germ line-coded pattern recognition receptors. Several families of pattern recognition receptors have been characterized, including Toll-like receptors and nucleotide-binding domain receptors. The identification of their ligands has driven the development of novel adjuvants many of which have been tested in vaccine clinical trials. Here, the authors review recent preclinical data and clinical trial results supporting the view that combinations of adjuvants are the way forward in vaccine design. Multiadjuvanted vaccines can stimulate the broad and robust protective immune responses required to fight chronic infectious diseases and cancer.

Flt3 ligand expands CD4+ FoxP3+ regulatory T cells in human subjects.

CD4(+) CD25(+) FoxP3(+) naturally occurring regulatory T (Treg) cells play a crucial role in the maintenance of immune tolerance and in preventing autoimmune pathology. Interventions that expand Treg cells are highly desirable, as they may offer novel treatment options in a variety of autoimmune and transplantation settings. Paralleling previous preclinical studies, we demonstrate here that administration of the hematopoietic growth factor Flt3L to human subjects increases the frequency and absolute number of Treg cells, and reduces the ratio of CD8(+) T cells to Treg cells in the peripheral blood. The increase in Treg cells was due to enhanced Treg-cell proliferation rather than release of Treg cells from the thymus. Further studies revealed that Flt3L-induced proliferation of Treg cells was an indirect effect that occurred via the interaction of Treg cells with the Flt3L-expanded pool of CD1c(+) myeloid dendritic cells. On the basis of these findings, Flt3L may represent a promising agent for promoting immune tolerance in a variety of clinical settings.

A novel HLA-B18 restricted CD8+ T cell epitope is efficiently cross-presented by dendritic cells from soluble tumor antigen.

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

Targeting Dendritic Cells in vivo for Cancer Therapy.

Monoclonal antibodies that recognize cell surface molecules have been used deliver antigenic cargo to dendritic cells (DC) for induction of immune responses. The encouraging anti-tumor immunity elicited using this immunization strategy suggests its suitability for clinical trials. This review discusses the complex network of DC, the functional specialization of DC subsets, the immunological outcomes of targeting different DC subsets and their cell surface receptors, and the requirements for the induction of effective anti-tumor CD4 and CD8 T cell responses that can recognize tumor-specific antigens. Finally, we review preclinical experiments and the progress toward targeting human DC in vivo.

ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway.

Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.

Immunoediting and persistence of antigen-specific immunity in patients who have previously been vaccinated with NY-ESO-1 protein formulated in ISCOMATRIX™.

BACKGROUND: NY-ESO-1 protein formulated in ISCOMATRIX™ results in CD4+, CD8+ T cell and antibody-mediated immunity. We evaluated persistence of immunity, relapse-free survival and tumour antigen expression upon relapse in patients vaccinated in an earlier trial. METHODS: Immunity was measured in 28 patients with resected NY-ESO-1-expressing tumours (melanoma 25, breast 3) 252-1,155 days (median = 681) after vaccination. In the earlier vaccination, trial patients received NY-ESO-1 with ISCOMATRIX™ adjuvant at three protein doses 10 µg, 30 µg or 100 µg (n = 14); 100 µg NY-ESO-1 protein (n = 8) or placebo (n = 6), together with 1 µg of intradermal (ID) NY-ESO-1 protein twice for DTH skin testing. Immune responses assessed in the current study included antibody titres, circulating NY-ESO-1-specific T cells and DTH reactivity 2 days after DTH skin testing with NY-ESO-1 protein (1 µg) or peptides (10 µg). Relapse-free survival was determined for 42 melanoma patients. On relapse NY-ESO-1 and HLA, class I was assessed by immunohistochemistry in 17. RESULTS: Persisting anti-NY-ESO-1 immunity was detected in 10/14 recipients who had previously received vaccine with ISCOMATRIX™ adjuvant. In contrast, immunity only persisted in 3/14 who received 100 µg un-adjuvanted NY-ESO-1 protein (3/8) or 2 µg DTH protein (0/6) P = 0.02. Hence, persisting NY-ESO-1 immunity was associated with prior adjuvant. Tumour NY-ESO-1 or HLA class I was downregulated in participants who relapsed suggesting immunoediting had occurred. CONCLUSION: Immunoediting suggests that a signal of anti-tumour activity was observed in high-risk resected melanoma patients vaccinated with NY-ESO-1/ISCOMATRIX™. This was associated with measurable persisting immunity in the majority of vaccinated subjects tested. A prospective randomised trial has been undertaken to confirm these results.

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells.

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.

An ISCOM vaccine combined with a TLR9 agonist breaks immune evasion mediated by regulatory T cells in an orthotopic model of pancreatic carcinoma.

Vaccines based on immune stimulatory complexes (ISCOM) induce T-cell responses against tumor antigen (Ag). However, immune responses are impaired in pancreatic cancer patients. We investigated the efficacy of an ISCOM vaccine in a murine pancreatic carcinoma model. Panc02 cells expressing OVA as a model Ag were induced subcutaneously or orthotopically in the pancreas of C57BL/6 mice. Treatment consisted of an OVA containing ISCOM vaccine, either used alone or in combination with the TLR9 agonist CpG. The ISCOM vaccine effectively induced Ag-specific CTL capable of killing tumor cells. However, in mice with established tumors CTL induction by the vaccine was inefficient and did not affect tumor growth. Lack of efficacy correlated with increased numbers of Treg. Depletion of Treg with anti-CD25 mAb restored CTL induction and prolonged survival. Adding low-dose CpG to the ISCOM vaccine reduced Treg numbers, enhanced CTL responses and induced regression of pancreatic tumors in a CD8(+) T cell-dependent manner. Mice cured from the primary tumor mounted a memory T-cell response against wild-type Panc02 tumors, indicative of epitope spreading. Combining ISCOM vaccines with TLR agonists is a promising strategy for breaking tumor immune evasion and deserves further evaluation for the treatment of pancreatic carcinoma.