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DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell-cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

Background: Usual vulvar intraepithelial neoplasia (uVIN) is a premalignancy caused by persistent infection with high-risk types of human papillomavirus (HPV), mainly type 16. Even though different treatment modalities are available (eg, surgical excision, laser evaporation or topical application of imiquimod), these treatments can be mutilating, patients often have recurrences and 2%-8% of patients develop vulvar carcinoma. Therefore, immunotherapeutic strategies targeting the pivotal oncogenic HPV proteins E6 and E7 are being explored to repress carcinogenesis., Method: In this phase I/II clinical trial, 14 patients with HPV16+ uVIN were treated with a genetically enhanced DNA vaccine targeting E6 and E7. Safety, clinical responses and immunogenicity were assessed. Patients received four intradermal HPV-16 E6/E7 DNA tattoo vaccinations, with a 2-week interval, alternating between both upper legs. Biopsies of the uVIN lesions were taken at screening and +3 months after last vaccination. Digital photography of the vulva was performed at every check-up until 12 months of follow-up for measurement of the lesions. HPV16-specific T-cell responses were measured in blood over time in ex vivo reactivity assays., Results: Vaccinations were well tolerated, although one grade 3 suspected unexpected serious adverse reaction was observed. Clinical responses were observed in 6/14 (43%) patients, with 2 complete responses and 4 partial responses (PR). 5/14 patients showed HPV-specific T-cell responses in blood, measured in ex vivo reactivity assays. Notably, all five patients with HPV-specific T-cell responses had a clinical response., Conclusions: Our results indicate that HPV-16 E6/E7 DNA tattoo vaccination is a biologically active and safe treatment strategy in patients with uVIN, and suggest that T-cell reactivity against the HPV oncogenes is associated with clinical benefit., Trial Registration Number: NTR4607., Competing Interests: Competing interests: JB is (part time) employee of Modra Pharmaceuticals and stockholder in Modra Pharmaceuticals. (not related to the manuscript). TS is advisor for Adaptive Biotechnologies, Allogene Therapeutics, Merus, Neogene Therapeutics, and Scenic Biotech; is a recipient of research support from Merck KgaA; is a stockholder in Allogene Therapeutics, Merus, Neogene Therapeutics and Scenic Biotech; and is venture partner at Third Rock Ventures, all not related to the current work. JH is advisor to Achilles Therapeutics, Bristol-Myers Squibb, BioNTech USA, Ipsen, Gadeta, Immunocore, MSD, Merck Serono, Molecular Partners, Neogene Therapeutics, Novartis, Pfizer, Roche/Genentech, Sanofi, Seattle Genetics, Third Rock Ventures, is stock holder in Neogene Therapeutics, and is a recipient of grant or research support from Bristol-Myers Squibb, MSD, Novartis and BioNTech USA. KDV reports research funding from Roche and is consultant for Third Rock Ventures, all outside the scope of this work. TDdG has served as advisor to TILT Biotherapeutics, LAVA Therapeutics, Macrophage Pharma and DCPrime, is a recipient of a grant from Idera Pharmaceuticals, and is co-founder and shareholder of LAVA Therapeutics. JHvdB is a recipient of grant or research support from BioNTech USA and Astra Zeneca, and is currently employed at CellPoint B.V., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Priming of CD8 + T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1 + antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14 + CD169 + monocytes and Axl + CD169 + DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169 + moDCs and Axl + CD169 + DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8 + T cells. Finally, Axl + CD169 + DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169 + DCs to drive antitumor T cell responses., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

In this study we developed a liposome-based vaccine containing palmitoylated synthetic long peptides (SLP) and alpha galactosylceramide (αGC) to specifically target dendritic cells (DC) for activation of both innate (invariant natural killer T-cells [iNKT]) and adaptive (CD8 + T-cells) players of the immune system. Combination of model tumor specific antigens (gp100/MART-1) formulated as a SLP and αGC in one liposome results in strong activation of CD8 + and iNKT, as measured by IFNγ secretion. Moreover, addition of lipo-Lewis Y (Le Y ) to the liposomes for C-type lectin targeting increased not only uptake by monocyte-derived dendritic cells (moDC), dermal dendritic cells and Langerhans cells but also enhanced gp100-specific CD8 + T- and iNKT cell activation by human skin-emigrated antigen presenting cells in an ex vivo explant model. Loading of moDC with liposomes containing Le Y also showed priming of MART-1 26-35L specific CD8 + T-cells. In conclusion, chemically linking a lipid tail to a glycan-based targeting moiety and SLP combined with αGC in one liposome allows for easy generation of vaccine formulations that target multiple skin DC subsets and induce tumor antigen specific CD8 + T- and iNKT cells. These liposomes present a new vaccination strategy against tumors., (Copyright © 2020 Stolk, de Haas, Vree, Duinkerken, Lübbers, van de Ven, Ambrosini, Kalay, Bruijns, van der Vliet, de Gruijl and van Kooyk.)

Dendritic cell (DC)-targeting vaccines show great promise in increasing antitumor immunity. Glycan-engineered vaccines facilitate both DC targeting and increased uptake by DCs for processing and presentation to CD4 + and CD8 + T cells to induce tumor-specific T-cell responses. However, the complexity of various DC subsets in skin tissues, expressing different glycan-binding receptors that can mediate vaccine uptake or drainage of vaccines via lymphatics directly to the lymph node-resident DCs, complicates the success of vaccines. Moreover, the influx of inflammatory immune cells to the site of vaccination, such as monocytes that differentiate to DCs and coexpress glycan-binding receptors, may contribute to the strength of DC-targeting glycovaccines for future clinical use., (Copyright © 2019. Published by Elsevier Ltd.)

The human skin is an attractive anti-tumor vaccination site due to the vast network of dendritic cell (DC) subsets that carry antigens to the draining lymph nodes and stimulate tumor specific CD4 + and CD8 + T cells in. Specific vaccine delivery to skin DC can be accomplished by targeting glycan coated antigens to C-type lectin receptors (CLRs) such as DC-SIGN expressed by human dermal DCs and Langerin expressed by Langerhans cells (LCs), which facilitate endocytosis and processing for antigen presentation and T cell activation. Although there are multiple human skin DC subsets, targeting individual DC subsets and receptors has been a focus in the past. However, the simultaneous targeting of multiple human skin DC subsets that mobilize the majority of the skin antigen presenting cells (APC) is preferred to accomplish more robust and efficient T cell stimulation. Dual CLR targeting using a single tumor vaccine has been difficult, as we previously showed Langerin to favor binding and uptake of monovalent glyco-peptides whereas DC-SIGN favors binding of larger multivalent glyco-particles such as glyco-liposomes. Methods: We used branched polyamidoamine (PAMAM) dendrimers as scaffold for melanoma specific gp100 synthetic long peptides and the common DC-SIGN and Langerin ligand Lewis Y (Le Y ), to create multivalent glyco-dendrimers with varying molecular weights for investigating dual DC-SIGN and Langerin targeting. Using DC-SIGN + monocyte derived DC (moDC) and Langerin + primary LC we investigated glyco-dendrimer CLR targeting properties and subsequent gp100 specific CD8 + T cell activation in vitro . In situ targeting ability to human dermal DC and LC through intradermal injection in a human skin explant model was elucidated. Results: Dual DC-SIGN and Langerin binding was achieved using glyco-dendrimers of approximately 100kD, thereby fulfilling our criteria to simultaneously target LCs and CD1a + and CD14 + dermal DC in situ . Both DC-SIGN and Langerin targeting by glyco-dendrimers resulted in enhanced internalization and gp100 specific CD8 + T cell activation. Conclusion: We designed the first glyco-vaccine with dual CLR targeting properties, thereby reaching multiple human skin DC subsets in situ for improved anti-tumor CD8 + T cell responses., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: In this study the toxicity and efficacy of an irradiated autologous tumor cell vaccine (ATV) co-injected with a class-B CpG oligodeoxynucleotide (CpG-B) and GM-CSF, followed by systemic CpG-B and IFN-α administration, were examined in patients with metastatic renal cell carcinoma (mRCC)., Methods: A single-arm Phase II trial was conducted, in which patients with mRCC were intradermally injected with a minimum of three whole-cell vaccines containing 0.7–1.3 × 107 irradiated autologous tumor cells (ATC), admixed with 1 mg CpG-B and 100 µg GM-CSF, followed by bi-weekly s.c. injections with 8 mg CpG-B and s.c. injections with 6 MU IFN-α three times per week., Results: Fifteen patients were treated according to the protocol. Treatment was well tolerated. Objective clinical responses occurred in three patients, including one long-term complete response. Disease stabilization occurred in another three patients. Positive delayed type hypersensitivity (DTH) responses to ATC were absent before treatment but present in 13 out of 15 patients during treatment. Immune monitoring revealed activation of plasmacytoid dendritic cells, non-classical monocytes and up-regulation of both PD-1 and CTLA4 on effector T cells upon treatment. Moreover, a pre-existing ex vivo IFN-γ response to ATC was associated with clinical response., Conclusions: ATV combined with systemic CpG-B and IFN-α is tolerable, safe, immunogenic and able to elicit anti-tumor responses in patients with mRCC. Immune activation and treatment-induced up-regulation of PD-1 and CTLA4 on circulating T cells further suggest an added benefit of combining this approach with immune checkpoint blockade [added]

In elderly acute myeloid leukemia (AML) patients post-remission treatment options are associated with high comorbidity rates and poor survival. Dendritic cell (DC)-based immunotherapy is a promising alternative treatment strategy. A novel allogeneic DC vaccine, DCP-001, was developed from an AML-derived cell line that uniquely combines the positive features of allogeneic DC vaccines and expression of multi-leukemia-associated antigens. Here, we present data from a phase I study conducted with DCP-001 in 12 advanced-stage elderly AML patients. Patients enrolled were in complete remission (CR1/CR2) (n = 5) or had smoldering disease (n = 7). All patients were at high risk of relapse and ineligible for post-remission intensification therapies. A standard 3 + 3 dose escalation design with extension to six patients in the highest dose was performed. Patients received four biweekly intradermal DCP-001 injections at different dose levels (10, 25, and 50 million cells DCP-001) and were monitored for clinical and immunological responses. Primary objectives of the study (feasibility and safety) were achieved with 10/12 patients completing the vaccination program. Treatment was well tolerated. A clear-cut distinction between patients with and without detectable circulating leukemic blasts during the vaccination period was noted. Patients with no circulating blasts showed an unusually prolonged survival [median overall survival 36 months (range 7-63) from the start of vaccination] whereas patients with circulating blasts, died within 6 months. Long-term survival was correlated with maintained T cell levels and induction of multi-functional immune responses. It is concluded that DCP-001 in elderly AML patients is safe, feasible and generates both cellular and humoral immune responses.

Introduction: Vascular endothelial growth factor (VEGF) is pivotal in tumor angiogenesis and therapies targeting the VEGF axis are widely used in the clinic for the treatment of cancer. We have developed a therapeutic vaccine targeting human (h)VEGF 165 . hVEGF 26-104 /RFASE is based on the truncated protein hVEGF 26-104 as antigen formulated in an oil-in-water emulsion containing the sulpholipopolysaccharide RFASE as adjuvant. Here we describe the toxicity and immunogenicity of this therapeutic vaccine in cynomolgus monkeys., Methods: In total 54 cynomolgus monkeys were used and divided in 7 groups. Groups 1-3 were control groups, either receiving PBS alone (group 1), RFASE alone (group 2) or hVEGF 26-104 alone (group 3). Animals allocated to groups 4-7 received hVEGF 26-104 together with RFASE, but with varying doses of the antigen or the adjuvant. All animals were immunized four times with 2-week intervals and safety and immunogenicity were monitored until 3 days after the final immunization., Results: Immunization induced an RFASE adjuvant dependent acute phase response. High titers of antibodies against hVEGF 26-104 and cross-reactive with hVEGF 165 , were found in monkey sera, 28 days after primer immunization. These antibodies were able to inhibit the binding of the monoclonal antibody bevacizumab with hVEGF 165 in a competition ELISA. Moreover, the biological activity of hVEGF 165 could be inhibited by the addition of immunized monkey serum in a VEGF specific bioassay. Importantly, no adverse events commonly observed with VEGF neutralization were observed throughout the study., Conclusion: These data show that hVEGF 26-104 /RFASE can be safely administered in cynomolgus monkeys, induces the desired immune response and therefore support the clinical development of this vaccine., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

There is a growing understanding of why certain patients do or do not respond to checkpoint inhibition therapy. This opens new opportunities to reconsider and redevelop vaccine strategies to prime an anticancer immune response. Combination of such vaccines with checkpoint inhibitors will both provide the fuel and release the brake for an efficient anticancer response. Here, we discuss vaccine strategies that use C-type lectin receptor (CLR) targeting of APCs, such as dendritic cells and macrophages. APCs are a necessity for the priming of antigen-specific cytotoxic and helper T cells. Because CLRs are natural carbohydrate-recognition receptors highly expressed by multiple subsets of APCs and involved in uptake and processing of Ags for presentation, these receptors seem particularly interesting for targeting purposes., (© The Author(s).)

The potency of human papillomavirus type 16 (HPV16)-encoded synthetic long peptides (SLP), conjugated to an optimized Toll-like receptor 2 ligand (TLR2-L), was assessed in ex vivo activation of HPV16+ cancer patient-derived T cells. Two highly immunogenic SLP sequences derived from the oncogenic E6 protein of HPV16 were selected and conjugated to a Pam3CSK4-based TLR2-L under GMP conditions. Both conjugates were able to mature human DCs in vitro and to activate human skin-derived antigen-presenting cells (APCs) upon intradermal injection in an ex vivo skin model, associated with induction of a favorable chemokine profile to attract and activate T cells. The conjugated SLPs were efficiently processed by APCs, since HPV16-specific CD4+ and CD8+ T-cell clones isolated from HPV16+ cervical tumors proliferated in response to both conjugates. The TLR2-L SLP conjugates significantly enhanced ex vivo T helper type 1 T-cell activation in cell suspensions obtained from tumor-draining lymph nodes (LN) resected during hysterectomy of HPV16+ cervical cancer patients. These results show that TLR2-L SLP conjugates can activate circulating or LN-derived tumor-specific T cells, a promising outcome for studying these two conjugates in a phase I/II clinical safety and immunogenicity trial.

Cancer immunotherapy induces a variety of autoinflammatory responses, including those against the thyroid gland, which can be exploited to predict clinical outcomes. Considering the paucity of information about thyroid autoimmunity in patients receiving cancer vaccines, we designed our study to assess the development of thyroglobulin antibodies (TgAbs) in patients treated with GVAX (vaccine made of a tumor cell type transfected with GM-CSF) and/or ipilimumab and correlated seroconversion with survival. Using both in house and commercial ELISA assays, we measured TgAbs in patients with pancreatic (No. = 53), prostate (No. = 35) or colon (No. = 8) cancer, before and after treatment with GVAX only (No. = 34), GVAX plus ipilimumab (No. = 42) or ipilimumab (No. = 20), and correlated their levels with patient's survival, disease status and T-cell surface markers. Antibodies to thyroperoxidase, myeloperoxidase, proteinase 3, insulin and actin were also measured. TgAbs specifically developed after GVAX, independent of the underlying cancer (81% in prostate, 75% colon cancer and 76% pancreatic cancer) and co-administration of ipilimumab (75% in GVAX only and 78% in GVAX plus ipilimumab). This TgAbs seroconversion could be detected mainly by the in house assay, suggesting that the thyroglobulin epitopes recognized by the antibodies induced by GVAX are different from the epitopes seen in the classic form of Hashimoto thyroiditis. Notably, TgAbs seroconversion was associated with significantly prolonged survival (p = 0.01 for pancreas and p = 0.005 for prostate cancer). In conclusion, GVAX immunotherapy induces the appearance of TgAbs that recognize a unique antigenic repertoire and associate with prolonged survival., (© 2014 UICC.)

Since few leukemia-associated antigens (LAA) are characterized for acute myeloid leukemia (AML), apoptotic tumor cells constitute an attractive LAA source for DC-based vaccines, as they contain both characterized and unknown LAA. However, loading DC with apoptotic tumor cells may interfere with DC function. Previously, it was shown in mice that apoptotic blebs induce DC maturation, whereas apoptotic cell remnants (ACR) do not. Here, we analyzed human monocyte-derived DC (MoDC) functionality in vitro, after ingesting either allogeneic AML-derived ACR or blebs. We show that MoDC ingest blebs to a higher extent and are superior in migrating toward CCL19, as compared to ACR-loaded MoDC. Although MoDC cytokine production was unaffected, co-culturing bleb-loaded MoDC with T cells led to an increased T cell proliferation and IFNγ production. Moreover, antigen-specific CD8(+) T cells frequencies increased to 0.63 % by priming with bleb-loaded MoDC, compared to 0.16 % when primed with ACR-loaded MoDC. Importantly, CD8(+) T cells primed by bleb-loaded MoDC recognized their specific epitope at one to two orders of magnitude lower concentrations compared to ACR-loaded MoDC. In conclusion, superior ingestion efficiency and migration, combined with favorable T cell cytokine release and CD8(+) T cell priming ability and avidity, point to blebs as the preferred component of apoptotic leukemic cells for LAA loading of DC for the immunotherapy of AML.

Invariant natural killer T cells (iNKT) and dendritic cells (DC) play a central role in tumor immunity through downstream activation of immune effector cells by pro-inflammatory cytokines. Evidence is accumulating that the CD1d-iNKT cell axis can be effectively used to potentiate DC-based cancer vaccines. Here, we provide a detailed methodology for the generation of (CD1d-expressing) monocyte-derived DC (moDC) and their subsequent loading with the iNKT cell agonist α-galactosylceramide (α-GalCer) or their direct ligation by agonistic anti-CD1d monoclonal antibodies.

Harvesting the potential of the immune system in order to eradicate (residual) acute myeloid leukemia (AML) cells is the long pursued goal of immunotherapy in AML. Strategies using apoptotic tumor cell vaccines have been explored for many years, without significant clinical improvements. In recent years insight has been gained into the mechanisms activating and interfering with tumor-directed immunity. With the arrival of novel immune-modulating agents allowing for the interference with regulatory molecules and interaction with immune-propelling mechanisms, new doors are opening for increasing vaccination efficacy. Combined with advances in the design of apoptotic tumor-based vaccines, we are on the verge of creating an effective AML vaccine strategy, offering a much needed novel therapeutic option for this devastating disease.

Background: New treatment modalities are needed for the treatment of cancers of the head and neck region (HNSCC). Survivin is important for the survival and proliferation of tumor cells and may therefore provide a target for immunotherapy. Here we focused on the ex vivo presence and in vitro induction of survivin specific T cells., Methods: Tetramer staining and ELIspot assays were used to document the presence of survivin specific T cells in patient derived material, and to monitor the presence and persistence of survivin specific T cells after repeated in vitro stimulation with autologous dendritic cells., Results: Ex vivo analysis showed the presence of survivin-specific T cells in the peripheral blood (by tetramer analysis) and in the draining lymph node (by ELIspot analysis) in a HNSCC and a locally advanced breast cancer patient respectively. However, we were unable to maintain isolated survivin specific T cells for prolonged periods of time. For the in vitro generation of survivin specific T cells, monocyte derived DC were electroporated with mRNA encoding full length survivin or a survivin mini-gene together with either IL21 or IL12 mRNA. Western blotting and immunohistochemical staining of dendritic cell cytospin preparations confirmed translation of the full length survivin protein. After repeated stimulation we observed an increase, followed by a decrease, of the number of survivin specific T cells. FACS sorted or limiting dilution cloned survivin specific T cells could not be maintained on feeder mix for prolonged periods of time. Protein expression analysis subsequently showed that activated, but not resting T cells contain survivin protein., Conclusions: Here we have shown that survivin specific T cells can be detected ex vivo in patient derived material. Furthermore, survivin specific T cells can be induced in vitro using autologous dendritic cells with enforced expression of survivin and cytokines. However, we were unable to maintain enriched or cloned survivin specific T cells for prolonged periods of time. Endogenous expression of survivin in activated T cells and subsequent fratricide killing might explain our in vitro observations. We therefore conclude that survivin, although it is a universal tumor antigen, might not be the ideal target for immunotherapeutic strategies for the treatment of cancer of the head and neck.

Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4(+) T cell differentiation, and CD4(+) and CD8(+) T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4(+)CTLA-4(+), CD4(+)PD-1(+), or differentiated (i.e., non-naive) CD8(+) T cells or low pre-treatment frequencies of differentiated CD4(+) or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4(+) in CD4(+) T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.

The ability of dendritic cells (DCs) to orchestrate innate and adaptive immune responses has been exploited to develop potent anti-cancer immunotherapies. Recent clinical trials exploring the efficacy of ex vivo modified autologous DC-based vaccines have reported some promising results. However, in vitro generation of autologous DCs for clinical administration, their loading with tumor associated antigens (TAAs) and their activation, is laborious and expensive, and, as a result of inter-individual variability in the personalized vaccines, remains poorly standardized. An attractive alternative approach is to load resident DCs in vivo by targeted delivery of TAAs, using viral vectors and activating them simultaneously. To this end, we have constructed genetically-modified adenoviral (Ad) vectors and bispecific adaptor molecules to retarget Ad vectors encoding TAAs to the CD40 receptor on DCs. Pre-clinical human and murine studies conducted so far have clearly demonstrated the suitability of a 'two-component' (i.e. Ad and adaptor molecule) configuration for targeted modification of DCs in vivo for cancer immunotherapy. This review summarizes recent progress in the development of CD40-targeted Ad-based cancer vaccines and highlights pre-clinical issues in the clinical translation of this approach., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: The granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells vaccine (GVAX) has antitumour activity against prostate cancer; preclinical studies have shown potent synergy when combined with ipilimumab, an antibody that blocks cytotoxic T-lymphocyte antigen 4. We aimed to assess the safety of combined treatment with GVAX and ipilimumab in patients with metastatic castration-resistant prostate cancer (mCRPC)., Methods: We did an open-labelled, single-centre, dose-escalation study of ipilimumab concurrent with a fixed dose of GVAX, with a subsequent expansion phase, both at the VU University Medical Centre (Amsterdam, Netherlands). Eligible patients had documented mCRPC and had not been previously treated with chemotherapy. All patients received a 5×10(8) cell priming dose of GVAX intradermally on day 1 with subsequent intradermal injections of 3×10(8) cells every 2 weeks for 24 weeks. The vaccinations were combined with intravenous ipilimumab every 4 weeks. We enrolled patients in cohorts of three; each cohort received an escalating dose of ipilimumab at 0·3, 1·0, 3·0, or 5·0 mg/kg. Our primary endpoint was safety. This study is registered with ClinicalTrials.gov, number NCT01510288., Findings: We enrolled 12 patients into our dose-escalation cohort. We did not record any severe immune-related adverse events at the first two dose levels. At the 3·0 mg/kg dose level, one patient had grade 2 and two patients grade 3 hypophysitis; at the 5·0 mg/kg dose level, two patients had grade 3 hypophysitis and one patient developed grade 4 sarcoid alveolitis (a dose-limiting toxic effect). Due to observed clinical activity and toxic events, we decided to expand the 3·0 mg/kg dose level, rather than enrol a further three patients at the 5·0 mg/kg level. 16 patients were enrolled in the expansion cohort, two of whom developed grade 2 hypophysitis, three colitis (one grade 1 and two grade 2), and one grade 3 hepatitis--all immune-related adverse events. The most common adverse events noted in all 28 patients were injection-site reactions (grade 1-2 events seen in all patients), fatigue (grade 1-2 in 20 patients, grade 3 in two), and pyrexia (grade 1-2 in 15 patients, grade 3 in one). 50% or greater declines in prostate-specific antigen from baseline was recorded in seven patients (25%); all had received 3·0 mg/kg or 5·0 mg/kg ipilimumab., Interpretation: GVAX combined with 3·0 mg/kg ipilimumab is tolerable and safe for patients with mCRPC. Further research on the combined treatment of patients with mCRPC with vaccination and ipilimumab is warranted., Funding: Cell Genesys Inc, Prostate Cancer Foundation, Dutch Cancer Society (KWF-VU 2006-3697), and Foundation Stichting VUmc Cancer Center Amsterdam., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

In situ delivery of tumor-associated antigen (TAA) genes into dendritic cells (DC) has great potential as a generally applicable tumor vaccination approach. Although adenoviruses (Ad) are an attractive vaccine vehicle in this regard, Ad-mediated transduction of DCs is hampered by the lack of expression of the Ad receptor CAR on the DC surface. DC activation also requires interaction of CD40 with its ligand CD40L to generate protective T-cell-mediated tumor immunity. Therefore, to create a strategy to target Ads to DCs in vivo, we constructed a bispecific adaptor molecule with the CAR ectodomain linked to the CD40L extracellular domain via a trimerization motif (CFm40L). By targeting Ad to CD40 with the use of CFm40L, we enhanced both transduction and maturation of cultured bone marrow-derived DCs. Moreover, we improved transduction efficiency of DCs in lymph node and splenic cell suspensions in vitro and in skin and vaccination site-draining lymph nodes in vivo. Furthermore, CD40 targeting improved the induction of specific CD8(+) T cells along with therapeutic efficacy in a mouse model of melanoma. Taken together, our findings support the use of CD40-targeted Ad vectors encoding full-length TAA for in vivo targeting of DCs and high-efficacy induction of antitumor immunity., (©2011 AACR.)

Adenovirus (Ad)-based vaccines are considered for cancer immunotherapy, yet, detailed knowledge on their mechanism of action and optimal delivery route for anti-tumor efficacy is lacking. Here, we compared the anti-tumor efficacy of an Ad-based melanoma vaccine after intradermal, intravenous, intranasal or intraperitoneal delivery in the B16F10 melanoma model. The intradermal route induced superior systemic anti-melanoma immunity which was MyD88 signaling-dependent. Predominant transduction of non-professional antigen-presenting cells at the dermal vaccination sites and draining lymph nodes, suggested a role for cross-presentation, which was confirmed in vitro. We conclude that the dermis provides an optimal route of entry for Ad-based vaccines for high-efficacy systemic anti-tumor immunization and that this immunization likely involves cross-priming events in the draining lymph nodes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Therapeutic vaccination with dendritic cells (DC) is an emerging investigational therapy for eradication of minimal residual disease in acute myeloid leukemia. Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g., monocyte-derived DC (MoDC) loaded with leukemia-associated antigens (LAA). However, the optimal source of LAA and the choice of DC-activating stimuli are still not well defined. Here, loading with leukemic cell preparations (harboring both unknown and known LAA) was explored in combination with a DC maturation-inducing cytokine cocktail (CC; IL-1β, IL-6, TNF-α, and PGE(2)) and Toll-like receptor ligands (TLR-L) to optimize uptake. Since heat shock induced apoptotic blasts were more efficiently taken up than lysates, we focused on uptake of apoptotic leukemic cells. Uptake of apoptotic blast was further enhanced by the TLR7/8-L R848 (20-30%); in contrast, CC-induced maturation inhibited uptake. CC, and to a lesser extent R848, enhanced the ability of MoDC to migrate and stimulate T cells. Furthermore, class II-associated invariant chain peptide expression was down-modulated after R848- or CC-induced maturation, indicating enhanced processing and presentation of antigenic peptides. To improve both uptake and maturation, leukemic cells and MoDC were co-incubated with R848 for 24 h followed by addition of CC. However, this approach interfered with CC-mediated MoDC maturation as indicated by diminished migratory and T cell stimulatory capacity, and the absence of IL-12 production. Taken together, our data demonstrate that even though R848 improved uptake of apoptotic leukemic cells, the sequential use of R848 and CC is counter-indicated due to its adverse effects on MoDC maturation.

Targeting viral vectors encoding tumor-associated antigens to dendritic cells (DCs) in vivo is likely to enhance the effectiveness of immunotherapeutic cancer vaccines. We have previously shown that genetic modification of adenovirus (Ad) 5 to incorporate CD40 ligand (CD40L) rather than native fiber allows selective transduction and activation of DCs in vitro. Here, we examine the capacity of this targeted vector to induce immune responses to the tumor antigen CEA in a stringent in vivo canine model. CD40-targeted Ad5 transduced canine DCs via the CD40-CD40L pathway in vitro, and following vaccination of healthy dogs, CD40-targeted Ad5 induced strong anti-CEA cellular and humoral responses. These data validate the canine model for future translational studies and suggest targeting of Ad5 vectors to CD40 for in vivo delivery of tumor antigens to DCs is a feasible approach for successful cancer therapy.

Dendritic cells (DC) are increasingly applied in the immunotherapy of cancer. As the development of a standardized DC vaccine product is often hampered by the limited availability of DC precursors and inter- and intra-donor variability, and the preparation of individual vaccines is labor-intensive, it would be preferable to use DC from a readily available and unlimited source, such as cell lines can provide. It has been described that leukemia-derived cell lines are able to differentiate into functional DC, creating possibilities for the development of highly reproducible DC vaccines and providing in vitro model systems for in-depth studies about DC physiology. This review discusses the different human DC cell line differentiation models described so far. Based on the available data, characteristics that determine the ability of leukemia cells to differentiate along the different precursor stages into functional DC will be formulated. In addition, evidence will be provided that the human CD34+ acute myeloid leukemia cell line MUTZ-3 provides DC that exhibit the functional properties that are crucial for the in vivo generation of CTL-mediated immunity and thus, currently, represents the most valuable, sustainable model system for myeloid DC differentiation and clinical DC vaccination studies.

The field of tumor vaccination is currently undergoing a shift in focus, from individualized tailor-made vaccines to more generally applicable vaccine formulations. Although primarily predicated by financial and logistic considerations, stemming from a growing awareness that clinical development for wide-scale application can only be achieved through backing from major pharmaceutical companies, these new approaches are also supported by a growing knowledge of the intricacies and minutiae of antigen presentation and effector T-cell activation. Here, the development of whole-cell tumor and dendritic cell (DC)-based vaccines from an individualized autologous set-up to a more widely applicable allogeneic approach will be discussed as reflected by translational studies carried out over the past two decades at our laboratories and clinics in the vrije universiteit medical center (VUmc) in Amsterdam, The Netherlands.

Dendritic cell (DC)-based immunotherapy faces new challenges because the efficacy of DC vaccines in clinical trials has been inconsistent. Strategies to improve immune responses induced by DC are currently being explored. We have recently shown the feasibility of generating fully functional DC from acute myeloid leukaemic (AML) blasts, but with varying expression levels of the important costimulatory molecule CD86. To overcome this variability, we developed a novel bispecific diabody that simultaneously and agonistically targeted CD40 on AML-DC and CD28 on naïve T cells. Beside optimization of CD28-mediated signalling, the resulting cellular cross-linking was also hypothesized to increase the strength and duration of T cell/AML-DC interactions, thus increasing T-cell responsiveness to AML antigens. The alphaCD40/alphaCD28-bispecific diabody was found to bind to its target antigens and provoked increased T-cell-DC cluster formation. The alphaCD40/alphaCD28 diabody is capable of increasing T-cell proliferation induced by AML-DC as well as the induction of DC maturation. Importantly, priming efficacy of tumour-specific cytotoxic T cells can also be improved by cross-linking AML-DC and T cells with the alphaCD40/alphaCD28 diabody. We propose that the alphaCD40/alphaCD28-bispecific diabody can serve as a potent therapeutic tool to effectively augment anti-tumour T-cell responses elicited by AML-DC.

Cancer vaccines have emerged as a potent strategy to improve cancer immunity, with or without the combination of checkpoint blockade. In our investigation, liposomal formulations containing synthetic long peptides and α-Galactosylceramide, along with a DC-SIGN-targeting ligand, Lewis Y (LeY), were studied for their anti-tumor potential. The formulated liposomes boosted with anti-CD40 adjuvant demonstrated robust invariant natural killer (iNKT), CD4+, and CD8+ T-cell activation in vivo. The incorporation of LeY facilitated the targeting of antigen-presenting cells expressing DC-SIGN in vitro and in vivo. Surprisingly, mice vaccinated with LeY-modified liposomes exhibited comparable tumor reduction and survival rates to those treated with untargeted counterparts despite a decrease in antigen-specific CD8+ T-cell responses. These results suggest that impaired induction of antigen-specific CD8+ T-cells via DC-SIGN targeting does not compromise anti-tumor potential, hinting at alternative immune activation routes beyond CD8+ T-cell activation. [ABSTRACT FROM AUTHOR]

Priming of CD8+ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+ CD169+ monocytes and Axl+ CD169+ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+ moDCs and Axl+ CD169+ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+ T cells. Finally, Axl+ CD169+ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+ DCs to drive antitumor T cell responses. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. While immunotherapy has been deemed a breakthrough treatment for various subtypes of cancer, its efficacy in PDAC is limited. This review discusses a wide range of immunotherapies, providing a general introduction to their working mechanism as well as current evidence on their clinical efficacy and immune eliciting abilities in PDAC. Utilizing combination (immuno)therapies to generate synergistic anti-tumor effects may provide the key to successful PDAC treatment. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with high mortality. The vast majority of patients present with unresectable, advanced stage disease, for whom standard of care chemo(radio)therapy may improve survival by several months. Immunotherapy has led to a fundamental shift in the treatment of several advanced cancers. However, its efficacy in PDAC in terms of clinical benefit is limited, possibly owing to the immunosuppressive, inaccessible tumor microenvironment. Still, various immunotherapies have demonstrated the capacity to initiate local and systemic immune responses, suggesting an immune potentiating effect. In this review, we address PDAC's immunosuppressive tumor microenvironment and immune evasion methods and discuss a wide range of immunotherapies, including immunomodulators (i.e., immune checkpoint inhibitors, immune stimulatory agonists, cytokines and adjuvants), oncolytic viruses, adoptive cell therapies (i.e., T cells and natural killer cells) and cancer vaccines. We provide a general introduction to their working mechanism as well as evidence of their clinical efficacy and immune potentiating abilities in PDAC. The key to successful implementation of immunotherapy in this disease may rely on exploitation of synergistic effects between treatment combinations. Accordingly, future treatment approaches should aim to incorporate diverse and novel immunotherapeutic strategies coupled with cytotoxic drugs and/or local ablative treatment, targeting a wide array of tumor-induced immune escape mechanisms. [ABSTRACT FROM AUTHOR]

Simple Summary: Metastatic pancreatic ductal adenocarcinoma has a dismal prognosis, and to date no curative treatment options exist. The image guided tumor ablation technique irreversible electroporation (IRE) employs high-voltage electrical pulses through the application of several needle electrodes in and around the tumor in order to induce cell death. IRE ablation of the primary tumor has the ability to reduce pancreatic tumor induced immune suppression while allowing the expansion of tumor specific effector T cells, hereby possibly shifting the pancreatic tumor microenvironment into a more immune permissive state. The addition of immune enhancing therapies to IRE might work synergistically and could potentially induce a clinically significant treatment effect. This study protocol describes the rationale and design of the PANFIRE-III trial that aims to assess the safety of the combination of IRE with IMO-2125 (toll-like receptor 9 ligand) and/or nivolumab in patients with metastatic pancreatic ductal adenocarcinoma. Irreversible electroporation (IRE) is a novel image-guided tumor ablation technique with the ability to generate a window for the establishment of systemic antitumor immunity. IRE transiently alters the tumor's immunosuppressive microenvironment while simultaneously generating antigen release, thereby instigating an adaptive immune response. Combining IRE with immunotherapeutic drugs, i.e., electroimmunotherapy, has synergistic potential and might induce a durable antitumor response. The primary objective of this study is to assess the safety of the combination of IRE with IMO-2125 (a toll-like receptor 9 ligand) and/or nivolumab in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). In this randomized controlled phase I clinical trial, 18 patients with mPDAC pretreated with chemotherapy will be enrolled in one of three study arms: A (control): nivolumab monotherapy; B: percutaneous IRE of the primary tumor followed by nivolumab; or C: intratumoral injection of IMO-2125 followed by percutaneous IRE of the primary tumor and nivolumab. Assessments include contrast enhanced computed tomography (ceCT), 18F-FDG and 18F-BMS-986192 (PD-L1) positron emission tomography (PET)-CT, biopsies of the primary tumor and metastases, peripheral blood samples, and quality of life and pain questionnaires. There is no curative treatment option for patients with mPDAC, and palliative chemotherapy regimens only moderately improve survival. Consequently, there is an urgent need for innovative and radically different treatment approaches. Should electroimmunotherapy establish an effective and durable anti-tumor response, it may ultimately improve PDAC's dismal prognosis. [ABSTRACT FROM AUTHOR]

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated apoptotic tumor cell-derived extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer. [ABSTRACT FROM AUTHOR]

In the original publication of the article the following abstract and keywords were inadvertently omitted. [ABSTRACT FROM AUTHOR]

Previous vaccination studies in patients with castration-resistant prostate cancer (CRPC) showed improved survival without prolongation of progression-free survival (PFS). This might be explained by enhanced efficacy of subsequent therapies because of heightened immune status. We therefore evaluated the efficacy of chemotherapy in CRPC patients after immunotherapy. We retrospectively analyzed 28 patients who were treated with ipilimumab and GVAX, an allogeneic vaccine, and 21 patients who were randomized to GVAX or no vaccination. To study whether immune status was related to the efficacy of chemotherapy, frequencies of myeloid and lymphocyte subsets were determined. Of 28 patients treated with GVAX and ipilimumab, 23 patients received docetaxel and 13 patients mitoxantrone. Median PFS after docetaxel was 6.4 mo (range 0.8–11.2), while median PFS after mitoxantrone was markedly longer than expected (4.8 mo; range 1.4–13.7). High CD8+ICOS+Tcell/Treg and pDC/mMDSC ratios were associated with relatively long PFS after mitoxantrone, suggesting a correlation between activated immune status and benefit of mitoxantrone. Analysis of 21 patients, randomized to GVAX or not, revealed a median PFS after docetaxel of 9.9 mo for vaccinated patients and 7.1 mo for unvaccinated patients. Interestingly, PFS after mitoxantrone (n = 14) was significantly longer in vaccinated patients as compared to controls (5.9 vs. 1.6 mo,p = 0.0048). In conclusion, mitoxantrone seems more effective in CRPC patients after immunotherapy, which may be related to the immune-stimulating effect of mitoxantrone in patients with heightened antitumor immunity. As this was a retrospective study with limited sample size, prospective studies are warranted to definitively show proof of principle. [ABSTRACT FROM AUTHOR]

Improvement of patient survival by anti-angiogenic therapy has proven limited. A vaccination approach inducing an immune response against the tumor vasculature combines the benefits of immunotherapy and anti-angiogenesis, and may overcome the limitations of current anti-angiogenic drugs. Strategies to use whole endothelial cell vaccines and DNA- or protein vaccines against key players in the VEGF signaling axis, as well as specific markers of tumor endothelial cells, have been tested in preclinical studies. Current clinical trials are now testing the promise of this specific anti-cancer vaccination approach. This review will highlight the state-of-the-art in this exciting field of cancer research. [ABSTRACT FROM AUTHOR]

TLR agonists are attractive candidate adjuvants for therapeutic cancer vaccines as they can induce a balanced humoral and T cell-mediated immune response. With a dense network of dendritic cells (DCs) and draining lymphatics, the skin provides an ideal portal for vaccine delivery. Beside direct DC activation, TLR agonists may also induce DC activation through triggering the release of inflammatory mediators by accessory cells in the skin microenvironment. Therefore, a human skin explant model was used to explore the in vivo potential of intradermally delivered TLR agonists to stimulate Langerhans cells and dermal DCs in their natural complex tissue environment. The skin-emigrated DCs were phenotyped and analyzed for T cell stimulatory capacity. We report that, of six tested TLR-agonists, the TLR2 and -3 agonists peptidoglycan (PGN) and polyribosinic-polyribocytidylic acid (Poly I:C) were uniquely able to enhance the T cell-priming ability of skin-emigrated DCs, which, in the case of PGN, was accompanied by Th1 polarization. The enhanced priming capacity of Poly I:C-stimulated DCs was associated with a strong upregulation of appropriate costimulatory molecules, including CD70, whereas that of PGN-stimulated DCs was associated with the release of a broad array of proinflammatory cytokines. Transcriptional profiling further supported the notion that the PGN- and Poly I:C-induced effects were mediated through binding to TLR2/nucleotide-binding oligomerization domain 2 and TLR3/MDA5, respectively. These data warrant further exploration of PGN and Poly I:C, alone or in combination, as DC-targeted adjuvants for intradermal cancer vaccines. [ABSTRACT FROM AUTHOR]